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WO2025233894A1 - Engineered meganucleases that target human mitochondrial genomes - Google Patents

Engineered meganucleases that target human mitochondrial genomes

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Publication number
WO2025233894A1
WO2025233894A1 PCT/IB2025/054863 IB2025054863W WO2025233894A1 WO 2025233894 A1 WO2025233894 A1 WO 2025233894A1 IB 2025054863 W IB2025054863 W IB 2025054863W WO 2025233894 A1 WO2025233894 A1 WO 2025233894A1
Authority
WO
WIPO (PCT)
Prior art keywords
promoter
engineered meganuclease
seq
cell
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2025/054863
Other languages
French (fr)
Inventor
Carlos T. MORAES
James Jefferson Smith
Ginger TOMBERLIN
John Morris
Wendy SHOOP
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Miami
Precision Biosciences Inc
Original Assignee
University of Miami
Precision Biosciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Miami, Precision Biosciences Inc filed Critical University of Miami
Publication of WO2025233894A1 publication Critical patent/WO2025233894A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present disclosure relates to recombinant meganucleases engineered to recognize and cleave recognition sequences found in the human mitochondrial genome.
  • the present disclosure further relates to the use of such recombinant meganucleases in methods for producing genetically-modified eukaryotic cells, and to a population of genetically- modified eukaryotic cells wherein the mitochondrial DNA has been modified.
  • BACKGROUND OF THE INVENTION In all organisms, mitochondria regulate cellular energy and metabolism under normal growth and development as well as in response to stress. Many of the proteins functioning in these roles are coded for in the mitochondrial genome. Thus, editing of the mitochondrial genome has diverse applications in both animals and plants.
  • mtDNA manipulation remains an underexplored area of science because of the inability to target mtDNA at high efficiencies and generate precise edits.
  • the mitochondrial genome is difficult to edit because it requires predictable repair mechanisms and delivery of an editing technology to this organelle.
  • there is an unmet need for precise editing of mtDNA which would open up an entire field of inquiry and opportunity in life sciences.
  • the ability to target and edit a defined region (preferably limited to just one gene) of the mitochondrial genome in a more predictable manner would be a clear benefit over currently available systems.
  • the invention provides an engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 in mitochondrial genomes of a eukaryotic cell, wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to a second recognition half- site of the recognition sequence and comprises a second hypervariable (HVR2) region.
  • HVR1 hypervariable
  • HVR2 hypervariable
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 7-10.
  • the HVR1 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-10.
  • the HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-10.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of any one of SEQ ID NOs: 7-10.
  • the HVR1 region comprises residues 215-270 of any one of SEQ ID NOs: 7-10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the HVR1 region comprises residues 215-270 of any one of SEQ ID NOs: 7-10.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of any one of SEQ ID NOs: 7-10.
  • the first subunit comprises a residue corresponding to residue 271 of any one of SEQ ID NOs: 7-10.
  • the first subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 9.
  • the first subunit comprises G, S, or A at a residue corresponding to residue 210 of any one of SEQ ID NOs: 7-10.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 271 of any one of SEQ ID NOs: 7-10.
  • the first subunit comprises residues 198-344 of any one of SEQ ID NOs: 7-10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the first subunit comprises residues 198-344 of any one of SEQ ID NOs: 7-10.
  • the first subunit comprises residues 196-354 of any one of SEQ ID NOs: 7-10.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs: 7-10.
  • the HVR2 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-10.
  • the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-10.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of any one of SEQ ID NOs: 7-10.
  • the HVR2 region comprises residues 24- 79 of any one of SEQ ID NOs: 7-10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the HVR2 region comprises residues 24-79 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 9.
  • the second subunit comprises a residue corresponding to residue 80 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises G, S, or A at a residue corresponding to residue 19 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 80 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises residues 7- 153 of any one of SEQ ID NOs: 7-10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the second subunit comprises residues 7-153 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises residues 6-153 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises residues 5-153 of any one of SEQ ID NOs: 7-10.
  • the second subunit comprises residues 4-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises residues 3-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises residues 2-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of any one of SEQ ID NOs: 7-10. Attorney Docket No.: P893391190WO (01242) In some embodiments, the second subunit comprises residues 1-153 of any one of SEQ ID NOs: 7-10.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.
  • the N-terminus of the linker is fused to the residue (i.e., a D residue) corresponding to residue 153 of any one of SEQ ID NOs: 7-10
  • the C-terminus of the linker is fused to the residue (i.e., a Y residue) corresponding to residue 196 of any one of SEQ ID NOs: 7- 10.
  • the linker comprises residues 154-195 of any one of SEQ ID NOs: 7-10.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of any one of SEQ ID NOs: 7-10.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of any one of SEQ ID NOs: 7-10.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 7-10.
  • the engineered meganuclease comprises an amino acid sequence of residues 4-343 of any one of SEQ ID NOs: 7-10.
  • the engineered meganuclease comprises an amino acid sequence of residues 3-343 of any one of SEQ ID NOs: 7-10.
  • the engineered meganuclease comprises an amino acid sequence of residues 2-343 of any one of SEQ ID NOs: 7-10.
  • the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 19-22.
  • the engineered meganuclease is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 19-22.
  • the engineered meganuclease comprises a mitochondrial transit peptide (MTP).
  • MTP mitochondrial transit peptide
  • Such engineered meganucleases described herein comprising an MTP are mitochondria-targeted engineered meganucleases.
  • the MTP comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence set forth in any one of SEQ ID NOs: 23-25.
  • the MTP comprises an amino acid sequence set forth in any one of SEQ ID NOs: 23-25.
  • the MTP is attached to the C-terminus of the engineered meganuclease.
  • the MTP is attached to the N-terminus of the engineered meganuclease.
  • the MTP is fused to the engineered meganuclease.
  • the MTP is attached to the engineered meganuclease by a polypeptide linker.
  • the engineered meganuclease is attached to a first MTP and a second MTP.
  • the first MTP and/or the second MTP comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence set forth in any one of SEQ ID NOs: 23-25.
  • the first MTP and/or the second MTP comprises an amino acid sequence set forth in any one of SEQ ID NOs: 23-25.
  • the first MTP and the second MTP are identical.
  • the first MTP and the second MTP are not identical.
  • the first MTP and/or the second MTP is fused to the engineered meganuclease.
  • the first MTP and/or the second MTP is attached to the engineered meganuclease by a polypeptide linker.
  • the engineered meganuclease is attached to a nuclear export sequence (NES).
  • the NES comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence set forth in SEQ ID NO: 26 or 27.
  • the NES comprises an amino acid sequence set forth in SEQ ID NO: 26 or 27.
  • the NES is attached at the N-terminus of the engineered meganuclease.
  • the NES is attached at the C-terminus of the engineered meganuclease. In some embodiments, the NES is fused to the engineered meganuclease. In some embodiments, the NES is attached to the engineered meganuclease by a polypeptide linker. In some embodiments, the engineered meganuclease comprises a first NES and a second NES. In some embodiments, the first NES is attached at the N-terminus of the engineered meganuclease, and the second NES is attached at the C-terminus of the engineered meganuclease.
  • the first NES and/or the second NES comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, Attorney Docket No.: P893391190WO (01242) 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence set forth in SEQ ID NO: 26 or 27.
  • the first NES and/or the second NES comprises an amino acid sequence set forth in SEQ ID NO: 26 or 27.
  • the first NES and the second NES are identical.
  • the first NES and the second NES are not identical.
  • the first NES and/or the second NES is fused to the engineered meganuclease. In some embodiments, the first NES and/or the second NES is attached to the engineered meganuclease by a polypeptide linker.
  • the invention provides a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein. In some embodiments, the polynucleotide is an mRNA. In another aspect, the invention provides a recombinant DNA construct comprising a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein.
  • the recombinant DNA construct encodes a recombinant virus genome comprising the polynucleotide.
  • the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adeno-associated virus (AAV).
  • the recombinant virus is a recombinant AAV.
  • the recombinant AAV is a muscle-tropic recombinant AAV or a central nervous system (CNS)-tropic recombinant AAV.
  • CNS central nervous system
  • the recombinant AAV has an AAV9 capsid, a MyoAAV capsid, or an AAVMYO capsid.
  • the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the engineered meganuclease.
  • the promoter is a constitutive promoter.
  • the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter.
  • the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter.
  • the promoter is an MHCK7 promoter, a truncated MCK (tMCK) promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter.
  • the promoter is a central nervous system (CNS) cell-specific promoter.
  • the promoter is a stem cell-specific promoter, a CD34+ HSC-specific promoter, a muscle-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell-specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, a kidney cell-specific promoter, a bone marrow cell-specific promoter, or an ear hair cell-specific promoter.
  • the recombinant DNA construct is a plasmid DNA.
  • Attorney Docket No.: P893391190WO (01242) the invention provides a recombinant virus comprising a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein.
  • the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant AAV.
  • the recombinant virus is a recombinant AAV.
  • the recombinant AAV is a muscle-tropic recombinant AAV or a central nervous system (CNS)-tropic recombinant AAV.
  • the recombinant AAV has an AAV9 capsid, a MyoAAV capsid, or an AAVMYO capsid.
  • the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the engineered meganuclease.
  • the promoter is a constitutive promoter.
  • the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter.
  • the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter.
  • the promoter is an MHCK7 promoter, a truncated MCK (tMCK) promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter.
  • the promoter is a central nervous system (CNS) cell-specific promoter.
  • CNS central nervous system
  • the promoter is a stem cell-specific promoter, a CD34+ HSC-specific promoter, a muscle-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell-specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, a kidney cell-specific promoter, a bone marrow cell-specific promoter, or an ear hair cell-specific promoter.
  • the cleavage site is repaired by non-homologous end joining, such that the recognition sequence comprises an insertion or deletion.
  • the mutant mitochondrial genomes comprising the recognition sequence are degraded in the genetically-modified eukaryotic cell. In some embodiments, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of mutant mitochondrial genomes comprising the recognition sequence are degraded in the genetically-modified eukaryotic cell.
  • the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising the recognition sequence increases in the genetically-modified eukaryotic cell. In some embodiments, the ratio increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more.
  • the percentage of wild-type mitochondrial genomes in the genetically-modified eukaryotic cell is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, of the total mitochondrial genomes in the genetically-modified eukaryotic cell.
  • the percentage of mutant mitochondrial genomes comprising the recognition sequence in the genetically-modified eukaryotic cell decreases by about Attorney Docket No.: P893391190WO (01242) 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more.
  • cellular respiration in the genetically-modified eukaryotic cell increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. In some embodiments, cellular respiration in the genetically-modified eukaryotic cell increases by about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more.
  • the invention provides a method for producing a population of eukaryotic cells comprising a plurality of genetically-modified cells, the method comprising introducing into a plurality of eukaryotic cells in the population: (a) a polynucleotide comprising a nucleic acid sequence encoding a mitochondria-targeted engineered meganuclease (e.g., comprising an MTP) described herein, wherein the mitochondria-targeted engineered meganuclease is expressed in the plurality of eukaryotic cells; or (b) a mitochondria-targeted engineered meganuclease described herein; wherein the mitochondria-targeted engineered meganuclease produces a cleavage site at a recognition sequence comprising SEQ ID NO: 3 in mutant mitochondrial genomes of the plurality of eukaryotic cells.
  • a mitochondria-targeted engineered meganuclease e.g., comprising an MTP
  • the cleavage site is repaired by non-homologous end joining, such that the recognition sequence comprises an insertion or deletion.
  • the mutant mitochondrial genomes comprising the recognition sequence are degraded in the plurality of genetically-modified eukaryotic cells. In some embodiments, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of mutant mitochondrial genomes comprising the recognition sequence are degraded in the plurality of genetically-modified eukaryotic cells.
  • the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising the recognition sequence increases in the plurality of genetically-modified eukaryotic cells. In some embodiments, the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising the recognition sequence increases in the population of eukaryotic cells.
  • the percentage of wild-type mitochondrial genomes in the plurality of Attorney Docket No.: P893391190WO (01242) genetically-modified eukaryotic cells increases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more.
  • the percentage of wild-type mitochondrial genomes in the population of eukaryotic cells increases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more.
  • the percentage of mutant mitochondrial genomes comprising the recognition sequence in the plurality of genetically-modified eukaryotic cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more.
  • the percentage of mutant mitochondrial genomes comprising the recognition sequence in the population of eukaryotic cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more.
  • cellular respiration in the plurality of genetically-modified eukaryotic cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more.
  • cellular respiration in the plurality of genetically-modified eukaryotic cells increases by about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more. In some embodiments, cellular respiration in the population of eukaryotic cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more.
  • cellular respiration in the population of eukaryotic cells increases by about 30-40%, about 40-50%, about 50-60%, about 60- 70%, about 70-80%, about 80-90%, about 90-100%, or more.
  • the recognition sequence is within a region of the mitochondrial DNA associated with a mitochondrial disorder.
  • the mitochondrial disorder is a mitochondrial DNA common deletion disorder.
  • the mutant mitochondrial genomes lack the nucleotides positioned between nucleotides 8,470 and 13,447 in a wild-type mitochondrial genome.
  • the method is performed in vivo. In some embodiments, the method is performed in vitro.
  • the polynucleotide is an mRNA.
  • the polynucleotide is any mRNA described herein. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is any recombinant DNA construct described herein. In some embodiments, the Attorney Docket No.: P893391190WO (01242) polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus. In some embodiments, the recombinant virus is any recombinant virus described herein.
  • the recombinant virus is a recombinant AAV.
  • the recombinant AAV is a muscle-tropic recombinant AAV or a CNS-tropic recombinant AAV.
  • the recombinant AAV has an AAV9 capsid, a MyoAAV capsid, or an AAVMYO capsid.
  • the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the mitochondria-targeted engineered meganuclease. In some embodiments, the promoter is a constitutive promoter.
  • the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter.
  • the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter.
  • the promoter is an MHCK7 promoter, a truncated MCK (tMCK) promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter.
  • the promoter is a central nervous system (CNS) cell-specific promoter.
  • CNS central nervous system
  • the promoter is a stem cell-specific promoter, a CD34+ HSC-specific promoter, a muscle-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell- specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell- specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, a kidney cell-specific promoter, a bone marrow cell-specific promoter, or an ear hair cell-specific promoter.
  • the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the human cell is a human muscle cell, a human muscle stem cell, or a human CNS cell.
  • the eukaryotic cell is a stem cell, a CD34+ HSC, a muscle cell, a skeletal muscle cell, a myotube cell, a muscle satellite cell, a neuron, an astrocyte, a microglia cell, an eye cell, a retinal cell, a retinal ganglion cell, a retinal pigmentary epithelium cell, a pancreatic cell, a pancreatic beta cell, a kidney cell, a bone marrow cell, or an ear hair cell.
  • the invention provides a genetically-modified eukaryotic cell, or a population of genetically-modified eukaryotic cells, produced by any method described herein for producing a genetically-modified eukaryotic cell or any method for producing a population of eukaryotic cells comprising a plurality of genetically-modified cells.
  • the invention provides a method for degrading mutant mitochondrial genomes in a target cell in a subject, or in a population of target cells in a subject, the method comprising delivering to the target cell or the population of target cells: (a) a polynucleotide Attorney Docket No.: P893391190WO (01242) comprising a nucleic acid sequence encoding a mitochondria-targeted engineered meganuclease (e.g., comprising an MTP) described herein, wherein the mitochondria-targeted engineered meganuclease is expressed in the target cell or the population of target cells; or (b) a mitochondria- targeted engineered meganuclease described herein; wherein the mitochondria-targeted engineered meganuclease produces a cleavage site in the mutant mitochondrial genomes at a recognition sequence comprising SEQ ID NO: 3, and wherein the mutant mitochondrial genomes are degraded.
  • the mutant mitochondrial genomes comprise a mtDNA common deletion. In some embodiments, the mutant mitochondrial genomes lack the nucleotides positioned between nucleotides 8,470 and 13,447 in a wild-type mitochondrial genome. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the target cell or the population of target cells are human muscle cells, human muscle stem cells, or human CNS cells.
  • the target cell is a stem cell, a CD34+ HSC, a muscle cell, a skeletal muscle cell, a myotube cell, a muscle satellite cell, a neuron, an astrocyte, a microglia cell, an eye cell, a retinal cell, a retinal ganglion cell, a retinal pigmentary epithelium cell, a pancreatic cell, a pancreatic beta cell, a kidney cell, a bone marrow cell, or an ear hair cell
  • the population of target cells is a population of stem cells, CD34+ HSCs, muscle cells, skeletal muscle cells, myotube cells, muscle satellite cells, neurons, astrocytes, microglia cells, eye cells, retinal cells, retinal ganglion cells, retinal pigmentary epithelium cells, pancreatic cells, pancreatic beta cells, kidney cells, bone marrow cells, or ear hair cells.
  • the polynucleotide is an mRNA. In some embodiments, the polynucleotide is any mRNA described herein. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is any recombinant DNA construct described herein. In some embodiments, the polynucleotide is introduced into the target cell, or the population of target cells, by a lipid nanoparticle. In some embodiments, the polynucleotide is introduced into the target cell, or the population of target cells, by a recombinant virus. In some embodiments, the recombinant virus is any recombinant virus described herein.
  • the recombinant virus is a recombinant AAV.
  • the recombinant AAV is a muscle-tropic recombinant AAV or a CNS-tropic recombinant AAV.
  • the recombinant AAV has an AAV9 capsid, a MyoAAV capsid, or an AAVMYO capsid.
  • the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the mitochondria-targeted engineered meganuclease. In some embodiments, the promoter is a constitutive promoter.
  • the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter.
  • the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter.
  • the promoter is an MHCK7 promoter, a truncated MCK (tMCK) promoter, a CK8 Attorney Docket No.: P893391190WO (01242) promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter.
  • the promoter is a central nervous system (CNS) cell-specific promoter.
  • the promoter is a stem cell-specific promoter, a CD34+ HSC-specific promoter, a muscle-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell-specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, a kidney cell-specific promoter, a bone marrow cell-specific promoter, or an ear hair cell-specific promoter.
  • CNS central nervous system
  • mutant mitochondrial genomes comprising the recognition sequence are degraded in the target cell or the population of the target cells.
  • the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising the recognition sequence increases in the target cell or the population of target cells.
  • the ratio increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more.
  • the percentage of wild-type mitochondrial genomes in the target cell or the population of target cells is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, of the total mitochondrial genomes in the target cell or the population of target cells.
  • the percentage of mutant mitochondrial genomes comprising the recognition sequence in the target cell or the population of target cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more.
  • cellular respiration in the target cell or the population of target cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more.
  • cellular respiration in Attorney Docket No.: P893391190WO (01242) the target cell or the population of target cells increases by about 30-40%, about 40-50%, about 50- 60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more.
  • the invention provides a method for treating a condition associated with a mitochondrial mtDNA common deletion in a subject, the method comprising administering to the subject: (a) a therapeutically-effective amount of a polynucleotide comprising a nucleic acid sequence encoding a mitochondria-targeted engineered meganuclease (e.g., comprising an MTP) described herein, wherein the polynucleotide is delivered to a target cell, or a population of target cells, in the subject, wherein the mitochondria-targeted engineered meganuclease is expressed in the target cell or the population of target cells; or (b) a therapeutically-effective amount of a mitochondria-targeted engineered meganuclease described herein, wherein the engineered meganuclease is delivered to a target cell, or a population of target cells, in the subject; wherein the mitochondria-targeted engineered meganuclease produces a cleavage site
  • the mutant mitochondrial genomes comprise the mtDNA common deletion. In some embodiments, the mutant mitochondrial genomes lack the nucleotides positioned between nucleotides 8,470 and 13,447 in a wild-type mitochondrial genome. In some embodiments, the method reduces or ameliorates one or more symptoms associated with the mtDNA common deletion. In some embodiments, the method comprises administering any pharmaceutical composition described herein that comprises a mitochondria- targeted meganuclease described herein, or a polynucleotide described herein that encodes a mitochondria-targeted engineered meganuclease described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.
  • the target cell or population of target cells are human muscle cells, human muscle stem cells, or human CNS cells.
  • the target cell is a stem cell, a CD34+ HSC, a muscle cell, a skeletal muscle cell, a myotube cell, a muscle satellite cell, a neuron, an astrocyte, a microglia cell, an eye cell, a retinal cell, a retinal ganglion cell, a retinal pigmentary epithelium cell, a pancreatic cell, a pancreatic beta cell, a kidney cell, a bone marrow cell, or an ear hair cell, or the population of target cells is a population of stem cells, CD34+ HSCs, muscle cells, skeletal muscle cells, myotube cells, muscle satellite cells, neurons, astrocytes, microglia cells, eye cells, retinal cells, retinal ganglion cells, retinal pigmentary epithelium cells, pancreatic cells, pancreatic beta cells, kidney cells, bone marrow cells, or an ear hair cell, or the
  • the condition is a condition of the bone marrow, the pancreas, muscle, skeletal muscle, central nervous system, the eye, or the ears.
  • the condition is Pearson Syndrome, Kearns-Sayre Syndrome (KSS), Progressive External Ophthalmoplegia (PEO), diabetes, or kidney dysfunction.
  • the polynucleotide is an mRNA.
  • the polynucleotide Attorney Docket No.: P893391190WO (01242) is any mRNA described herein.
  • the polynucleotide is a recombinant DNA construct.
  • the polynucleotide is any recombinant DNA construct described herein.
  • the polynucleotide is delivered to the target cell, or the population of target cells, by a lipid nanoparticle. In some embodiments, the polynucleotide is delivered to the target cell, or the population of target cells, by a recombinant virus. In some embodiments, the recombinant virus is any recombinant virus described herein. In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV is a muscle-tropic recombinant AAV or a CNS-tropic recombinant AAV.
  • the recombinant AAV has an AAV9 capsid, a MyoAAV capsid, or an AAVMYO capsid.
  • the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the mitochondria-targeted engineered meganuclease.
  • the promoter is a constitutive promoter.
  • the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter.
  • the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter.
  • the promoter is an MHCK7 promoter, a truncated MCK (tMCK) promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter.
  • the promoter is a central nervous system (CNS) cell-specific promoter.
  • the promoter is a stem cell-specific promoter, a CD34+ HSC-specific promoter, a muscle-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell- specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, a kidney cell-specific promoter, a bone marrow cell-specific promoter, or an ear hair cell-specific promoter.
  • mutant mitochondrial genomes comprising the recognition sequence are degraded in the target cell or the population of the target cells.
  • the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising the recognition sequence increases in the target cell or the population of target cells.
  • the ratio increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about Attorney Docket No.: P893391190WO (01242) 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more.
  • the percentage of wild-type mitochondrial genomes in the target cell or the population of target cells is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, of the total mitochondrial genomes in the target cell or the population of target cells.
  • the percentage of mutant mitochondrial genomes comprising the recognition sequence in the target cell or population of target cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more.
  • cellular respiration in the target cell or the population of target cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more.
  • cellular respiration in the target cell or the population of target cells increases by about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more.
  • FIGURES Figure 1A shows the MIT 9-10 recognition sequence (SEQ ID NO: 3) present in mutant mitochondrial genomes comprising the common deletion.
  • the MIT 9-10 recognition sequence targeted by engineered meganucleases of the invention comprises two recognition half-sites. Each recognition half-site comprises 9 base pairs, separated by a 4 base pair central sequence.
  • the MIT Attorney Docket No.: P893391190WO (01242) 9-10 recognition sequence comprises two recognition half-sites referred to as MIT9 and MIT10.
  • Figure 1B illustrates the deletion of nucleotides from human mitochondrial DNA that is referred to as the common deletion. The common deletion results in the removal of a 4977 basepair region of the mitochondrial DNA, reducing the size of the genome from 16,569 basepairs to 11,592 basepairs.
  • the MIT 9-10 recognition sequence (SEQ ID NO: 3) is not present in wild- type mitochondrial DNA but is generated following the common deletion.
  • Figure 2 shows that the engineered meganucleases described herein comprise two subunits, wherein the first subunit comprising the HVR1 region binds to a first recognition half-site (e.g., MIT9) and the second subunit comprising the HVR2 region binds to a second recognition half-site (e.g., MIT10).
  • the first subunit comprising the HVR1 region can be positioned as either the N- terminal or C-terminal subunit.
  • the second subunit comprising the HVR2 region can be positioned as either the N-terminal or C-terminal subunit.
  • Figure 3 shows an alignment of MIT 9-10 meganucleases exemplified herein, including MIT 9-10x.3 (SEQ ID NO: 7), MIT 9-10L.90 (SEQ ID NO: 8), MIT 9-10L.209 (SEQ ID NO: 9), and MIT 9-10L.210 (SEQ ID NO: 10).
  • Figure 4 shows a schematic of reporter assay in CHO cells for evaluating engineered meganucleases targeting the MIT 9-10 recognition sequence (SEQ ID NO: 3) or a recognition sequence present in wild-type human mitochondrial DNA (SEQ ID NO: 5) that differs from MIT 9- 10 by only a single nucleotide.
  • a CHO cell line was produced in which a reporter cassette was integrated stably into the genome of the cell.
  • the reporter cassette comprised, in 5' to 3' order: an SV40 Early Promoter; the 5' 2/3 of the GFP gene; the recognition sequence for an engineered meganuclease described herein (e.g., the MIT 9- 10 recognition sequence or the corresponding wild-type sequence); the recognition sequence for the CHO-23/24 meganuclease (WO/2012/167192); and the 3' 2/3 of the GFP gene.
  • Cells stably transfected with this cassette did not express GFP in the absence of a DNA break-inducing agent.
  • Meganucleases were introduced by transduction of an mRNA encoding each meganuclease.
  • the duplicated regions of the GFP gene recombined with one another to produce a functional GFP gene.
  • the percentage of GFP-expressing cells could then be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the meganucleases.
  • HELA cells were transfected with a plasmid coding for the MIT 9-10x.3 meganuclease that comprised a mitochondrial transit peptide (MTP) at its N-terminus.
  • Immunocytological staining was achieved with anti-flag antibody (Meganucelase marker), and Mitotracker Red, which stains mitochondria.
  • Cells were viewed under 40x magnification with Zeiss LSM710 confocal microscope.
  • Figure 7 depicts the development of mitochondria-targeted engineered meganucleases specific for the human mtDNA common deletion, and a comparison to previously developed mitoTALENs.
  • Figure 7A provides an illustration exemplifying the approach to specifically eliminate the common deletion mtDNA in living cells.
  • the mitoTALEN monomers bind to regions that are too far apart in the wild-type mtDNA for FokI nuclease to dimerize and cleave the DNA. Only after the common deletion occurs are the mitoTALEN monomers close enough to allow the FokI nuclease to dimerize and cleave the genome.
  • the mitochondria-targeted MIT 9-10 meganucleases described herein are monomeric and bind to the MIT 9-10 recognition sequence that spans the breakpoint after the common deletion occurs. The MIT 9-10 recognition sequence is not present until after the common deletion occurs.
  • Figure 7B depicts the common deletion breakpoint region showing the DNA binding for mitoTALEN monomers and the MIT 9-10 meganuclease.
  • the box in the center sequence illustrates the 13-bp direct repeat believed to mediate common deletion formation.
  • the MIT 9-10 nuclease binding region avoids part of this direct repeat.
  • Figure 7C shows relative levels of wild-type genomes were increased, using a 3-primer PCT, in sorted cells expressing both monomers (yellow) in the case of mitoTALEN and in “green” cells in the case of the mitochondria-targeted MIT 9-10 meganuclease.
  • the arrows represent the amplifications depicted in panel A.
  • Figure 7D provides results of experiments using the same 3-primer PCR technique in which sorted cells were analyzed after transfection with a plasmid coding for the MIT 9-10 meganuclease (co-transfected with a GFP-expressing plasmid). Sorted cells were “Black” (negative) and “Green” (positive). The lane marked “GFP” was transfected with GFP plasmid only. The lane marked “MitoMega” was transfected with the MIT 9-10 meganuclease but not sorted.
  • Figure 8 shows the percentage (%) indel formation at either the mutant MIT 9-10 site or the corresponding wild-type site in cells transfected with either GFP mRNA at 500ng (negative control), or mRNA encoding the MIT 9-10x.3 or MIT 9-10L.90 engineered meganucleases at 500ng, 50ng, or 5ng, respectively.
  • Each of the tested meganucleases were fused to a mitochondrial transit peptide (MTP) at their N-terminus.
  • MTP mitochondrial transit peptide
  • Figure 9 shows flow cytometry for GFP positive cells in a FlpIn CHO cell reporter system, wherein cells comprising either the MIT 9-10 recognition sequence or the corresponding wild-type sequence were transfected with mRNA encoding the indicated MIT 9-10 meganucleases or a mock negative control at low and high doses of mRNA.
  • Figure 10 shows the immunocytochemistry imaging of COS-7 cells transfected with the MIT 9-10L.209, MIT 9-10L.210, or MIT 9-10L.90 meganucleases that each comprised an MTP at the N-terminus and were fused to a FLAG tag.
  • FIG. 11 shows the shift in mitochondrial heteroplasmic DNA levels in BH10.9 cells clone 1-C-7-65% co-transfected with the MIT9-10 L.209, MIT9-10L.210 and MIT9-10L.90 meganucleases, each comprising an N-terminal MTP, and a GFP plasmid.
  • FIG. 12 shows the shift in mitochondrial heteroplasmic DNA levels in BH10.9 cells clone 1-C-7-65% co-transfected with the MIT9-10 L.209, MIT9-10L.210 and MIT9-10L.90 meganucleases, each comprising an N-terminal MTP, and a GFP plasmid. Cells were sorted for GFP 48 hours after transfection and the percentage (%) change in levels of mutant mtDNA compared to untransfected control cells was calculated.
  • Figure 13 provides semiquantitative PCR results of experiments where BH10.9 cells clone 1-C-7-65% were co-transfected with the MIT9-10 L.209, MIT9-10L.210, and MIT9-10L.90 meganucleases, each comprising an N-terminal MTP, and a GFP plasmid.
  • Cells were sorted for GFP expression and a 3-primer semi-quantitative PCR technique was used to determine levels of WT or mutant mtDNA in untransfected “black cells” (Bl) and in transfected “green cells” (Gr).
  • the upper band is the WT mtDNA sequence and the lower band is the mutant mtDNA sequence.
  • Figure 14 shows PCR analysis of heteroplasmic shift after 7 days and 14 days in cybrid cells carrying approximately 80% mutant mtDNA, that were electroporated with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each comprising an MTP at their N-terminus, or a GFP mRNA control.
  • Figure 15 shows a primer strategy for PCR analysis of wild-type mitochondrial DNA and mitochondrial DNA comprising the common deletion in cybrid cells, which carry approximately 80% mutant mtDNA.
  • the cybrid cells were electroporated with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each comprising an MTP at their N-terminus, or a GFP mRNA control.
  • Figure 16 shows a PCR analysis of wild-type mitochondrial DNA and mitochondrial DNA comprising the common deletion in cybrid cells, which carry approximately 80% mutant mtDNA.
  • the cybrid cells were electroporated with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each comprising an MTP at their N-terminus, or a GFP mRNA control.
  • Figure 17 shows a PCR analysis of mitochondrial DNA copy number in cybrid cells, which carry approximately 80% mutant mtDNA.
  • the cybrid cells were electroporated with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each comprising an MTP at their N- terminus, or a GFP mRNA control.
  • PCR analysis was conducted on days 4 and 14.
  • Figure 18 shows cellular respiration of cybrids cells comprising the common deletion in mitochondrial DNA 21 days following electroporation with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each linked to an MTP at their N-terminus, or GFP mRNA as a control.
  • Figure 18A shows changes in the normalized oxygen consumption rate (OCR).
  • Figure 18B shows changes in the extracellular acidification rate (ECAR).
  • OCR normalized oxygen consumption rate
  • ECAR extracellular acidification rate
  • Figure 19 shows basal and maximal cellular respiration rates of cybrids cells comprising the common deletion in mitochondrial DNA 21 days following electroporation with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each linked to an MTP at their N-terminus, or GFP mRNA as a control.
  • SEQ ID NO: 1 sets forth the amino acid sequence of the I-CreI meganuclease found in Chlamydomonas reinhardtii.
  • SEQ ID NO: 2 sets forth the amino acid sequence of a LAGLIDADG motif.
  • SEQ ID NO: 3 sets forth the nucleic acid sequence of the MIT 9-10 recognition sequence (sense).
  • SEQ ID NO: 4 sets forth the nucleic acid sequence of the MIT 9-10 recognition sequence reverse complement.
  • SEQ ID NO: 5 sets forth the nucleic acid sequence of nucleotides 13,445-13,466 of wild- type human mitochondrial DNA (sense).
  • SEQ ID NO: 6 sets forth the nucleic acid sequence of the reverse complement of nucleotides 13,445-13,466 of wild-type human mitochondrial DNA.
  • SEQ ID NO: 7 sets forth the nucleic acid sequence of the MIT 9-10x.3 engineered meganuclease.
  • SEQ ID NO: 8 sets forth the amino acid sequence of the MIT 9-10.L90 engineered meganuclease.
  • SEQ ID NO: 9 sets forth the amino acid sequence of the MIT 9-10.L209 engineered meganuclease.
  • SEQ ID NO: 10 sets forth the amino acid sequence of the MIT 9-10.L210 engineered meganuclease.
  • SEQ ID NO: 11 sets forth the amino acid sequence of the MIT 9-10x.3 MIT9-binding subunit.
  • SEQ ID NO: 12 sets forth the amino acid sequence of the MIT 9-10.L90 MIT9- binding subunit.
  • SEQ ID NO: 13 sets forth the amino acid sequence of the MIT 9-10.L209 MIT9- binding subunit.
  • SEQ ID NO: 14 sets forth the amino acid sequence of the MIT 9-10.L210 MIT9- binding subunit.
  • SEQ ID NO: 15 sets forth the amino acid sequence of the MIT 9-10x.3 MIT10- binding subunit.
  • SEQ ID NO: 16 sets forth the amino acid sequence of the MIT 9-10.L90 MIT10- binding subunit.
  • SEQ ID NO: 17 sets forth the amino acid sequence of the MIT 9-10.L209 MIT10- binding subunit.
  • SEQ ID NO: 18 sets forth the amino acid sequence of the MIT 9-10.L210 MIT10- binding subunit.
  • SEQ ID NO: 19 sets forth a nucleic acid sequence encoding the MIT 9-10x.3 engineered meganuclease.
  • SEQ ID NO: 20 sets forth a nucleic acid sequence encoding the MIT 9-10.L90 engineered meganuclease.
  • SEQ ID NO: 21 sets forth a nucleic acid sequence encoding the MIT 9-10.L209 engineered meganuclease.
  • SEQ ID NO: 22 sets forth a nucleic acid sequence encoding the MIT 9-10.L210 engineered meganuclease.
  • SEQ ID NO: 23 sets forth the amino acid sequence of the COX VIII MTP .
  • SEQ ID NO: 24 sets forth the amino acid sequence of the SU9 MTP.
  • SEQ ID NO: 25 sets forth the amino acid sequence of the COX VIII-SU9 MTP.
  • SEQ ID NO: 26 sets forth the amino acid sequence of the MVMp NS2 NES sequence.
  • SEQ ID NO: 27 sets forth the amino acid sequence of the NES sequence.
  • SEQ ID NO: 28 sets forth the nucleic acid sequence of the PCR primer used to determine WT or mutant mtDNA frequency.
  • SEQ ID NO: 29 sets forth the nucleic acid sequence of the PCR primer used to determine WT or mutant mtDNA frequency.
  • SEQ ID NO: 30 sets forth the nucleic acid sequence of the PCR primer used to determine WT or mutant mtDNA frequency.
  • SEQ ID NO: 31 sets forth the nucleic acid sequence of a ddPCR primer F1.
  • SEQ ID NO: 32 sets forth the nucleic acid sequence of a ddPCR primer R1.
  • SEQ ID NO: 33 sets forth the nucleic acid sequence of a ddPCR primer P1.
  • SEQ ID NO: 34 sets forth the nucleic acid sequence of a ddPCR primer P2.
  • SEQ ID NO: 35 sets forth the nucleic acid sequence of a ddPCR primer P3.
  • SEQ ID NO: 36 sets forth the nucleic acid sequence of a ddPCR primer P3.
  • SEQ ID NO: 37 sets forth the nucleic acid sequence of a ddPCR primer P3.
  • SEQ ID NO: 38 sets forth the nucleic acid sequence of a ddPCR primer hND4-TAMRA.
  • SEQ ID NO: 39 sets forth the nucleic acid sequence of a ddPCR primer PrF.
  • SEQ ID NO: 40 sets forth the nucleic acid sequence of a ddPCR primer PrB.
  • SEQ ID NO: 41 sets forth the nucleic acid sequence of a ddPCR primer hCOM deletion- FAM.
  • SEQ ID NO: 42 sets forth the nucleic acid sequence of a ddPCR primer PrF.
  • SEQ ID NO: 43 sets forth the nucleic acid sequence of a ddPCR primer PrB.
  • SEQ ID NO: 44 sets forth the nucleic acid sequence of a ddPCR primer hND1-HEX.
  • SEQ ID NO: 45 sets forth the nucleic acid sequence of a ddPCR primer PrF.
  • SEQ ID NO: 46 sets forth the nucleic acid sequence of a ddPCR primer PrB.
  • SEQ ID NO: 47 sets forth the nucleic acid sequence of a ddPCR primer mus18s-Cy5.
  • SEQ ID NO: 48 sets forth the nucleic acid sequence of a ddPCR primer PrF.
  • SEQ ID NO: 49 sets forth the nucleic acid sequence of a ddPCR primer PrB.
  • the term "5’ cap” (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the “front” or 5’ end of a eukaryotic messenger RNA shortly after the start of transcription.
  • the 5’ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other.
  • the 5’ end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase.
  • This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.
  • the capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
  • allele refers to one of two or more variant forms of a gene.
  • the term “allogeneic” or, alternatively, “allogenic,” refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced.
  • two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.
  • allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
  • constitutive promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • a control or “a control cell” refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a genetically-modified cell.
  • a control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the genetically-modified cell; (b) a cell of the same genotype as the genetically-modified cell but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest); or, (c) a cell genetically identical to the genetically-modified cell but which is not exposed to conditions or stimuli or further genetic modifications that would induce expression of altered genotype or phenotype.
  • a control or control cell of the instant invention can be a cell or population of cells that does not comprise an engineered meganuclease or a polynucleotide having an amino acid sequence encoding an engineered meganuclease.
  • the term “corresponding to” with respect to modifications of two proteins or amino acid sequences is used to indicate that a specified modification in the first protein is a substitution of the same amino acid residue as in the modification in the second protein, and that the amino acid position of the modification in the first protein corresponds to or aligns with the amino acid position of the modification in the second protein when the two proteins are subjected to standard sequence alignments (e.g., using the BLASTp program) and aligned for maximum sequence identity across the entire subunit or protein.
  • residues X and Y correspond to each other in a sequence alignment and despite the fact that X and Y may be at different position relative to the N-terminus or C-terminus.
  • the term “disrupted” or “disrupts” or “disrupts expression” or “disrupting a target sequence” refers to the introduction of a mutation (e.g., frameshift mutation) that interferes with the gene function and prevents expression and/or function of the polypeptide/expression product encoded thereby.
  • nuclease-mediated disruption of a gene can result in the expression of a truncated protein and/or expression of a protein that does not retain its wild-type function.
  • introduction of a donor template into a gene can result in no expression of an encoded protein, expression of a truncated protein, and/or expression of a protein that does not retain its wild-type function.
  • the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or Attorney Docket No.: P893391190WO (01242) other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • the term “endogenous” in reference to a nucleotide sequence or protein is intended to mean a sequence or protein that is naturally comprised within or expressed by a cell.
  • the terms “exogenous” or “heterologous” in reference to a nucleotide sequence or amino acid sequence are intended to mean a sequence that is purely synthetic, that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • expression refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • viruses e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses
  • the term “genetically-modified” refers to a cell or organism in which, or in an ancestor of which, a genomic DNA sequence has been deliberately modified by recombinant technology.
  • a “genetically-modified” cell may refer to a cell wherein the mitochondrial DNA has been deliberately modified by recombinant technology.
  • the term “homologous recombination” or “HR” refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g., Cahill et al. (2006), Front. Biosci.11:1958-1976).
  • the homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell.
  • homology arms or “sequences homologous to sequences flanking a nuclease cleavage site” refer to sequences flanking the 5' and 3' ends of a nucleic acid molecule, which promote insertion of the nucleic acid molecule into a cleavage site generated by a nuclease.
  • homology arms can have a length of at least 50 base pairs, preferably at least 100 base pairs, and up to 2000 base pairs or more, and can have at least 90%, preferably at least Attorney Docket No.: P893391190WO (01242) 95%, or more, sequence homology to their corresponding sequences in the genome. In some embodiments, the homology arms are about 500 base pairs.
  • in vitro transcribed RNA refers to RNA, preferably mRNA, which has been synthesized in vitro.
  • the in vitro transcribed RNA is generated from an in vitro transcription vector.
  • the in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • the term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector.
  • lipid nanoparticle refers to a lipid composition having a typically spherical structure with an average diameter between 10 and 1000 nanometers.
  • lipid nanoparticles can comprise at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid.
  • Lipid nanoparticles known in the art that are suitable for encapsulating nucleic acids, such as mRNA, are contemplated for use in the invention.
  • the term “modification” with respect to recombinant proteins means any insertion, deletion, or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., a wild-type or a native sequence).
  • a reference sequence e.g., a wild-type or a native sequence.
  • NHEJ non-homologous end-joining
  • DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair.
  • cleavage at a target recognition sequence results in NHEJ at a target recognition site.
  • Nuclease-induced cleavage of a target site in the coding sequence of a gene followed by DNA repair by NHEJ can introduce mutations into the coding sequence, such as frameshift mutations, that disrupt gene function.
  • engineered nucleases can be used to effectively knock-out a gene in a population of cells.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino Attorney Docket No.: P893391190WO (01242) acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns.
  • operably linked is intended to mean a functional linkage between two or more elements.
  • an operable linkage between a nucleic acid sequence encoding a nuclease as disclosed herein and a regulatory sequence is a functional link that allows for expression of the nucleic acid sequence encoding the nuclease.
  • Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame.
  • the word “or” is used in the inclusive sense of “and/or” and not the exclusive sense of “either/or.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • reduced or decreased refers to a reduction in the percentage of cells or ratio of cells in a population of cells that comprise mutant mitochondrial genomes having the mtDNA common deletion when compared to a population of control cells.
  • “reduced” or “decreased” refers to a reduction in the percentage of mutant mitochondrial genomes or ratio of mutant mitochondrial genomes to wild-type mitochondrial genomes in a single cell or in a population of cells. Such a reduction is up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100%. Accordingly, the term “reduced” encompasses both a partial reduction and a complete reduction of mutant mtDNA.
  • sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/), and are described in, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet.3:266-272; Madden et al. (1996), Meth. Enzymol.266:131-141; Altschul et al. (1997), Nucleic Acids Res.25:3389-3402); Zhang et al. (2000), J. Comput.
  • poly(A) is a series of adenosines attached by polyadenylation to the mRNA.
  • the 3’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • the terms “recombinant” or “engineered,” with respect to a protein means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein and cells or organisms that express the protein. With respect to a nucleic acid, the term “recombinant” or “engineered” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques.
  • Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation, and other gene transfer technologies; homologous recombination; site- directed mutagenesis; and gene fusion.
  • a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host is not considered recombinant or engineered.
  • the term “recombinant DNA construct,” “recombinant construct,” “expression cassette,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA fragment” are used interchangeably herein and are single or double-stranded polynucleotides.
  • tissue-specific promoter or “cell-specific promoter” refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • transfected or “transformed” or “transduced” or “nucleofected” refer to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • the term “transfer vector” refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “transfer vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like.
  • viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • transient refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
  • vector or “recombinant DNA vector” may be a construct that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art.
  • Vectors can include, without limitation, plasmid vectors and recombinant AAV vectors, or any other vector known in the art suitable for delivering a gene to a target cell. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleotides or nucleic acid sequences of the invention.
  • a “vector” also refers to a viral vector.
  • Viral vectors can include, without limitation, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (AAV).
  • wild-type refers to the most common naturally occurring allele (i.e., polynucleotide sequence) in the allele population of the same type of gene, wherein a polypeptide encoded by the wild-type allele has its original functions.
  • wild-type also refers to a polypeptide encoded by a wild-type allele.
  • wild-type can also refer to a cell, an organism, and/or a subject which possesses a wild-type allele of a particular gene, or a cell, an organism, and/or a subject used for comparative purposes.
  • attorney Docket No.: P893391190WO (01242) As used herein, the term “altered specificity,” when referencing to a nuclease, means that a nuclease binds to and cleaves a recognition sequence, which is not bound to and cleaved by a reference nuclease (e.g., a wild-type) under physiological conditions, or that the rate of cleavage of a recognition sequence is increased or decreased by a biologically significant amount (e.g., at least 2 ⁇ , or 2 ⁇ -10 ⁇ ) relative to a reference nuclease.
  • a biologically significant amount e.g., at least 2 ⁇ , or 2 ⁇ -10 ⁇
  • center sequence refers to the four base pairs separating half-sites in the meganuclease recognition sequence. These bases are numbered +1 through +4. The center sequence comprises the four bases that become the 3' single-strand overhangs following meganuclease cleavage. “Center sequence” can refer to the sequence of the sense strand or the antisense (opposite) strand. Meganucleases are symmetric and recognize bases equally on both the sense and antisense strand of the center sequence.
  • the sequence A+1A+2A+3A+4 on the sense strand is recognized by a meganuclease as T+1T+2T+3T+4 on the antisense strand and, thus, A+1A+2A+3A+4 and T+1T+2T+3T+4 are functionally equivalent (e.g., both can be cleaved by a given meganuclease).
  • the sequence C+1T+2G+3C+4 is equivalent to its opposite strand sequence, G+1C+2A+3G+4 due to the fact that the meganuclease binds its recognition sequence as a symmetric homodimer.
  • cleave or “cleavage” refer to the hydrolysis of phosphodiester bonds within the backbone of a recognition sequence within a target sequence that results in a double-stranded break within the target sequence, referred to herein as a “cleavage site”.
  • DNA-binding affinity or “binding affinity” means the tendency of a nuclease to non-covalently associate with a reference DNA molecule (e.g., a recognition sequence or an arbitrary sequence). Binding affinity is measured by a dissociation constant, Kd.
  • a nuclease has “altered” binding affinity if the Kd of the nuclease for a reference recognition sequence is increased or decreased by a statistically significant percent change relative to a reference nuclease.
  • the term “hypervariable region” refers to a localized sequence within a meganuclease monomer or subunit that comprises amino acids with relatively high variability.
  • a hypervariable region can comprise about 50-60 contiguous residues, about 53-57 contiguous residues, or preferably about 56 residues.
  • the residues of a hypervariable region may correspond to positions 24-79 or positions 215-270 of any one of SEQ ID NOs: 7-10.
  • a hypervariable region can comprise one or more residues that contact DNA bases in a recognition sequence and can be modified to alter base preference of the monomer or subunit.
  • a hypervariable region can also comprise one or more residues that bind to the DNA backbone when the meganuclease associates with a double-stranded DNA recognition sequence. Such residues can be modified to alter the binding affinity of the meganuclease for the DNA backbone and the target Attorney Docket No.: P893391190WO (01242) recognition sequence.
  • a hypervariable region may comprise between 1-20 residues that exhibit variability and can be modified to influence base preference and/or DNA-binding affinity.
  • a hypervariable region comprises between about 15-20 residues that exhibit variability and can be modified to influence base preference and/or DNA-binding affinity.
  • variable residues within a hypervariable region correspond to one or more of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-10.
  • variable residues within a hypervariable region correspond to one or more of positions 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-10.
  • linker refers to an exogenous peptide sequence used to join two nuclease subunits into a single polypeptide.
  • a linker may have a sequence that is found in natural proteins or may be an artificial sequence that is not found in any natural protein.
  • a linker may be flexible and lacking in secondary structure or may have a propensity to form a specific three- dimensional structure under physiological conditions.
  • a linker can include, without limitation, those encompassed by U.S. Patent Nos.8,445,251, 9,340,777, 9,434,931, and 10,041,053, each of which is incorporated by reference in its entirety.
  • a linker may have an amino acid sequence that sets forth residues 154-195 of any one of SEQ ID NOs: 7-10.
  • the term “meganuclease” refers to an endonuclease that binds double- stranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments, the recognition sequence for a meganuclease of the present disclosure is 22 base pairs.
  • a meganuclease can be an endonuclease that is derived from I-CreI (SEQ ID NO: 1) and can refer to an engineered variant of I-CreI that has been modified relative to natural I-CreI with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties. Methods for producing such modified variants of I-CreI are known in the art (e.g., WO 2007/047859, incorporated by reference in its entirety).
  • a meganuclease as used herein binds to double-stranded DNA as a heterodimer.
  • a meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains is joined into a single polypeptide using a peptide linker.
  • the term “homing endonuclease” is synonymous with the term “meganuclease.”
  • Meganucleases of the present disclosure are substantially non-toxic when expressed in the targeted cells as described herein such that cells can be transfected and maintained at 37°C without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.
  • mitochondrial transit peptide refers to an engineered meganuclease attached to a peptide, such as a mitochondrial transit peptide, or other molecule that is capable of directing the engineered meganuclease to the mitochondria such that the Attorney Docket No.: P893391190WO (01242) engineered meganuclease is capable of binding and cleaving mitochondrial DNA within the mitochondrial organelle.
  • mitochondrial transit peptide or “MTP” refers to a peptide or fragment of amino acids that can be attached to a separate molecule in order to transport the molecule in the mitochondria.
  • an MTP can be attached to a nuclease, such as an engineered meganuclease, in order to transport the engineered meganuclease into the mitochondria.
  • MTPs can consist of an alternating pattern of hydrophobic and positively charged amino acids to form what is called amphipathic helix.
  • nuclease and “endonuclease” are used interchangeably to refer to naturally-occurring or engineered enzymes, which cleave a phosphodiester bond within a polynucleotide chain.
  • the term “recognition half-site,” “recognition sequence half-site,” or simply “half-site” means a nucleic acid sequence in a double-stranded DNA molecule that is recognized and bound by a monomer of a homodimeric or heterodimeric meganuclease or by one subunit of a single-chain meganuclease.
  • the terms “recognition sequence” or “recognition site” refers to a DNA sequence that is bound and cleaved by a nuclease. In the case of a meganuclease, a recognition sequence comprises a pair of inverted, 9 basepair “half sites” which are separated by four basepairs.
  • the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site.
  • Cleavage by a meganuclease produces four basepair 3’ overhangs.
  • “Overhangs,” or “sticky ends” are short, single-stranded DNA segments that can be produced by endonuclease cleavage of a double- stranded DNA sequence.
  • the overhang comprises bases 10-13 of the 22 basepair recognition sequence.
  • single-chain meganuclease refers to a polypeptide comprising a pair of nuclease subunits joined by a linker.
  • a single-chain meganuclease has the organization: N-terminal subunit – Linker – C-terminal subunit.
  • the two meganuclease subunits will generally be non-identical in amino acid sequence and will bind non-identical DNA sequences.
  • single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences.
  • a single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric.
  • the term “meganuclease” can refer to a dimeric or single-chain meganuclease.
  • the term “specificity” means the ability of a nuclease to bind and cleave double-stranded DNA molecules only at a particular sequence of base pairs referred to as the recognition sequence, or only at a particular set of recognition sequences.
  • the set of recognition Attorney Docket No.: P893391190WO (01242) sequences will share certain conserved positions or sequence motifs but may be degenerate at one or more positions.
  • a highly-specific nuclease is capable of cleaving only one or a very few recognition sequences. Specificity can be determined by any method known in the art.
  • the terms “target site” or “target sequence” refers to a region of the chromosomal DNA of a cell comprising a recognition sequence for a nuclease.
  • a “vector” can also refer to a viral vector (i.e. a recombinant virus).
  • Viral vectors can include, without limitation, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (AAV).
  • serotype or “capsid” refers to a distinct variant within a species of virus, such as recombinant adeno-associated viruses, that is determined based on the viral cell surface antigens.
  • a “control” or “control cell” refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a genetically-modified cell.
  • a control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the genetically-modified cell; (b) a cell of the same genotype as the genetically-modified cell but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest); or, (c) a cell genetically identical to the genetically-modified cell but which is not exposed to conditions, stimuli, or further genetic modifications that would induce expression of altered genotype or phenotype.
  • the term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • an effective amount of the engineered meganuclease comprises about 1x10 10 gc/kg to about 1x10 14 gc/kg (e.g., 1x10 10 gc/kg, 1x10 11 gc/kg, 1x10 12 gc/kg, 1x10 13 gc/kg, or 1x10 14 gc/kg) of a nucleic acid encoding the engineered meganuclease.
  • an effective amount of a nucleic acid encoding an engineered meganuclease, or a pharmaceutical composition comprising a nucleic acid encoding an engineered meganuclease disclosed herein reduces at least one symptom of a disease in a subject.
  • the term “effective dose”, “effective amount”, “therapeutically effective dose”, or “therapeutically effective amount,” as used herein, refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • the term “gc/kg” or “gene copies/kilogram” refers to the number of copies of a nucleic acid encoding an engineered meganuclease or the number of copies of a template Attorney Docket No.: P893391190WO (01242) nucleic acid described herein per weight in kilograms of a subject that is administered the nucleic acid encoding the engineered meganuclease.
  • the term “preventing” refers to the prevention of the disease or condition in the patient.
  • the term “prophylaxis” means the prevention of or protective treatment for a disease or disease state.
  • the term “reduced” refers to any reduction in the symptoms or severity of a disease. Such a reduction may be up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100%. Accordingly, the term “reduced” encompasses both a partial reduction and a complete reduction of a disease state.
  • the term “muscle stem cell” refers to a progenitor cell capable of developing into a muscle cell. Muscle stem cells can include, for example, muscle satellite cells.
  • the recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range.
  • variable which is inherently discrete can be equal to any integer value within the numerical range, including the end-points of the range.
  • variable variable which is inherently continuous can be equal to any real value within the numerical range, including the end-points of the range.
  • a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values ⁇ 0 and ⁇ 2 if the variable is inherently continuous.
  • the present disclosure provides compositions and methods for binding and cleaving a recognition sequence on the mitochondrial genome without impacting the surrounding regions in the mitochondrial genome.
  • engineered meganucleases such as homing endonucleases, attached to MTPs such that DSBs can be generated in the mtDNA.
  • the present invention demonstrates that engineered meganucleases can be directed into the mitochondria organelle and facilitate precise editing of mtDNA, thus opening up an entire field of prospects and opportunities in life sciences.
  • Engineered meganucleases described herein can be attached to a mitochondrial transit peptide (MTP) to generate a mitochondria-targeted engineered meganuclease that can effectively traffic from the cytoplasm of a eukaryotic cell into the mitochondria.
  • MTP mitochondrial transit peptide
  • the engineered meganuclease can bind and cleave a recognition sequence in the mitochondrial genome.
  • NHEJ can produce mutagenesis at the cleavage site, resulting in inactivation of the allele.
  • NHEJ-associated mutagenesis may inactivate an allele via generation of early stop codons, frameshift mutations producing aberrant non-functional proteins, or could trigger mechanisms such as nonsense-mediated mRNA decay.
  • nucleases to induce mutagenesis via NHEJ can be used to target a specific mutation or a sequence present in a wild-type allele. Further, the use of nucleases to induce a double-strand break in a target locus is known to stimulate homologous recombination, particularly of transgenic DNA sequences flanked by sequences that are homologous to the genomic target. In this manner, exogenous nucleic acid sequences can be inserted into a target locus. Such exogenous nucleic acids can encode any sequence or polypeptide of interest.
  • a site-specific nuclease can cleave a recognition sequence in the mitochondrial genome that results in degradation of the mitochondrial genome from the cleaved ends created by the site-specific nuclease.
  • the nucleases used to practice the invention are meganucleases.
  • the meganucleases used to practice the invention are single-chain meganucleases.
  • a single-chain meganuclease comprises an N-terminal subunit and a C-terminal subunit joined by a linker peptide.
  • Each of the two domains recognizes and binds to half of the recognition sequence (i.e., a recognition half-site) and the site of DNA cleavage is at the middle of the recognition sequence Attorney Docket No.: P893391190WO (01242) near the interface of the two subunits.
  • DNA strand breaks are offset by four base pairs such that DNA cleavage by a meganuclease generates a pair of four base pair, 3' single-strand overhangs.
  • an engineered meganuclease of the invention has been engineered to bind and cleave an MIT 9-10 recognition sequence (SEQ ID NO: 3).
  • MIT 9-10 meganuclease Such an engineered meganuclease is referred to herein as “MIT 9-10 meganuclease” or “MIT 9-10 nuclease.”
  • the MIT 9-10 meganuclease is attached to an MTP to form a mitochondria- targeting engineered meganuclease that cleaves the MIT 9-10 recognition sequence of SEQ ID NO: 3.
  • MIT 9-10 meganucleases comprising an MTP can further comprise an NES to further decrease localization to the nucleus and thus reduce any potential cleavage in the nuclear genome.
  • Engineered meganucleases of the invention can comprise a first subunit, comprising a first hypervariable (HVR1) region, and a second subunit, comprising a second hypervariable (HVR2) region. Further, the first subunit can bind to a first recognition half-site in the recognition sequence (e.g., the MIT9 half-site), and the second subunit can bind to a second recognition half-site in the recognition sequence (e.g., the MIT10 half-site).
  • HVR1 hypervariable
  • HVR2 hypervariable region
  • the first subunit can bind to a first recognition half-site in the recognition sequence (e.g., the MIT9 half-site)
  • the second subunit can bind to a second recognition half-site in the recognition sequence (e.g., the MIT10 half-site).
  • the first and second subunits can be oriented such that the first subunit, which comprises the HVR1 region and binds the first half-site, is positioned as the N-terminal subunit, and the second subunit, which comprises the HVR2 region and binds the second half-site, is positioned as the C-terminal subunit.
  • the first and second subunits can be oriented such that the first subunit, which comprises the HVR1 region and binds the first half-site, is positioned as the C-terminal subunit, and the second subunit, which comprises the HVR2 region and binds the second half-site, is positioned as the N-terminal subunit.
  • Exemplary MIT 9-10 meganucleases described herein are provided in Tables 1 and 2 and are further described below.
  • MIT9 MIT9 *MIT9 MIT10 MIT10 *MIT10 Meganuclease SEQ Subunit Subunit Subunit Subunit Subunit Subunit Subunit Subunit Subunit Subunit Subunit Subunit Subunit Subunit Subunit ID Residues SEQ ID % Residues SEQ ID % MIT9-10x.3 7 198-344 11 100 7-153 15 100 MIT9-10.L90 8 198-344 12 100 7-153 16 95.92 MIT9-10.L209 9 198-344 13 99.32 7-153 17 95.24 MIT9-10.L210 10 198-344 14 100 7-153 18 95.92 *“MIT9 Subunit %” and “MIT10 Subunit %” represent the amino acid sequence identity between the MIT9-binding and MIT10-binding subunit regions of each meganuclease and the MIT9-binding and MIT10-binding subunit regions, respectively, of the MIT9-10x.3 meganuclease.
  • the engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 3 within mitochondrial genome, wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region.
  • HVR1 hypervariable
  • HVR2 hypervariable hypervariable
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7.
  • the HVR1 region comprises one or more residues corresponding to residues 215, Attorney Docket No.: P893391190WO (01242) 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7.
  • the HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 7.
  • the HVR1 region comprises residues 215-270 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the HVR1 region comprises residues 215-270 of SEQ ID NO: 7.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 7.
  • the first subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 7.
  • the first subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 7.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 7.
  • the first subunit comprises residues 198-344 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the first subunit comprises residues 198-344 of SEQ ID NO: 7.
  • the first subunit comprises residues 196-354 of SEQ ID NO: 7.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7.
  • the HVR2 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the HVR2 region comprises residues 24-79 of SEQ ID NO: 7.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 7.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of SEQ ID NO: 7.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of SEQ ID NO: 7.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of SEQ ID NO: 7.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 7.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 7.
  • the second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 7.
  • the second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 7.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 7.
  • the second subunit comprises residues 7-153 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the second subunit comprises residues 7-153 of SEQ ID NO: 7.
  • the second subunit comprises residues 6-153 of SEQ ID NO: 7.
  • the second subunit comprises residues 5-153 of SEQ ID NO: 7.
  • the second subunit comprises residues 4-153 of SEQ ID NO: 7.
  • the second subunit comprises residues 3-153 of SEQ ID NO: 7.
  • the second subunit comprises residues 2-153 of SEQ ID NO: 7.
  • the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 1-153 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.
  • the N-terminus of the linker is fused to the residue (i.e., a D residue) corresponding to residue 153 of SEQ ID NO: 7, and the C-terminus of the linker is fused to the residue (i.e., a Y residue) corresponding to residue 196 of SEQ ID NO: 7.
  • the linker comprises residues 154-195 of SEQ ID NO: 7.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of SEQ ID NO: 7.
  • the engineered Attorney Docket No.: P893391190WO (01242) meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of SEQ ID NO: 7.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 7.
  • the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 4-343 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 3-343 of SEQ ID NO: 7.
  • the engineered meganuclease comprises an amino acid sequence of residues 2-343 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence of SEQ ID NO: 19.
  • the engineered meganuclease is encoded by a nucleic acid sequence of SEQ ID NO: 19.
  • MIT 9-10L.90 SEQ ID NO: 8
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 8.
  • the HVR1 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 8.
  • the HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 8.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 8.
  • the HVR1 region comprises residues 215-270 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the HVR1 region comprises residues 215-270 of SEQ ID NO: 8.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 8.
  • the first subunit comprises a residue Attorney Docket No.: P893391190WO (01242) corresponding to residue 271 of SEQ ID NO: 8.
  • the first subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 8.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 8.
  • the first subunit comprises residues 198-344 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the first subunit comprises residues 198-344 of SEQ ID NO: 8.
  • the first subunit comprises residues 196-354 of SEQ ID NO: 8.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 8.
  • the HVR2 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the HVR2 region comprises residues 24-79 of SEQ ID NO: 8.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 8.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of SEQ ID NO: 8.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of SEQ ID NO: 8.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 8.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, Attorney Docket No.: P893391190WO (01242) 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 8.
  • the second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 8.
  • the second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 8.
  • the second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 8.
  • the second subunit comprises residues 7-153 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the second subunit comprises residues 7-153 of SEQ ID NO: 8.
  • the second subunit comprises residues 6-153 of SEQ ID NO: 8.
  • the second subunit comprises residues 5-153 of SEQ ID NO: 8.
  • the second subunit comprises residues 4-153 of SEQ ID NO: 8.
  • the second subunit comprises residues 3-153 of SEQ ID NO: 8.
  • the second subunit comprises residues 2-153 of SEQ ID NO: 8.
  • the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 1-153 of SEQ ID NO: 8.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.
  • the N-terminus of the linker is fused to the residue (i.e., a D residue) corresponding to residue 153 of SEQ ID NO: 8
  • the C-terminus of the linker is fused to the residue (i.e., a Y residue) corresponding to residue 196 of SEQ ID NO: 8.
  • the linker comprises residues 154-195 of SEQ ID NO: 8.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of SEQ ID NO: 8.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 8.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8.
  • the engineered meganuclease comprises an amino acid sequence of residues 4-343 of SEQ ID NO: 8.
  • the engineered Attorney Docket No.: P893391190WO (01242) meganuclease comprises an amino acid sequence of residues 3-343 of SEQ ID NO: 8.
  • the engineered meganuclease comprises an amino acid sequence of residues 2-343 of SEQ ID NO: 8.
  • the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of SEQ ID NO: 20.
  • the HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 9.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 9.
  • the HVR1 region comprises residues 215-270 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the HVR1 region comprises residues 215-270 of SEQ ID NO: 9.
  • the first subunit comprises residues 198-344 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the first subunit comprises residues 198-344 of SEQ ID NO: 9.
  • the first subunit comprises residues 196-354 of SEQ ID NO: 9.
  • the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 9.
  • the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 9.
  • the HVR2 region comprises residues 24-79 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the HVR2 region comprises residues 24-79 of SEQ ID NO: 9.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of SEQ ID NO: 9.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of SEQ ID NO: 9.
  • the second subunit comprises residues 7-153 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino Attorney Docket No.: P893391190WO (01242) acid substitutions.
  • the second subunit comprises residues 7-153 of SEQ ID NO: 9.
  • the second subunit comprises residues 6-153 of SEQ ID NO: 9.
  • the second subunit comprises residues 5-153 of SEQ ID NO: 9.
  • the second subunit comprises residues 4-153 of SEQ ID NO: 9.
  • the second subunit comprises residues 3-153 of SEQ ID NO: 9.
  • the second subunit comprises residues 2-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 9. In some embodiments, the second subunit comprises residues 1-153 of SEQ ID NO: 9.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.
  • the N-terminus of the linker is fused to the residue (i.e., a D residue) corresponding to residue 153 of SEQ ID NO: 9, and the C-terminus of the linker is fused to the residue (i.e., a Y residue) corresponding to residue 196 of SEQ ID NO: 9.
  • the linker comprises residues 154-195 of SEQ ID NO: 9.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of SEQ ID NO: 9.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 9.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 9.
  • the engineered meganuclease comprises an amino acid sequence of residues 4-343 of SEQ ID NO: 9.
  • the engineered meganuclease comprises an amino acid sequence of residues 3-343 of SEQ ID NO: 9.
  • the engineered meganuclease comprises an amino acid sequence of residues 2-343 of SEQ ID NO: 9.
  • the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more Attorney Docket No.: P893391190WO (01242) sequence identity to a nucleic acid sequence of SEQ ID NO: 21.
  • the engineered meganuclease is encoded by a nucleic acid sequence of SEQ ID NO: 21.
  • MIT 9-10L.210 SEQ ID NO: 10.
  • the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 10.
  • the HVR1 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 10.
  • the HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 10.
  • the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 10.
  • the HVR1 region comprises residues 215-270 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.
  • the HVR1 region comprises residues 215-270 of SEQ ID NO: 10.
  • the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 10.
  • the first subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 10.
  • the first subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 10.
  • the first subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 10.
  • the first subunit comprises residues 198-344 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the first subunit comprises residues 198-344 of SEQ ID NO: 10.
  • the first subunit comprises residues 196-354 of SEQ ID NO: 10.
  • the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 10.
  • the HVR2 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 10.
  • the HVR2 region comprises residues 24-79 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or Attorney Docket No.: P893391190WO (01242) 11 amino acid substitutions.
  • the HVR2 region comprises residues 24-79 of SEQ ID NO: 10.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 10.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of SEQ ID NO: 10.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of SEQ ID NO: 10.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of SEQ ID NO: 10.
  • the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 10.
  • the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 10.
  • the second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 10.
  • the second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 10. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 6-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 5-153 of SEQ ID NO: 10.
  • the second subunit comprises residues 4-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 3-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 2-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 1-153 of SEQ ID NO: 10.
  • the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.
  • the N-terminus of the linker is fused to the residue (i.e., a D residue) corresponding to residue 153 of SEQ ID NO: 10
  • the C-terminus of the linker is fused to the residue (i.e., a Y residue) corresponding to residue 196 of SEQ ID NO: 10.
  • the linker comprises residues 154-195 of SEQ ID NO: 10.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of SEQ ID NO: 10.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 10.
  • the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 4-343 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 3-343 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 2-343 of SEQ ID NO: 10.
  • the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of SEQ ID NO: 22.
  • MTPs for directing the engineered meganuclease into the mitochondria can be from 10-100 amino acids in length.
  • the MTP is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, or more amino acids long.
  • MTPs can contain additional signals that subsequently target Attorney Docket No.: P893391190WO (01242) the protein to different regions of the mitochondria, such as the mitochondrial matrix.
  • Non limiting examples of MTPs for use in the compositions and methods disclose herein include, Neurospora crassa F0 ATPase subunit 9 (SU9) MTP, human cytochrome c oxidase subunit VIII (CoxVIII or Cox8) MTP, the P1 isoform of subunit c of human ATP synthase MTP, aldehyde dehydrogenase targeting sequence MTP, Glutaredoxin 5 MTP, Pyruvate dehydrogenase MTP, Peptidyl-prolyl isomerase MTP, Acetyltransferase MTP, Isocitrate dehydrogenase MTP, cytochrome oxidase MTP, and the subunits of the FA portion of ATP synthase MTP, CPN60/No GGlinker MTP, Superoxide dismutase (SOD) MTP, Superoxide dismutase doubled(2SOD) MTP, Superoxide
  • the MTP comprises a combination of at least two MTPs.
  • the combination of MTPs can be a combination of identical MTPs or a combination of different MTPs.
  • the MTP comprises the Cox VIII MTP (SEQ ID NO: 23) and the SU9 MTP (SEQ ID NO: 24) combined into a single MTP represented by SEQ ID NO: 25.
  • an MTP can be attached by any appropriate means to an engineered meganuclease disclosed herein.
  • the MTP can be attached to the N-terminus of the engineered meganuclease.
  • the MTP can be attached to the C-terminus of the engineered meganuclease. In some embodiments multiple MTPs can be attached to a single engineered meganuclease. For example, a first MTP can be attached to the N-terminus of the engineered meganuclease and a second MTP can be attached to the C-terminus of the engineered meganuclease. In some embodiments, the first and second MTP are identical and in other embodiments, the first and second MTP not identical.
  • the MTP(s) can be attached by any means that allows for transport of the engineered meganuclease into the mitochondria of a cell.
  • the MTP is attached by fusing the MTP to the N- or C-terminus of the engineered meganuclease.
  • the MTP can also be attached to the engineered meganuclease by a peptide linker.
  • the linker can be, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20 amino acids.
  • the MTP is attached to a peptide linker at the N- or C-terminus of the engineered meganuclease.
  • a mitochondria-targeted engineered meganuclease described herein is attached to a nuclear export sequence (NES) in order to help prevent the engineered meganuclease from cleaving the nuclear genome.
  • NES nuclear export sequence
  • the NES comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 26 or 27.
  • the NES may Attorney Docket No.: P893391190WO (01242) comprise the amino acid sequence of SEQ ID NO: 26 or 27.
  • the NES is attached at the N-terminus of the engineered meganuclease. In other embodiments, the NES is attached at the C-terminus of the engineered meganuclease.
  • the NES is fused to the engineered meganuclease.
  • the NES is attached to the engineered meganuclease by a polypeptide linker.
  • more than one NES is attached to the engineered meganuclease.
  • an engineered meganuclease disclosed herein can comprise a first NES and a second NES.
  • the first NES is attached at the N-terminus of the engineered meganuclease
  • the second NES is attached at the C-terminus of the engineered meganuclease.
  • the first NES and/or the second NES comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26 or 27.
  • the first NES and/or the second NES may comprise the amino acid sequence set forth in SEQ ID NO: 26 or 27.
  • the first NES and the second NES are identical. In other embodiments, the first NES and the second NES are not identical.
  • the NES can be attached to the engineered meganuclease by any appropriate means known in the art.
  • the first NES and/or the second NES can be fused to the engineered meganuclease.
  • the first NES and/or the second NES is attached to the engineered meganuclease by a polypeptide linker.
  • An engineered meganuclease with an NES may have reduced or decreased transport to the nucleus of a target cell or target cell population (e.g., a eukaryotic cell or eukaryotic cell population), compared to an engineered meganuclease without an NES.
  • nuclear transport of an engineered meganuclease with an NES may be less than that of an engineered meganuclease without an NES, by about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60- 70%, 70-80%, 80-90%, 90-100%, or more (e.g., by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more).
  • an engineered meganuclease with an NES may induce fewer nuclear indels (i.e., less cleavage and resulting deletion in nuclear genome of a target cell or target cell population) compared to an engineered meganuclease without an NES.
  • nuclear indels induced by an engineered meganuclease with an NES may be less than that induced by an engineered meganuclease without an NES, by about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, or more.
  • the first subunit i.e., comprising HVR1
  • the second subunit i.e., comprising HVR2
  • the first subunit i.e., the C-terminal subunit
  • the first subunit can lack residues at its N-terminus that correspond to residues 1-4 of wild-type I-CreI because the binding site of the polypeptide linker is at the Y residue corresponding to position 5 of wild-type I-CreI.
  • the first subunit can further comprise residues at its C-terminus that correspond to residues 154-163 of wild-type I-CreI.
  • the second subunit i.e., the N-terminal subunit
  • the second subunit can lack residues at its C-terminus that correspond to residues 154-163 of wild-type I-CreI because the binding site of the polypeptide linker is at the D residue corresponding to position 153 of wild-type I-CreI.
  • the second subunit can further comprise one or more residues at its N-terminus that correspond to one or more of residues 1-6 of wild-type I-CreI (e.g., residues 1-6, 2-6, 3-6, 4-6, or 5-6).
  • the first subunit i.e., comprising HVR1
  • the second subunit i.e., comprising HVR2
  • the first subunit can lack residues at its C-terminus that correspond to residues 154-163 of wild-type I-CreI because the binding site of the polypeptide linker is at the D residue corresponding to position 153 of wild-type I-CreI.
  • the first subunit can further comprise one or more residues at its N-terminus that correspond to one or more of residues 1-6 of wild-type I-CreI (e.g., residues 1-6, 2-6, 3-6, 4-6, or 5-6).
  • the second subunit i.e., the C-terminal subunit
  • the second subunit can lack residues at its N- terminus that correspond to residues 1-4 of wild-type I-CreI because the binding site of the polypeptide linker is at the Y residue corresponding to position 5 of wild-type I-CreI.
  • the second subunit can further comprise residues at its C-terminus that correspond to residues 154-163 of wild- type I-CreI.
  • the disclosed engineered meganucleases comprise (i) an inactivating amino acid in the N-terminal subunit that reduces or abolishes cleavage activity; (ii) an inactivating amino acid in the C-terminal subunit that reduces or abolishes cleavage activity; or (iii) an inactivating amino acid in the N-terminal subunit that reduces or abolishes cleavage activity and an inactivating amino acid in the C-terminal subunit that reduces or abolishes cleavage activity.
  • an inactivating amino acid that “reduces” cleavage activity of an engineered meganuclease inactivates only the subunit comprising that amino acid, while not affecting the ability of the other subunit to cleave its DNA strand.
  • the other subunit remains active and the engineered meganuclease becomes a nickase that remains capable of cleaving one strand of the double-stranded DNA.
  • both subunits comprise an inactivating amino acid that reduces cleavage activity, neither subunit is active, the engineered meganuclease Attorney Docket No.: P893391190WO (01242) does not comprise any cleavage activity, and it cannot generate a single-strand or double-strand break in the DNA.
  • an inactivating amino acid that “abolishes” cleavage activity of an engineered meganuclease can be present in only one subunit but will inactivate both subunits of the engineered meganuclease, such that it does not comprise any cleavage activity and cannot generate a single-strand or double-strand break in the DNA.
  • the inactivating amino acid is an A at a position corresponding to position 20 (i.e., D20A) or position 211 (i.e., D211A) of SEQ ID NOs: 7-10.
  • the inactivating amino acid is an E at a position corresponding to position 47 (i.e., Q47E) or position 238 (i.e., Q238E) of SEQ ID NOs: 7-10.
  • the N-terminal subunit comprises an E at a position corresponding to position 47 (i.e., Q47E) of SEQ ID NOs: 7-10
  • the C-terminal subunit comprises an E at a position corresponding to position 238 (Q238E) of SEQ ID NOs: 7-10, wherein the engineered meganuclease does not comprise cleavage activity (i.e., activity is abolished).
  • the N-terminal subunit comprises an A at a position corresponding to position 20 (i.e., D20A) of SEQ ID NOs: 7-10
  • the C-terminal subunit comprises an A at a position corresponding to position 211 (i.e., D211A) of SEQ ID NOs: 7-10, wherein the engineered meganuclease does not comprise cleavage activity (i.e., activity is abolished).
  • the N-terminal subunit comprises an E at a position corresponding to position 47 (i.e., Q47E) of SEQ ID NOs: 7-10 and the C-terminal subunit does not comprise an inactivating amino acid, wherein the engineered meganuclease is a nickase that is only capable of cleaving the antisense strand of a dsDNA target site.
  • the C-terminal subunit comprises an E at a position corresponding to position 238 (i.e., Q238E) of SEQ ID NOs: 7-10 and the N-terminal subunit does not comprise an inactivating amino acid, wherein the engineered meganuclease is a nickase that is only capable of cleaving the sense strand of a dsDNA target site.
  • engineered meganuclease does not comprise cleavage activity (i.e., activity is abolished) due to one or more inactivating amino acid modifications
  • engineered meganucleases are capable of binding to a double-stranded DNA comprising the recognition sequence of SEQ ID NO: 3 (i.e., MIT 9-10) without cleaving the double-stranded DNA.
  • engineered meganuclease comprises an inactivating amino acid modification such that only one subunit has cleavage activity
  • the engineered meganuclease is a nickase
  • engineered meganucleases are capable of binding to a double- stranded DNA comprising the recognition sequence of SEQ ID NO: 3 (i.e., MIT 9-10) and cleaving either the sense or antisense strand of the DNA.
  • the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an engineered meganuclease described herein, or a pharmaceutically acceptable carrier and a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein.
  • pharmaceutical compositions are provided that comprise a pharmaceutically acceptable carrier and a therapeutically effective amount of a nucleic acid encoding an engineered meganuclease described herein.
  • Such pharmaceutical compositions can be prepared in accordance with known techniques.
  • nuclease polypeptides or DNA/RNA encoding the same or cells expressing the same
  • a pharmaceutically acceptable carrier typically admixed with a pharmaceutically acceptable carrier, and the resulting composition is administered to a subject.
  • the carrier must be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject.
  • pharmaceutical compositions of the invention can further comprise one or more additional agents or biological molecules useful in the treatment of a disease in the subject.
  • the pharmaceutical composition comprises a recombinant virus (i.e., a viral vector) comprising a polynucleotide (e.g., a viral genome) comprising a nucleic acid sequence encoding an engineered meganuclease described herein.
  • a recombinant virus i.e., a viral vector
  • a polynucleotide e.g., a viral genome
  • recombinant viruses are known in the art and include recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, and recombinant adeno-associated viruses (AAV) (reviewed in Vannucci, et al.
  • Recombinant AAVs useful in the invention can have any capsid or serotype that allows for transduction of the virus into a target cell type and expression of the engineered meganuclease by the target cell.
  • recombinant AAV has a capsid of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVHSC, or other AAVs known in the art.
  • the recombinant virus is injected directly into target tissues.
  • the recombinant virus is delivered systemically via the circulatory system.
  • the recombinant AAV has an AAV9 capsid, a MyoAAV capsid (e.g., Attorney Docket No.: P893391190WO (01242) Tabebordbar, et al. (2021) Cell.184: 4919-4938), or an AAVMYO capsid (e.g., Weinmann et al., (2020) Nature Communications.11:5432; Andari et al., (2022) Science Advances.8(38)).
  • AAV9 capsid e.g., Attorney Docket No.: P893391190WO (01242) Tabebordbar, et al. (2021) Cell.184: 4919-4938
  • AAVMYO capsid e.g., Weinmann et al., (2020) Nature Communications.11:5432; Andari et al., (2022) Science Advances.8(38)
  • AAVs can also be self-complementary such that they do not require second-strand DNA synthesis in the host cell (McCarty, et al. (2001) Gene Ther.8:1248-54).
  • Nucleic acids delivered by recombinant AAVs can include left (5') and right (3') inverted terminal repeats.
  • the pharmaceutical composition comprises one or more mRNAs described herein (e.g., mRNAs encoding an engineered meganuclease) formulated within lipid nanoparticles.
  • lipid nanoparticles for use in the method of the invention can be prepared by various techniques which are presently known in the art.
  • Nucleic acid-lipid particles and their method of preparation are disclosed in, for example, U.S. Patent Publication Nos.20040142025 and 20070042031, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Selection of the appropriate size of lipid nanoparticles must take into consideration the site of the target cell and the application for which the lipid nanoparticles is being made. Generally, the lipid nanoparticles will have a size within the range of about 25 to about 500 nm. In some embodiments, the lipid nanoparticles have a size from about 50 nm to about 300 nm or from about 60 nm to about 120 nm.
  • the size of the lipid nanoparticles may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421 ⁇ 150 (1981), incorporated herein by reference.
  • QELS quasi-electric light scattering
  • a variety of methods are known in the art for producing a population of lipid nanoparticles of particular size ranges, for example, sonication or homogenization. One such method is described in U.S. Pat. No.4,737,323, incorporated herein by reference.
  • Some lipid nanoparticles contemplated for use in the invention comprise at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid.
  • lipid nanoparticles can comprise from about 50 mol % to about 85 mol % of a cationic lipid, from about 13 mol % to about 49.5 mol % of a non-cationic lipid, and from about 0.5 mol % to about 10 mol % of a lipid conjugate, and are produced in such a manner as to have a non- lamellar (i.e., non-bilayer) morphology.
  • lipid nanoparticles can Attorney Docket No.: P893391190WO (01242) comprise from about 40 mol % to about 85 mol % of a cationic lipid, from about 13 mol % to about 49.5 mol % of a non-cationic lipid, and from about 0.5 mol % to about 10 mol % of a lipid conjugate, and are produced in such a manner as to have a non-lamellar (i.e., non-bilayer) morphology.
  • Cationic lipids can include, for example, one or more of the following: palmitoyi-oleoyl- nor-arginine (PONA), MPDACA, GUADACA, ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate) (MC3), LenMC3, CP-LenMC3, ⁇ -LenMC3, CP- ⁇ - LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide, Pan-MC3, Pan-MC4 and Pan MC5, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoeth
  • the cationic lipid can also be DLinDMA, DLin-K-C2-DMA (“XTC2”), MC3, LenMC3, CP-LenMC3, ⁇ -LenMC3, CP- ⁇ -LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide, Pan-MC3, Pan-MC4, Pan MC5, or mixtures thereof.
  • XTC2 DLin-K-C2-DMA
  • the cationic lipid comprises from about 50 mol % to about 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, or from about 50 mol % to about 60 mol % of the total lipid present in the particle.
  • the cationic lipid comprises from about 40 mol % to about 90 mol %, from about 40 mol % to about 85 mol %, from about 40 mol % to about 80 mol %, from about 40 mol % to about 75 mol %, from about 40 mol % to about 70 mol %, from about 40 mol % to about 65 mol %, or from about 40 mol % to about 60 mol % of the total lipid present in the particle.
  • the non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids.
  • the non-cationic lipid comprises one of the following neutral lipid components: (1) cholesterol or a derivative thereof; (2) a phospholipid; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.
  • cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2′- hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, and mixtures thereof.
  • the phospholipid may be a neutral lipid including, but not limited to, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphati
  • the phospholipid is DPPC, DSPC, or mixtures thereof.
  • the non-cationic lipid e.g., one or more phospholipids and/or cholesterol
  • the non-cationic lipid comprises from about 10 mol % to about 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol % to about 60 mol %, from about 25 mol % to about 60 mol %, from about 30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, from about 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %, from about 25 mol % to about 55 mol %, from about 30 mol % to about 55 mol %, from about 13 mol % to about 50 mol %, from about 15 mol % to about 50 mol % or from about 20 mol % to about 50 mol % of the total lipid present
  • the non-cationic lipid is a mixture of a phospholipid and cholesterol or a Attorney Docket No.: P893391190WO (01242) cholesterol derivative
  • the mixture may comprise up to about 40, 50, or 60 mol % of the total lipid present in the particle.
  • the conjugated lipid that inhibits aggregation of particles may comprise, e.g., one or more of the following: a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, a cationic-polymer-lipid conjugates (CPLs), or mixtures thereof.
  • the nucleic acid-lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate.
  • the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL.
  • the conjugated lipid that inhibits aggregation of particles may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or mixtures thereof.
  • the PEG-DAA conjugate may be PEG- di lauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18), or mixtures thereof.
  • Additional PEG-lipid conjugates suitable for use in the invention include, but are not limited to, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT Application No. PCT/US08/88676.
  • PEG-lipid conjugates suitable for use in the invention include, without limitation, 1-[8′-(1,2-dimyristoyl-3- propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl- ⁇ -methyl-poly(ethylene glycol) (2KPEG- DMG).
  • 2KPEG-DMG The synthesis of 2KPEG-DMG is described in U.S. Pat. No.7,404,969.
  • the conjugated lipid that inhibits aggregation of particles may comprise from about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7 mol %, from about 1 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or any fraction thereof
  • the PEG moiety has an average molecular weight of about 2,000 Daltons.
  • the conjugated lipid that inhibits aggregation of particles may comprise from about 5.0 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the PEG moiety has an average molecular weight of about 750 Daltons.
  • the composition comprises amphoteric liposomes, which contain at least one positive and at least one negative charge carrier, which differs from the positive one, the Attorney Docket No.: P893391190WO (01242) isoelectric point of the liposomes being between 4 and 8. This objective is accomplished owing to the fact that liposomes are prepared with a pH-dependent, changing charge. Liposomal structures with the desired properties are formed, for example, when the amount of membrane-forming or membrane-based cationic charge carriers exceeds that of the anionic charge carriers at a low pH and the ratio is reversed at a higher pH.
  • strongly cationic compounds can include, for example: DC-Chol 3- ⁇ -[N-(N′,N′-dimethylmethane) carbamoyl] cholesterol, TC-Chol 3- ⁇ -[N- (N′, N′, N′-trimethylaminoethane) carbamoyl cholesterol, BGSC bisguanidinium-spermidine- cholesterol, BGTC bis-guadinium-tren-cholesterol, DOTAP (1,2-dioleoyloxypropyl)-N,N,N- trimethylammonium chloride, DOSPER (1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylarnide, DOTMA (1,2-dioleoyloxypropyl)-N,N,N-trimethylamronium chloride) (Lipofectin®), DORIE 1,2-dioleoyloxypropyl)-3-dimethylhydroxyethylammonium bro
  • Examples of weakly cationic compounds include, without limitation: His-Chol (histaminyl- cholesterol hemisuccinate), Mo-Chol (morpholine-N-ethylamino-cholesterol hemisuccinate), or histidinyl-PE.
  • Examples of neutral compounds include, without limitation: cholesterol, ceramides, phosphatidyl cholines, phosphatidyl ethanolamines, tetraether lipids, or diacyl glycerols.
  • Anionic compounds useful for amphoteric liposomes include those non-cationic compounds previously described herein.
  • weakly anionic compounds can include: CHEMS (cholesterol hemisuccinate), alkyl carboxylic acids with 8 to 25 carbon atoms, or diacyl glycerol hemisuccinate.
  • Additional weakly anionic compounds can include the amides of aspartic acid, or glutamic acid and PE as well as PS and its amides with glycine, alanine, glutamine, asparagine, serine, cysteine, threonine, tyrosine, glutamic acid, aspartic acid or other amino acids or aminodicarboxylic acids.
  • the esters of hydroxycarboxylic acids or hydroxydicarboxylic acids and PS are also weakly anionic compounds.
  • the neutral lipids comprise from about 10 mol % to about 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol % to about 60 mol %, from about 25 mol % to about 60 mol %, from about 30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, from about 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %, from about 25 mol % to about 55 mol %, from about 30 mol % to about 55 mol %, from about 13 mol % to about 50 mol %, from about 15 mol % to about 50 mol % or from about 20 mol % to about 50 mol % of the total lipid present in the particle.
  • the PEG moiety has an average molecular weight of about 2,000 Daltons.
  • the conjugated lipid that inhibits aggregation of particles may comprise from about 5.0 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the lipid nanoparticles have a composition that specifically enhances delivery and uptake in the liver or specifically within hepatocytes. In certain embodiments, the lipid nanoparticles have a composition that specifically enhances delivery and uptake in a nerve cell.
  • the invention provides recombinant viruses (e.g., recombinant AAVs) for use in the methods of the invention. Recombinant AAVs are typically produced in mammalian cell lines such as HEK-293.
  • the viral cap and rep genes are removed from the vector to prevent its self-replication to make room for the therapeutic gene(s) to be delivered (e.g. the nuclease gene), it is necessary to provide these in trans in the packaging cell line. In addition, it is necessary to provide the “helper” (e.g., adenoviral) components necessary to support replication (Cots et al. (2013), Curr. Gene Ther.13(5): 370-81).
  • helper e.g., adenoviral
  • recombinant AAVs are produced using a triple-transfection in which a cell line is transfected with a first plasmid encoding the “helper” components, a second plasmid comprising the cap and rep genes, and a third plasmid comprising the viral ITRs containing the intervening DNA sequence to be packaged into the virus.
  • Viral particles comprising a genome (ITRs and intervening gene(s) of interest) encased in a capsid are then isolated from cells by freeze-thaw cycles, sonication, detergent, or other means known in the art.
  • Particles are then purified using cesium-chloride density gradient centrifugation or affinity chromatography and subsequently delivered to the gene(s) of interest to cells, tissues, or an organism such as a human patient. Because recombinant AAVs are typically produced (manufactured) in cells, precautions must be taken in practicing the current invention to ensure that the engineered meganuclease is not expressed in the packaging cells. Because the viral genomes of the invention may comprise a recognition sequence for the nuclease, any nuclease expressed in the packaging cell line may be capable of cleaving the viral genome before it can be packaged into viral particles. This will result in reduced packaging efficiency and/or the packaging of fragmented genomes.
  • Engineered meganucleases described herein can be placed under the control of any promoter suitable for expression of the engineered meganuclease.
  • the promoter is a constitutive promoter.
  • the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter.
  • the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter.
  • the promoter is a central nervous system (CNS) cell-specific promoter.
  • CNS central nervous system
  • the promoter is a constitutive promoter, or the promoter is a tissue- specific promoter such as, for example, a muscle cell-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell-specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, or a pancreatic beta cell-specific promoter.
  • a muscle cell-specific promoter such as, for example, a muscle cell-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific
  • the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter.
  • the engineered meganuclease can be placed under control of a tissue-specific promoter that is not active in the packaging cells.
  • a tissue-specific promoter that is not active in the packaging cells.
  • a muscle cell-specific promoter can be used. Examples of muscle cell-specific promoters include C5-12 (Liu, et al. (2004) Hum Gene Ther.15:783-92), the muscle-specific creatine kinase (MCK) promoter (Yuasa, et al.
  • SM22 smooth muscle 22
  • tMCK truncated MCK
  • SPc-512 SP-301 promoter
  • MH promoter an MH promoter
  • Sk-CRM4/DES promoter an MH promoter
  • desmin promoter a desmin promoter.
  • CNS (neuron)-specific promoters include the NSE, Synapsin, and MeCP2 promoters (Lentz, et al. (2012) Neurobiol Dis.48:179-88).
  • liver-specific promoters examples include albumin promoters (such as Palb), human ⁇ 1-antitrypsin (such as Pa1AT), and hemopexin (such as Phpx) (Kramer et al., (2003) Mol. Therapy 7:375-85), hybrid liver-specific promoter (hepatic locus control region from ApoE gene (ApoE-HCR) and a liver- specific alpha1-antitrypsin promoter), human thyroxine binding globulin (TBG) promoter, and apolipoprotein A-II promoter.
  • eye-specific promoters examples include opsin, and corneal epithelium-specific K12 promoters (Martin et al.
  • tissue-specific promoters are not highly-active in HEK-293 cells and, thus, will not be expected to yield significant levels of nuclease gene expression in packaging cells when incorporated into viral vectors of the present invention.
  • the viral vectors of the present invention contemplate the use of other cell lines with the use of incompatible tissue specific promoters (i.e., the well-known HeLa cell line (human epithelial cell) and using the liver-specific hemopexin promoter).
  • tissue-specific promoters include: synovial sarcomas PDZD4 (cerebellum), C6 (liver), ASB5 (muscle), PPP1R12B (heart), SLC5A12 (kidney), cholesterol regulation APOM (liver), ADPRHL1 (heart), and monogenic malformation syndromes TP73L (muscle).
  • PDZD4 corthelial sarcomas PDZD4 (cerebellum), C6 (liver), ASB5 (muscle), PPP1R12B (heart), SLC5A12 (kidney), cholesterol regulation APOM (liver), ADPRHL1 (heart), and monogenic malformation syndromes TP73L (muscle).
  • the recombinant virus can be packaged in cells from a different species in Attorney Docket No.: P893391190WO (01242) which the nuclease is not likely to be expressed.
  • viral particles can be produced in microbial, insect, or plant cells using mammalian promoters, such as the well-known cytomegalovirus- or SV40 virus-early promoters, which are not active in the non-mammalian packaging cells.
  • mammalian promoters such as the well-known cytomegalovirus- or SV40 virus-early promoters, which are not active in the non-mammalian packaging cells.
  • viral particles are produced in insect cells using the baculovirus system as described by Gao, et al. (Gao et al. (2007), J. Biotechnol.131(2):138-43). A nuclease under the control of a mammalian promoter is unlikely to be expressed in these cells (Airenne et al. (2013), Mol. Ther.21(4):739-49).
  • insect cells utilize different mRNA splicing motifs than mammalian cells.
  • a mammalian intron such as the human growth hormone (HGH) intron or the SV40 large T antigen intron
  • HGH human growth hormone
  • SV40 large T antigen intron a mammalian intron
  • insect cells will not express a functional nuclease and will package the full-length genome.
  • mammalian cells to which the resulting recombinant AAV particles are delivered will properly splice the pre-mRNA and will express functional nuclease protein.
  • Haifeng Chen has reported the use of the HGH and SV40 large T antigen introns to attenuate expression of the toxic proteins barnase and diphtheria toxin fragment A in insect packaging cells, enabling the production of recombinant AAV vectors carrying these toxin genes (Chen, H (2012) Mol Ther Nucleic Acids.1(11): e57).
  • the engineered meganuclease gene can be operably linked to an inducible promoter such that a small-molecule inducer is required for nuclease expression. Examples of inducible promoters include the Tet-On system (Clontech; Chen et al.
  • Practicing the current invention using such ligand-inducible transcription activators includes: 1) placing the nuclease gene under the control of a promoter that responds to the corresponding transcription factor, the nuclease gene having (a) binding site(s) for the transcription factor; and 2) including the gene encoding the transcription factor in the packaged viral genome.
  • the latter step is necessary because the nuclease will not be expressed in the target cells or tissues following recombinant AAV delivery if the transcription activator is not also provided to the same cells.
  • the transcription activator then induces nuclease gene expression only in cells or tissues that are treated with the cognate small- molecule activator.
  • recombinant AAVs are produced in a mammalian cell line that expresses a transcription repressor that prevents expression of the nuclease.
  • Transcription repressors are known in the art and include the Tet-Repressor, the Lac-Repressor, the Cro repressor, and the Lambda-repressor.
  • packaging cells are transfected/transduced with a vector encoding a transcription repressor and the nuclease gene in the viral genome (packaging vector) is operably linked to a promoter that is modified to comprise binding sites for the repressor such that the repressor silences the promoter.
  • the gene encoding the transcription repressor can be placed in a variety of positions.
  • It can be encoded on a separate vector; it can be incorporated into the packaging vector outside of the ITR sequences; it can be incorporated into the cap/rep vector or the adenoviral helper vector; or it can be stably integrated into the genome of the packaging cell such that it is expressed constitutively.
  • Methods to modify common mammalian promoters to incorporate transcription repressor sites are known in the art. For example, Chang and Roninson modified the strong, constitutive CMV and RSV promoters to comprise operators for the Lac repressor and showed that gene expression from the modified promoters was greatly attenuated in cells expressing the repressor (Chang and Roninson (1996), Gene 183:137-42).
  • the use of a non- human transcription repressor ensures that transcription of the nuclease gene will be repressed only in the packaging cells expressing the repressor and not in target cells or tissues transduced with the resulting recombinant AAV.
  • the invention provides methods for producing genetically-modified cells, both in vitro and in vivo, using engineered meganucleases comprising an MTP (i.e., mitochondria-targeted engineered meganucleases) that bind and cleave recognition sequences found within mtDNA, such as human mtDNA.
  • MTP i.e., mitochondria-targeted engineered meganucleases
  • Cleavage at such recognition sequences can allow for NHEJ at the cleavage site, insertion of an exogenous sequence via homologous recombination, or degradation of the mtDNA.
  • the invention includes that an engineered meganuclease described herein, or a nucleic acid encoding an engineered meganuclease described herein, can be delivered (i.e., introduced) into cells, such as eukaryotic cells (e.g., human cells).
  • Engineered meganucleases described herein can be delivered into a cell in the form of protein or, preferably, as a nucleic acid encoding the engineered meganuclease.
  • nucleic acids can be DNA (e.g., circular or linearized plasmid DNA or PCR products) or RNA (e.g., mRNA).
  • RNA e.g., mRNA
  • polynucleotides are provided herein that comprise a nucleic acid sequence encoding an engineered meganuclease disclosed herein.
  • the polynucleotide is an mRNA.
  • the polynucleotides encoding an engineered meganuclease disclosed herein can be operably linked to a promoter.
  • expression cassettes comprise a promoter operably linked to a polynucleotide having a nucleic acid sequence encoding a engineered meganuclease disclosed herein.
  • the engineered meganuclease coding sequence is delivered in DNA form, it should be operably linked to a promoter to facilitate transcription of the engineered meganuclease-encoding sequence.
  • Mammalian promoters suitable for the invention include constitutive promoters such as the cytomegalovirus early (CMV) promoter (Thomsen et al.
  • SV40 early promoter (Benoist and Chambon (1981), Nature.290(5804):304-10), a CAG promoter, an EF1 alpha promoter, or a UbC promoter, as well as inducible promoters such as the tetracycline-inducible promoter (Dingermann et al. (1992), Mol Cell Biol.12(9):4038-45).
  • An engineered meganuclease described herein can also be operably linked to a synthetic promoter.
  • Synthetic promoters can include, without limitation, the JeT promoter (WO 2002/012514).
  • a nucleic acid sequence encoding an engineered meganuclease described herein is operably linked to a tissue or cell-specific promoter, such as a muscle cell-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte- specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell- specific promoter, a retinal ganglion cell-specific promoter, a retinal pigmentary epithelium- specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, or other promoters described herein.
  • a tissue or cell-specific promoter such as a muscle cell-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte- specific promoter
  • a nucleic acid sequence encoding an engineered meganuclease is delivered on a recombinant DNA construct or expression cassette.
  • the recombinant DNA construct can comprise an expression cassette (i.e., “cassette”) comprising a promoter and a polynucleotide having a nucleic acid sequence encoding an engineered meganuclease described herein.
  • the polynucleotides provided herein can be mRNA or DNA.
  • the polynucleotides further comprise a sequence encoding a selectable marker.
  • the selectable marker can be any marker that allows selection of cells or organisms (e.g., bacteria, eukaryotic cells, mammalian cells, plant cells, plants, and/or plant parts) that contain a polynucleotide disclosed herein.
  • the selectable marker is an antibiotic resistance gene.
  • mRNA encoding the engineered meganuclease is delivered to a cell because this reduces the likelihood that the gene encoding the engineered meganuclease will integrate into the genome of the cell.
  • Attorney Docket No.: P893391190WO (01242) Such mRNA encoding an engineered meganuclease can be produced using methods known in the art such as in vitro transcription.
  • the mRNA is 5' capped using 7- methyl-guanosine, anti-reverse cap analogs (ARCA) (US 7,074,596), CLEANCAP® analogs such as Cap 1 analogs (Trilink, San Diego, CA), or enzymatically capped using vaccinia capping enzyme or similar.
  • the mRNA may be polyadenylated.
  • the mRNA may contain various 5’ and 3’ untranslated sequence elements to enhance expression the encoded engineered meganuclease and/or stability of the mRNA itself. Such elements can include, for example, posttranslational regulatory elements such as a woodchuck hepatitis virus posttranslational regulatory element.
  • the mRNA may contain nucleoside analogs or naturally- occurring nucleosides, such as pseudouridine, 5-methylcytidine, N6-methyladenosine, 5- methyluridine, or 2-thiouridine. Additional nucleoside analogs include, for example, those described in US 8,278,036.
  • Purified engineered meganucleases can be delivered into cells to cleave mitochondrial DNA by a variety of different mechanisms known in the art, including those further detailed herein.
  • a nucleic acid encoding an engineered meganuclease described herein is introduced into the cell using a single-stranded DNA template.
  • the single- stranded DNA can further comprise a 5' and/or a 3' AAV inverted terminal repeat (ITR) upstream and/or downstream of the sequence encoding the engineered meganuclease.
  • the single-stranded DNA can further comprise a 5' and/or a 3' homology arm upstream and/or downstream of the sequence encoding the engineered meganuclease.
  • genes encoding an engineered meganuclease described herein are introduced into a cell using a linearized DNA template. Such linearized DNA templates can be produced by methods known in the art.
  • a plasmid DNA encoding a nuclease can be digested by one or more restriction enzymes such that the circular plasmid DNA is linearized prior to being introduced into a cell.
  • Purified engineered meganucleases, or nucleic acids encoding engineered meganucleases can be delivered into cells to cleave mitochondrial DNA by a variety of different mechanisms known in the art, including those further detailed herein below.
  • engineered meganucleases, DNA/mRNA encoding engineered meganucleases, or cells expressing engineered meganucleases are formulated for systemic administration, or administration to target tissues, in a pharmaceutically acceptable carrier in accordance with known techniques.
  • proteins/RNA/mRNA/cells are typically admixed with a pharmaceutically acceptable carrier.
  • the carrier must, of course, be acceptable in Attorney Docket No.: P893391190WO (01242) the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient.
  • the carrier can be a solid or a liquid, or both, and can be formulated with the compound as a unit-dose formulation.
  • the engineered meganucleases, or DNA/mRNA encoding the engineered meganucleases are coupled to a cell penetrating peptide or targeting ligand to facilitate cellular uptake.
  • cell penetrating peptides known in the art include poly-arginine (Jearawiriyapaisarn, et al. (2008) Mol Ther.16:1624-9), TAT peptide from the HIV virus (Hudecz et al. (2005), Med. Res. Rev.25: 679-736), MPG (Simeoni, et al. (2003) Nucleic Acids Res. 31:2717–2724), Pep-1 (Deshayes et al.
  • engineered meganucleases or DNA/mRNA encoding engineered meganucleases, are coupled covalently or non-covalently to an antibody that recognizes a specific cell-surface receptor expressed on target cells such that the engineered meganuclease protein/DNA/mRNA binds to and is internalized by the target cells.
  • engineered meganuclease protein/DNA/mRNA can be coupled covalently or non-covalently to the natural ligand (or a portion of the natural ligand) for such a cell- surface receptor.
  • engineered meganucleases or DNA/mRNA encoding engineered meganucleases, are encapsulated within biodegradable hydrogels.
  • Hydrogels can provide sustained and tunable release of the therapeutic payload to the desired region of the target tissue without the need for frequent injections, and stimuli-responsive materials (e.g., temperature- and pH-responsive hydrogels) can be designed to release the payload in response to environmental or externally applied cues (Kang Derwent et al. (2008) Trans Am Ophthalmol Soc.106:206-214).
  • engineered meganuclease, or DNA/mRNA encoding engineered meganucleases are coupled covalently or, preferably, non-covalently to a nanoparticle or encapsulated within such a nanoparticle using methods known in the art (Sharma, et al. (2014) Biomed Res Int.2014).
  • a nanoparticle is a nanoscale delivery system whose length scale is ⁇ 1 ⁇ m, preferably ⁇ 100 nm.
  • Such nanoparticles may be designed using a core composed of metal, lipid, polymer, or biological macromolecule, and multiple copies of the nuclease proteins, mRNA, or DNA can be attached to or encapsulated with the nanoparticle core. This increases the copy number of the protein/mRNA/DNA that is delivered to each cell and, so, increases the intracellular expression of each engineered meganuclease to maximize the likelihood that the target recognition sequences will be cut.
  • Nanoparticles may be further modified with polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) to form a core-shell nanoparticle whose Attorney Docket No.: P893391190WO (01242) surface confers additional functionalities to enhance cellular delivery and uptake of the payload (Jian et al. (2012) Biomaterials.33(30): 7621-30).
  • Nanoparticles may additionally be advantageously coupled to targeting molecules to direct the nanoparticle to the appropriate cell type and/or increase the likelihood of cellular uptake. Examples of such targeting molecules include antibodies specific for cell-surface receptors and the natural ligands (or portions of the natural ligands) for cell surface receptors.
  • the engineered meganucleases or DNA/mRNA encoding the engineered meganucleases are encapsulated within liposomes or complexed using cationic lipids (see, e.g., LIPOFECTAMINETM, Life Technologies Corp., Carlsbad, CA; Zuris et al. (2015) Nat Biotechnol.33: 73-80; Mishra et al. (2011) J Drug Deliv.2011:863734).
  • the liposome and lipoplex formulations can protect the payload from degradation, enhance accumulation and retention at the target site, and facilitate cellular uptake and delivery efficiency through fusion with and/or disruption of the cellular membranes of the target cells.
  • engineered meganucleases or DNA/mRNA encoding engineered meganucleases, are encapsulated within polymeric scaffolds (e.g., PLGA) or complexed using cationic polymers (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv.2(4): 523-536).
  • Polymeric carriers can be designed to provide tunable drug release rates through control of polymer erosion and drug diffusion, and high drug encapsulation efficiencies can offer protection of the therapeutic payload until intracellular delivery to the desired target cell population.
  • engineered meganucleases or DNA/mRNA encoding engineered meganucleases, are combined with amphiphilic molecules that self-assemble into micelles (Tong et al. (2007) J Gene Med.9(11): 956-66).
  • Polymeric micelles may include a micellar shell formed with a hydrophilic polymer (e.g., polyethyleneglycol) that can prevent aggregation, mask charge interactions, and reduce nonspecific interactions.
  • engineered meganucleases or DNA/mRNA encoding engineered meganucleases, are formulated into an emulsion or a nanoemulsion (i.e., having an average particle diameter of ⁇ 1nm) for administration and/or delivery to the target cell.
  • emulsion refers to, without limitation, any oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersions or droplets, including lipid structures that can form as a result of hydrophobic forces that drive apolar residues (e.g., long hydrocarbon chains) away from water and polar head groups toward water, when a water immiscible phase is mixed with an aqueous phase.
  • lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases.
  • Emulsions are composed of an aqueous phase and a lipophilic phase (typically containing an oil and an organic solvent). Emulsions also frequently contain one or more surfactants. Nanoemulsion formulations are well known, e.g., as described in Attorney Docket No.: P893391190WO (01242) US Pat. Nos.6,015,832, 6,506,803, 6,635,676, 6,559,189, and 7,767,216, each of which is incorporated herein by reference in its entirety.
  • engineered meganucleases or DNA/mRNA encoding engineered meganucleases, are covalently attached to, or non-covalently associated with, multifunctional polymer conjugates, DNA dendrimers, and polymeric dendrimers (Mastorakos et al. (2015) Nanoscale.7(9): 3845-56; Cheng et al. (2008) J Pharm Sci.97(1): 123-43).
  • the dendrimer generation can control the payload capacity and size, and can provide a high payload capacity.
  • display of multiple surface groups can be leveraged to improve stability, reduce nonspecific interactions, and enhance cell-specific targeting and drug release.
  • polynucleotides having nucleic acid sequences encoding an engineered meganuclease are introduced into a cell using a recombinant virus.
  • recombinant viruses include recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, and recombinant AAVs (reviewed in Vannucci, et al. (2013 New Microbiol.36:1-22).
  • Recombinant AAVs useful in the invention can have any capsid or serotype that allows for transduction of the virus into a target cell type and expression of the engineered meganuclease by the target cell.
  • a recombinant AAV has a capsid of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVHSC, or other capsids known in the art.
  • the recombinant virus is injected directly into target tissues.
  • the recombinant virus is delivered systemically via the circulatory system. It is known in the art that different AAVs tend to localize to different tissues, and one could select an appropriate AAV capsid/serotype for preferential delivery to a particular tissue.
  • the recombinant AAV has an AAV9 capsid, a MyoAAV capsid (e.g., Tabebordbar, et al. (2021) Cell.184: 4919- 4938), or an AAVMYO capsid (e.g., Weinmann et al., (2020) Nature Communications.11:5432; Andari et al., (2022) Science Advances.8(38)).
  • AAVs can also be self-complementary such that they do not require second-strand DNA synthesis in the host cell (McCarty, et al. (2001) Gene Ther.8:1248-54).
  • Nucleic acids delivered by recombinant AAVs can include left (5') and right (3') inverted terminal repeats.
  • a recombinant virus used for delivery of a polynucleotide having nucleic acid sequences encoding an engineered meganuclease is a self-limiting recombinant virus.
  • a self-limiting recombinant virus can have limited persistence time in a cell or organism due to the presence of a recognition sequence for an engineered meganuclease within the viral genome.
  • a self-limiting recombinant virus can be engineered to provide coding for a promoter, an engineered meganuclease described herein, and a meganuclease recognition site within the ITRs.
  • the self-limiting recombinant virus delivers the meganuclease gene to a cell, tissue, or organism, Attorney Docket No.: P893391190WO (01242) such that the engineered meganuclease is expressed and able to cut the genome of the cell at an endogenous recognition sequence within the genome.
  • the delivered engineered meganuclease will also find its target site within the self-limiting recombinant virus itself, and cut the viral genome at this target site.
  • the polynucleotides having nucleic acid sequences encoding an engineered meganuclease are delivered in DNA form (e.g. plasmid) and/or via a viral vector (e.g. AAV) they must be operably linked to a promoter, such as those promoters described elsewhere herein.
  • a promoter such as those promoters described elsewhere herein.
  • this can be a viral promoter such as endogenous promoters from the viral vector (e.g. the LTR of a lentiviral vector) or constitutive or tissue-specific promoters described elsewhere herein.
  • polynucleotides having nucleic acid sequences encoding an engineered meganuclease are operably linked to a promoter that drives gene expression preferentially in the target cells or tissues.
  • methods for producing a genetically-modified eukaryotic cell or a genetically-modified eukaryotic cell population by introducing into the eukaryotic cell or eukaryotic cell population a polynucleotide of the present disclosure, such as a polynucleotide containing a nucleic acid sequence that encodes an engineered meganuclease described herein.
  • the engineered meganuclease Upon expression in the eukaryotic cell or eukaryotic cell population, the engineered meganuclease localizes to the mitochondria, binds a recognition sequence in the mitochondrial genome, and generates a cleavage site.
  • the cleavage site generated by the engineered meganuclease can be repaired by NHEJ repair pathway which may result in a nucleic acid insertion or deletion at the cleavage site.
  • the cleavage site generated by the engineered meganuclease in the mitochondrial genome of the eukaryotic cell or eukaryotic cell population can be repaired by alternative nonhomologous end-joining (Alt-NHEJ) or microhomology-mediated end joining (MMEJ).
  • the NHEJ or Alt-NHEJ/MMEJ can result in insertion and/or deletion of a nucleic acid at the cleavage site.
  • the NHEJ or Alt- NHEJ/MMEJ can result in insertion and/or deletion of 1-1000 (e.g., 1-10, 10-100, 100-200, 200- 300, 300-400, 400-500, 500-600, 600-700, 700-80, 800-900, or 900-1000) nucleotides, such as about 1, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 nucleotides at the cleavage site.
  • mitochondrial genomes in a genetically-modified eukaryotic cell disclosed herein or a genetically-modified eukaryotic cell population disclosed herein can be degraded.
  • the percentage of mitochondrial genomes comprising the recognition sequence is decreased by about 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Attorney Docket No.: P893391190WO (01242) 99%, or 100%, or can be degraded by about can be degraded by about 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, or more, compared to a control cell.
  • mutant mitochondrial genomes comprising the recognition sequence of SEQ ID NO: 3 are degraded.
  • the overall ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes will increase following administration or expression of an engineered meganuclease disclosed herein.
  • the ratio of wild-type to mutant mitochondrial genomes in a single genetically-modified eukaryotic cell disclosed herein or a population genetically-modified eukaryotic cells increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more.
  • the percentage of wild-type genomes in a single genetically- modified eukaryotic cell disclosed herein, or a population of genetically-modified eukaryotic cells disclosed herein can increase as mutant mitochondrial genomes comprising SEQ ID NO: 3 are recognized, cleaved, and degraded by the engineered meganuclease.
  • the percentage of wild-type mitochondrial genomes in a genetically-modified eukaryotic cell or genetically modified cell population disclosed herein can be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, of the total mitochondrial genomes in the genetically-modified eukaryotic cell or genetically modified cell population when compared to a eukaryotic cell or eukaryotic cell population that does not express an engineered meganuclease disclosed herein.
  • mutant mitochondrial genomes comprising the recognition sequence of SEQ ID NO: 3 in the genetically-modified eukaryotic cell or genetically-modified cell population can decrease by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more when compared to a eukaryotic cell or eukaryotic cell population that does not express an engineered meganuclease disclosed herein.
  • mitochondrial respiration in a genetically-modified eukaryotic cell or a genetically-modified eukaryotic cell population disclosed herein can increase by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, Attorney Docket No.: P893391190WO (01242) about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more when compared to a eukaryotic cell that does not express an engineered meganuclease disclosed herein.
  • Mitochondrial respiration in a genetically-modified eukaryotic cell or a genetically-modified eukaryotic cell population disclosed herein can be increased by about 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, or more when compared to a eukaryotic cell or eukaryotic cell population that does not express an engineered meganuclease disclosed herein.
  • the recognition sequence is within a region of the mitochondrial genome associated with a mitochondrial disorder.
  • the recognition sequence can be within a region of the mitochondrial genome associated with human mtDNA common deletion.
  • This mutation removes all or part of the genes encoding four complex I subunits, one complex IV subunit, two complex V subunits and five tRNA genes, which are indispensable for maintaining normal mitochondrial function.
  • the mitochondrial common deletion has attracted tremendous interest as it is associated with several sporadic diseases including myopathies, Alzheimer disease, Pearson’ s syndrome, photoaging of the skin, Kearns-Sayre syndrome (KSS) and chronic progressive external ophthalmoplegia (CPEO). Furthermore, this deletion also accumulates in many tissues during aging, and has been used as an indication of mtDNA oxidative damage.
  • the mitochondrial common deletion is a deletion of 4,977bp between nucleotides 8,470 and 13,447 of the mitochondrial genome.
  • both normal and mutated mtDNA can exist in the same cell, a situation known as heteroplasmy.
  • the number of defective mitochondria may be out-numbered by the number of normal mitochondria. Symptoms may not appear in any given generation until the mutation affects a significant proportion of mtDNA.
  • the uneven distribution of normal and mutant mtDNA in different tissues can affect different organs in members of the same family. This can result in a variety of symptoms in affected family members.
  • the recognition sequence disclosed herein in the mitochondrial genome of the eukaryotic cell or eukaryotic cell population is positioned between nucleotides 8,470 and 13,447 of the mitochondrial genome.
  • the recognition sequence in the mitochondrial genome of the eukaryotic cell or eukaryotic cell population is positioned between nucleotides 8,460 and 8,580 or between nucleotides 13,437 and 13,457.
  • the recognition sequence of SEQ ID NO: 3 is located only on mutant mitochondrial genomes.
  • the engineered meganuclease can localize to the mitochondria, bind the recognition sequence in the mitochondrial genome, and generate a cleavage site.
  • the genomes can be cleaved and subsequently degraded.
  • mutant mitochondrial genomes can be used to Attorney Docket No.: P893391190WO (01242) help treat or alleviate the symptoms of the mitochondrial common deletion (i.e., mitochondrial common deletion disorder). Accordingly, methods are provided herein for degrading mutant mitochondrial genomes in a target cell or a population of target cells by delivering to the target cell or population a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease or an engineered meganuclease disclosed herein.
  • the target cell or population of target cells comprise mutant mitochondrial genomes having the mitochondrial common deletion, and the engineered meganuclease recognizes and cleaves the recognition sequence of SEQ ID NO: 3.
  • the target cell or target cell population can be in a mammalian subject, such as a human subject.
  • the target cell is a muscle cell, a skeletal muscle cell, a myotube cell, a muscle stem cell or satellite cell, a neuron, an astrocyte, a microglia cell, an eye cell, a retinal cell, a retinal ganglion cell, a retinal pigmentary epithelium cell, a pancreatic cell, or a pancreatic beta cell, or wherein said population of target cells is a population of muscle cells, skeletal muscle cells, myotube cells, muscle stem cells or satellite cells, neurons, astrocytes, microglia cells, eye cells, retinal cells, retinal ganglion cells, retinal pigmentary epithelium cells, pancreatic cells, or pancreatic beta cells.
  • Such methods include administering to a subject a therapeutically- effective amount of a polynucleotide having a nucleic acid sequence encoding an engineered meganuclease described herein, or a therapeutically-effective amount of an engineered meganuclease described herein, wherein the engineered meganuclease produces a cleavage site at the recognition sequence of SEQ ID NO: 3 in mutant mitochondrial genomes having the common deletion.
  • the cleavage site produced in mutant mitochondrial genomes can lead to degradation of the mutant mitochondrial genomes.
  • treating comprises reducing or alleviating at least one symptom of a condition associated with the mtDNA common deletion.
  • Symptoms of the mtDNA common deletion include but are not limited to any symptom of myopathies, Alzheimer disease, Pearson’ s syndrome, photoaging of the skin, Kearns-Sayre syndrome (KSS), or chronic progressive external ophthalmoplegia (CPEO).
  • symptoms of the mtDNA common deletion can include pigmentary retinopathy, and PEO, cerebellar ataxia, impaired intellect (intellectual disability, dementia, or both), sensorineural hearing loss, ptosis, oropharyngeal and esophageal dysfunction, exercise intolerance, muscle weakness, cardiac conduction block, endocrinopathy, sideroblastic anemia and exocrine pancreas dysfunction, ptosis, impaired eye movements due to paralysis of the extraocular muscles (ophthalmoplegia), oropharyngeal weakness, or variably severe proximal limb weakness with exercise intolerance.
  • impaired intellect intellectual disability, dementia, or both
  • sensorineural hearing loss ptosis
  • oropharyngeal and esophageal dysfunction exercise intolerance
  • muscle weakness muscle weakness
  • cardiac conduction block endocrinopathy
  • sideroblastic anemia and exocrine pancreas dysfunction ptosis
  • the condition is a condition of the bone marrow, the pancreas, muscle, skeletal Attorney Docket No.: P893391190WO (01242) muscle, central nervous system, the eye, or the ears.
  • the condition is Pearson Syndrome, Kearns-Sayre Syndrome (KSS), Progressive External Ophthalmoplegia (PEO), diabetes, or kidney dysfunction.
  • the methods of treating a condition associated with a mtDNA common deletion in a subject involve administration of a pharmaceutical composition disclosed herein.
  • a subject is administered a pharmaceutical composition disclosed herein at a dose of about 1x10 10 gc/kg to about 1x10 14 gc/kg (e.g., 1x10 10 gc/kg, 1x10 11 gc/kg, 1x10 12 gc/kg, 1x10 13 gc/kg, or 1x10 14 gc/kg) of a nucleic acid encoding an engineered meganuclease.
  • a pharmaceutical composition disclosed herein at a dose of about 1x10 10 gc/kg to about 1x10 14 gc/kg (e.g., 1x10 10 gc/kg, 1x10 11 gc/kg, 1x10 12 gc/kg, 1x10 13 gc/kg, or 1x10 14 gc/kg) of a nucleic acid encoding an engineered meganuclease.
  • a subject is administered a pharmaceutical composition at a dose of at least about 1x10 10 gc/kg, at least about 1x10 11 gc/kg, at least about 1x10 12 gc/kg, at least about 1x10 13 gc/kg, or at least about 1x10 14 gc/kg of a nucleic acid encoding an engineered meganuclease.
  • a subject is administered a pharmaceutical composition at a dose of about 1x10 12 gc/kg to about 9x10 13 gc/kg (e.g., about 1x10 12 gc/kg, about 2x10 12 gc/kg, about 3x10 12 gc/kg, about 4x10 12 gc/kg, about 5x10 12 gc/kg, about 6x10 12 gc/kg, about 7x10 12 gc/kg, about 8x10 12 gc/kg, about 9x10 12 gc/kg, about 1x10 13 gc/kg, about 2x10 13 gc/kg, about 3x10 13 gc/kg, about 4x10 13 gc/kg, about 5x10 13 gc/kg, about 6x10 13 gc/kg, about 7x10 13 gc/kg, about 8x10 13 gc/kg, or about 9x10 13 gc/kg) of a nucleic acid encoding
  • the subject is administered a lipid nanoparticle formulation at a dose of within about 0.1 mg/kg to about 0.25 mg/kg, about 0.25 mg/kg to about 0.5 mg/kg, about 0.5 mg/kg to about 0.75 mg/kg, about 0.75 mg/kg to about 1.0 mg/kg, about 1.0 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 2.0 mg/kg, about 2.0 mg/kg to about 2.5 mg/kg, or about 2.5 mg/kg to about 3.0 mg/kg of mRNA encoding an engineered meganuclease.
  • the target tissue(s) for delivery of engineered meganucleases disclosed herein, or nucleic acids encoding engineered meganucleases disclosed herein include without limitation, nerve tissue, muscle tissue, neuromuscular tissue, pancreatic tissue, and ocular/retinal tissue.
  • the target cell for delivery is a muscle cell, a skeletal muscle cell, a myotube cell, a muscle satellite cell, a neuron, an astrocyte, a microglia cell, an eye cell, a retinal cell, a retinal ganglion cell, a retinal pigmentary epithelium cell, a pancreatic cell, or a pancreatic beta cell, or wherein said population of target cells is a population of muscle cells, skeletal muscle cells, myotube cells, muscle stem cells or satellite cells, neurons, astrocytes, microglia cells, eye cells, retinal cells, retinal ganglion cells, retinal pigmentary epithelium cells, pancreatic cells, or pancreatic beta cells or the population of target cells is a population of muscle cells, skeletal muscle cells, myotube cells, muscle stem cells or satellite cells, neurons, astrocytes, microglia cells, eye cells, retinal cells, retinal ganglion cells, retinal pigmentary epithelium cells, pancreatic cells, or pancreatic beta cells or
  • the one or more engineered meganucleases, polynucleotides encoding such engineered meganucleases, or recombinant viruses comprising one or more polynucleotides encoding such engineered meganucleases, as described herein can be administered via any suitable route of administration known in the art.
  • the one or more engineered meganucleases, polynucleotides encoding such engineered meganucleases, or recombinant viruses comprising one or more polynucleotides encoding such engineered meganucleases, as described herein may be administered by an administration route comprising intravenous, intramuscular, intraperitoneal, subcutaneous, intrahepatic, transmucosal, transdermal, intraarterial, and sublingual.
  • engineered meganucleases or mRNA, or DNA vectors encoding such engineered meganucleases, are supplied to target cells (e.g., nerve cells, muscle cells, pancreatic cells, ocular cells, etc.) via injection directly to the target tissue.
  • target cells e.g., nerve cells, muscle cells, pancreatic cells, ocular cells, etc.
  • Other suitable routes of administration of the engineered meganucleases, polynucleotides encoding such engineered meganucleases, or recombinant viruses comprising one or more polynucleotides encoding such engineered meganucleases may be readily determined by the treating physician as necessary.
  • a therapeutically effective amount of an engineered meganuclease described herein is administered to a subject in need thereof.
  • the dosage or dosing frequency of the engineered meganuclease may be adjusted over the course of the treatment, based on the judgment of the administering physician. Appropriate doses will depend, among other factors, on the specifics of any AAV chosen (e.g., serotype, etc.), on the route of administration, on the subject being treated (i.e., age, weight, sex, and general condition of the subject), and the mode of administration. Thus, the appropriate dosage may vary from patient to patient. An appropriate effective amount can be readily determined by one of skill in the art. Dosage treatment may be a single dose schedule or a multiple dose schedule. Moreover, the subject may be administered as many doses as appropriate.
  • AAV e.g., serotype, etc.
  • Exogenous nucleic acid molecules of the invention may be introduced into a cell and/or delivered to a subject by any of the means previously discussed.
  • exogenous nucleic acid molecules are introduced by way of a recombinant virus, such as a lentivirus, retrovirus, adenovirus, or a recombinant AAV.
  • Recombinant AAVs useful for introducing an exogenous nucleic acid molecule can have any serotype that allows for transduction of the virus into the cell and insertion of the exogenous nucleic acid molecule sequence into the cell genome, including those serotypes/capsids previously described herein.
  • the recombinant AAVs can also be self-complementary such that they do not require second-strand DNA synthesis in the host cell.
  • Exogenous nucleic acid molecules introduced using a recombinant AAV can be flanked by a 5' (left) and 3' (right) inverted terminal repeat.
  • an exogenous nucleic acid molecule can be introduced into a cell using a single-stranded DNA template.
  • the single-stranded DNA can comprise the exogenous nucleic acid molecule and, in particular embodiments, can comprise 5' and 3' homology arms to promote insertion of the nucleic acid sequence into the nuclease cleavage site by homologous recombination.
  • the single-stranded DNA can further comprise a 5' AAV inverted terminal repeat (ITR) sequence 5' upstream of the 5' homology arm, and a 3' AAV ITR sequence 3' downstream of the 3' homology arm.
  • ITR inverted terminal repeat
  • polynucleotides comprising nucleic acid sequences encoding engineered meganucleases of the invention and/or an exogenous nucleic acid molecule of the invention can be introduced into a cell by transfection with a linearized DNA template.
  • a plasmid DNA encoding an engineered meganuclease and/or an exogenous nucleic acid molecule can, for example, be digested by one or more restriction enzymes such that the circular plasmid DNA is linearized prior to transfection into the cell.
  • an exogenous nucleic acid of the invention When delivered to a cell, an exogenous nucleic acid of the invention can be operably linked to any promoter suitable for expression of the encoded polypeptide in the cell, including those mammalian promoters and inducible promoters previously discussed. An exogenous nucleic acid of the invention can also be operably linked to a synthetic promoter.
  • the present invention encompasses variants of the polypeptide and polynucleotide sequences described herein. Attorney Docket No.: P893391190WO (01242) As used herein, “variants” is intended to mean substantially similar sequences.
  • a “variant” polypeptide is intended to mean a polypeptide derived from the “native” polypeptide by deletion or addition of one or more amino acids at one or more internal sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native polypeptide.
  • a “native” polynucleotide or polypeptide comprises a parental sequence from which variants are derived.
  • Variant polypeptides encompassed by the embodiments are biologically active. That is, they continue to possess the desired biological activity of the native protein; for example, the ability to bind and cleave recognition sequences found in mtDNA (e.g., human mtDNA), such as MIT 9-10 recognition sequence (SEQ ID NO: 3).
  • biologically active variants of a native polypeptide of the embodiments will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, sequence identity to the amino acid sequence of the native polypeptide, native subunit, native HVR1, or native HVR2 as determined by sequence alignment programs and parameters described elsewhere herein.
  • a biologically active variant of a polypeptide or subunit of the embodiments may differ from that polypeptide or subunit by as few as about 1-40 amino acid residues, as few as about 1-20, as few as about 1-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • the polypeptides of the embodiments may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci.
  • engineered meganucleases of the invention can comprise variants of the HVR1 and HVR2 regions disclosed herein.
  • Parental HVR regions can comprise, for example, residues 24-79 or residues 215-270 of the exemplified engineered meganucleases.
  • variant Attorney Docket No.: P893391190WO (01242) HVRs can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to an amino acid sequence corresponding to residues 24-79 or residues 215-270 of the engineered meganucleases exemplified herein, such that the variant HVR regions maintain the biological activity of the engineered meganuclease (i.e., binding to and cleaving the recognition sequence).
  • a variant HVR1 region or variant HVR2 region can comprise residues corresponding to the amino acid residues found at specific positions within the parental HVR.
  • “corresponding to” means that an amino acid residue in the variant HVR is the same amino acid residue (i.e., a separate identical residue) present in the parental HVR sequence in the same relative position (i.e., in relation to the remaining amino acids in the parent sequence).
  • a parental HVR sequence comprises a serine residue at position 26
  • a variant HVR that “comprises a residue corresponding to” residue 26 will also comprise a serine at a position that is relative (i.e., corresponding) to parental position 26.
  • engineered meganucleases of the invention comprise an HVR1 that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 7-10.
  • engineered meganucleases of the invention comprise an HVR2 that has 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7-10.
  • an engineered meganuclease monomer or subunit described herein can comprise a G, S, or A at a residue corresponding to position 19 of I-CreI (WO 2009001159), a Y, R, K, or D at a residue corresponding to position 66 of I-CreI and/or an E, Q, or K at a residue corresponding to position 80 of I-CreI (US8021867).
  • a “variant” comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide.
  • variants of the nucleic acids of the embodiments will be constructed such that the open reading frame is maintained.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the embodiments.
  • Variant polynucleotides include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode an engineered meganuclease, or an exogenous nucleic acid molecule, or template nucleic acid of the embodiments.
  • variants of a particular polynucleotide of the embodiments will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
  • Variants of a particular polynucleotide of the embodiments can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.
  • the deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide.
  • EXAMPLE 1 Reporter Assay for MIT 9-10 Meganuclease Activity The purpose of this experiment was to determine whether a MIT 9-10 meganuclease could bind and cleave the human MIT 9-10 recognition sequence (SEQ ID NO: 3) in mammalian cells, and to determine whether the MIT 9-10 meganuclease could discriminate against the corresponding sequence found on wild-type mitochondrial DNA (mtDNA) (SEQ ID NO: 5) that spans nucleotides 13,445-13,466.
  • mtDNA wild-type mitochondrial DNA
  • MIT 9-10x.3 SEQ ID NO: 7
  • GFP Green Fluorescent Protein
  • the GFP gene in each cell line contains a direct sequence duplication separated by a pair of recognition sequences such that intracellular cleavage of either recognition sequence by the meganuclease would stimulate a homologous recombination event resulting in a functional GFP gene.
  • two recognition sequences were inserted into the GFP gene.
  • One recognition sequence was for the human MIT 9-10 recognition sequence – either the mutant (SEQ ID NO: 3) or wild-type (SEQ ID NO: 5) sequence, which only differ by one base.
  • Cell line number 1 (mutant) contained the mutant allele
  • cell line number 2 wild-type
  • the second recognition sequence inserted into both lines was a CHO-23/24 recognition sequence, which is recognized and cleaved by a control meganuclease called “CHO-23/24.”
  • the CHO-23/24 recognition sequence is used as a positive control and standard measure of activity.
  • the CHO reporter cells detailed above were transfected with mRNA encoding the MIT 9- 10x.3 meganuclease.
  • a control sample of CHO reporter cells were transfected with mRNA encoding the CHO-23/24 meganuclease.
  • 5e4 CHO reporter cells were transfected with 2.5ng (low dose) of mRNA in a 96-well plate using LIPOFECTAMINE® Attorney Docket No.: P893391190WO (01242) MESSENGERMAX (ThermoFisher) according to the manufacturer’s instructions.
  • the transfected CHO cells were evaluated by image cytometry at 2 days post transfection to determine the percentage of GFP-positive cells compared to an untransfected negative control. This was done for both the wild-type and mutant cell lines to determine specificity of the nuclease for the mutant sequence.
  • the MIT 9-10x.3 meganuclease was able to bind and cleave the MIT 9-10 recognition sequence in the mutant reporter line ( Figure 5). Additionally, the MIT 9-10x.3 nuclease was highly specific for the mutant site versus the corresponding wild-type site. Specifically, the MIT 9-10 nuclease yielded ⁇ 80% GFP+ cells when tested against the intended (deletion breakpoint) target site but ⁇ 5% GFP+ cells when tested against the wild-type target sites ( Figure 5). In summary, these studies demonstrated that the early-generation MIT 9-10x.3 meganuclease could efficiently and selectively bind and cleave the MIT 9-10 recognition sequence in cells.
  • EXAMPLE 2 Localization of Mitochondria-Targeted MIT 9-10 Meganucleases The purpose of this experiment was to visualize MIT 9-10x.3 localization when a mitochondrial transit peptide (MTP) was fused to the N-terminus.
  • MTP mitochondrial transit peptide
  • the MIT 9-10 protein was further modified to comprise a FLAG tag on its C-terminus.
  • 6e5 HELA cells were nucleofected with 600ng MTP-MIT 9-10x.3-FLAG mRNA using the Lonza 4D-NucleofectorTM (SE buffer, condition CM-150).
  • MIT 9-10x.3 staining appears punctate and overlays with MitoTracker staining ( Figure 6). There does not appear to be any nuclear localization of the MIT 9-10x.3 nuclease when attached to the MTP.
  • the cell line used is a cybrid (i.e., cytoplasmic hybrid) that contains both wild-type and mutant mtDNA comprising the common deletion.
  • the cell line is 91% mutant – that is, 91% of the mtDNA population contains the mutant allele and 9% contains the wildtype allele.
  • 8e5 cybrid cells were nucleofected with MTP-MIT 9-10x.3 mRNA across a dose titration using the Lonza 4D-NucleofectorTM (SF buffer, condition CA-137).
  • the MTP-MIT 9-10x.3 mRNA doses started at 1e5 RNA copies/cell; this translates to 8e10 RNA copies total, or 94.8ng of RNA.
  • the mRNA was then serially diluted 1:10 down to 1e2 RNA copies/cell.
  • Cells were collected at one day post-nucleofection for gDNA extraction and evaluated for transfection efficiency using a Beckman Coulter CytoFlex S cytometer. Transfection efficiency exceeded 95%.
  • gDNA was isolated using the Macherey Nagel NucleoSpin Blood QuickPure kit. mitoTALEN are expressed from two plasmids, each coding for a mitoTALEN monomer. The plasmids also express either eGFP or mCherry.
  • Transfected cells were sorted 48 hours after transfection and populations expressing eGFP and mCherry (yellow cells), only eGFP (green) or neither fluorescent marker (Blafigck cells) was isolated and had their DNA extracted and analyzed by the 3-primer PCR as illustrated in Figure 7A (see arrows) with one forward-primer and two backward primers of the mtDNA Primer B1 corresponds to a mtDNA region inside the common deletion, whereas primers F1 and B2 flank the deleted region. Primers F1 and B1 only amplify WT–mtDNAs, and primers F and B2 amplify ⁇ – mtDNAs.
  • EXAMPLE 4 Evaluation of Mitochondria-Targeted MIT 9-10 Meganucleases in FlpIn CHO Cells
  • the purpose of this experiment was to evaluate several MIT 9-10 meganucleases for (1) activity against the mutant target site and (2) specificity against the corresponding wild-type sequence in an in vitro model. This was done using two FlpIn CHO cell lines that contain a portion of the human mitochondrial genome integrated onto the nuclear chromosome. The integrated sequence contains either the wild-type recognition sequence (SEQ ID NO: 5) or the mutant recognition sequence (i.e., MIT 9-10; SEQ ID NO: 3), as well as surrounding mtDNA sequence.
  • SEQ ID NO: 5 wild-type recognition sequence
  • MIT 9-10 SEQ ID NO: 3
  • the mutant and wild-type binding sites only differ by one nucleotide and therefore meganuclease specificity is paramount, as the objective is to generate an engineered meganuclease that can cleave the mutant sequence at high efficiency while not cleaving the wild-type sequence.
  • Specificity and potency were evaluated by droplet digital PCR (ddPCR) by calculating insertion/deletion (indel) formation at each site.
  • ddPCR droplet digital PCR
  • indel insertion/deletion
  • the engineered meganucleases compared in this study were MIT 9-10x.3 (SEQ ID NO: 7) and MIT 9-10L.90 (SEQ ID NO: 8), an engineered meganuclease that was further optimized from the MIT 9-10x.3 sequence.
  • FlpIn CHO lines were made using the Flp-InTM system from ThermoFisher Scientific.
  • the integration cassette contained either the MIT 9-10 recognition sequence or the wild-type sequence, as well as surrounding mtDNA sequence.
  • 6e5 FlpIn CHO cells were nucleofected using the Lonza 4D- NucleofectorTM at MIT 9-10 meganuclease mRNA doses of either 500ng, 50ng, or 5ng (SF buffer, condition EN-138). Cells were collected at two days post-nucleofection for gDNA extraction and evaluated for transfection efficiency using a Beckman Coulter CytoFlex S cytometer.
  • Transfection efficiency exceeded 95% for both cell lines.
  • gDNA was isolated using the Macherey Nagel NucleoSpin Blood QuickPure kit.
  • Droplet digital PCR (ddPCR) was utilized to determine indel frequency at both the MIT 9- 10 mutant and wild-type sites using P1/P2, F1, and R1 to generate an amplicon surrounding the binding site, as well as P3, F2, R2 to generate a reference amplicon that acts as a genomic counter.
  • the ratio of the two amplicons should be equal in an un-treated population and drop relative to indel formation at the binding site in treated samples.
  • Amplifications were multiplexed in a 24 ⁇ L reaction containing 1x ddPCR Supermix for Probes (no dUTP, BioRad), 250nM of each probe, Attorney Docket No.: P893391190WO (01242) 900nM of each primer, 20 U/ ⁇ L Kpn-I HF (NEB), and 150ng cellular gDNA. Droplets were generated using a QX100 droplet generator (BioRad).
  • Cycling conditions were as follows: 1 cycle of 95 ⁇ C (2 ⁇ C/s ramp) for 10 minutes, 45 cycles of 94 ⁇ C (2 ⁇ C/s ramp) for 10 seconds, 59.2 ⁇ C (2 ⁇ C/s ramp) for 30 seconds, 72C (0.2 ⁇ C/s ramp) for 1 minute 30 seconds, 1 cycle of 98 ⁇ C for 10 minutes, 4 ⁇ C hold.
  • Droplets were analyzed using a QX200 droplet reader (BioRad) and QuantaSoft analysis software (BioRad) was used to acquire and analyze data.
  • F1 CGTATGGCCCACCATAAT (SEQ ID NO: 31)
  • R1 AGTAGAAACCTGTGAGGAAAG (SEQ ID NO: 32)
  • P1 (mutant) AATGGTGAGGGAGGTAGGTGGTA (SEQ ID NO: 33)
  • P2 (WT) TACCACCAACCTCCCT (SEQ ID NO: 34)
  • P3 AGCAGTTCTACCGTACAACCCTAACA (SEQ ID NO: 35)
  • F2 GGCAGTTGAGGTGGATTA (SEQ ID NO: 36)
  • R2 GGAATGCGGTAGTAGTTAGG (SEQ ID NO: 37)
  • the two MIT 9-10 meganucleases were evaluated for indel formation in both the mutant and wild- type sequences at three mRNA doses.
  • MIT 9-10 meganucleases exhibited activity at the wild-type site at a very high mRNA dose (500ng) ( Figure 8).
  • MIT 9-10x.3 generated 78.7% indels in the mutant cell line and only 2.7% indels in the wild-type cell line.
  • MIT 9-10L.90 generated 91.1% indels in the mutant line and 16.2% indels in the wild-type line.
  • MIT 9-10x.3 generated 29.2% indels in the mutant line and 1.9% indels in the wild-type line.
  • MIT 9-10L.90 generated 69.6% indels in the mutant line and 3.2% indels in the wild-type line. Together, these data suggest that while MIT 9-10L.90 is a more potent nuclease, MIT 9- 10x.3 appears to be more specific for the mutant recognition sequence found with the common deletion.
  • MIT 9-10L.209 and MIT 9-10L.210 were each optimized from the MIT 9-10L.90 meganuclease. To do this, each of the engineered meganucleases was evaluated using the CHO cell reporter assay depicted in Figure 3 and described in Example 1. The CHO reporter cells were transfected with mRNA encoding each of the MIT 9-10L.90, MIT 9-10L.209, and MIT 9-10L.210 meganucleases. A control sample of CHO reporter cells were transfected with mRNA encoding the CHO-23/24 meganuclease.
  • 5e4 CHO reporter cells comprising the MIT 9-10 recognition sequence (SEQ ID NO: 3) were transfected with 2.5ng (low dose) or 90 ng (high dose) of mRNA in a 96-well plate using LIPOFECTAMINE® MESSENGERMAX (ThermoFisher) according to the manufacturer’s instructions.
  • CHO reporter cells comprising the corresponding wild-type sequence (SEQ ID NO: 5) (shown in Figure 9 as “9A-10”) were transfected with 90 ng (high dose) of mRNA only.
  • the transfected CHO cells were evaluated by image cytometry at 2 days post transfection to determine the percentage of GFP- positive cells compared to an untransfected negative control.
  • the MIT 9- 10L.209 meganuclease exhibited similar activity against the MIT 9-10 recognition sequence as the earlier-generation MIT 9-10L.90 meganuclease but exhibited substantially lower activity against the wild-type sequence.
  • the MIT 9-10L.210 meganuclease exhibited much greater activity against the MIT 9-10 recognition sequence than the MIT 9-10L.90 meganuclease, and while it also exhibited lower activity against the wild-type sequence, the MIT 9-10L.209 meganuclease exhibited the lowest activity of the three meganucleases evaluated.
  • MIT 9-10 meganucleases were tested to determine if they could shift mtDNA heteroplasmy in human cells. Initially, to determine if these meganucleases could appropriately localize to the mitochondria where the heteroplasmic mtDNA is located, Cos-7 cells were transfected with C-terminal FLAG-tagged MIT 9-10L.209, MIT 9-10L.210, and MIT 9-10L.90 meganucleases that each comprised an N-terminal MTP.
  • BH10.9 cells were transfected with two independent plasmids: 25 ug of plasmids encoding the MIT 9-10L.209, MIT 9-10L.210, or MIT 9-10L.90 meganucleases each together with 5 ug of a plasmid carrying GFP (pLenti-GFP). Transfection was carried out with the GenJet DNA in vitro transfection reagent version II (#SL100489; SignaGen Laboratories. Each of the MIT 9-10 meganucleases in this experiment comprised an N-terminal MTP (Cox8Su9; SEQ ID NO: 25) and a C-terminal FLAG Tag.
  • cells were FACS sorted for eGFP expression using a FACSAria IIu by gating on single cell fluorescence using 561 nm laser and 600LP with a 530/30 filter set for eGFP.
  • Cells expressing the green marker were labeled as “green” because they were expected to co-express the MIT 9-10 meganucleases together with the plasmid carrying GFP.
  • Cells were also isolated that did not express the fluorescent markers, which were labeled as “black” and therefore would not be expected to have any meganuclease expression.
  • Cells were expanded and DNA was extracted at day 0 (day of the sorting) and days 4, 8 and 12 days after sorting from both green and black populations.
  • Figure 11 shows the summary of three independent experiments demonstrating a shift in mtDNA heteroplasmy, which is indicated by the percentage of change in the mutant in green cells normalized to black cells (non-transfected).
  • the reduction in heteroplasmy was approximately 40% in cells treated with the MIT 9-10L.209 meganuclease, which persisted over days 4, 8, and 12.
  • the MIT 9-10L.210 and MIT 9-10L.90 meganucleases also showed a decrease in the mutant mtDNA but to a lesser extent.
  • Figure 12 shows a corresponding shift in heteroplasmy in the green cells normalized to control heteroplasmic cells prior to transfection.
  • EXAMPLE 7 Evaluation of MIT 9-10 Meganucleases For Shifting mtDNA Heteroplasmy after mRNA Electroporation of Cybrid Cells
  • Cybrid cells carrying approximately 80% mutant mtDNA were electroporated with mRNA coding for 2 different meganucleases, MIT 9-10x.3 or MIT 9-10L.209, or GFP-mRNA as a control. Two different concentrations of mRNA were used: 1e4 and 1e5 RNA copies/cell. Electroporation was done at 1250V, 30sec, 1 pulse with Neon transfection system (Invitrogen).
  • Oxygen consumption rates were measured using the Seahorse XFe96 Analyzer (Agilent Technologies). OCR values were normalized to proteins levels and data analysis was conducted using Wave software (Agilent Technologies). A clear recovery was observed in the cells treated with mRNA encoding the mitochondria- targeted MIT 9-10x.3 or MIT 9-10L.209 meganucleases compared to mRNA GFP (1e4 copies/cell) (Figure 18A). Changes in the extracellular acidification rate (ECAR) over time are shown in Figure Attorney Docket No.: P893391190WO (01242) 18B. Figure 19 shows that both basal and maximal respiration values were significantly increased in cells treated with the mitochondria-targeted MIT 9-10 meganucleases.
  • ECAR extracellular acidification rate

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Abstract

Disclosed herein are engineered meganucleases engineered to recognize and cleave a recognition sequence present in human mitochondrial DNA (mtDNA). The disclosure further relates to the use of such engineered meganucleases in methods for producing genetically-modified eukaryotic cells, and to a population of genetically-modified eukaryotic cells, wherein mutant mtDNA is modified or degraded.

Description

Attorney Docket No.: P893391190WO (01242) ENGINEERED MEGANUCLEASES THAT TARGET HUMAN MITOCHONDRIAL GENOMES STATEMENT REGARDING THE SEQUENCE LISTING The Sequence Listing associated with this application is provided in ST.26 (XML) format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The XML copy named P89339_1190WO_Seq_List.xml is 50,811 bytes in size, was created on May 5, 2025 and is being submitted electronically via USPTO Patent Center. FIELD OF THE INVENTION The present disclosure relates to the field of molecular biology and recombinant nucleic acid technology. In particular, the present disclosure relates to recombinant meganucleases engineered to recognize and cleave recognition sequences found in the human mitochondrial genome. The present disclosure further relates to the use of such recombinant meganucleases in methods for producing genetically-modified eukaryotic cells, and to a population of genetically- modified eukaryotic cells wherein the mitochondrial DNA has been modified. BACKGROUND OF THE INVENTION In all organisms, mitochondria regulate cellular energy and metabolism under normal growth and development as well as in response to stress. Many of the proteins functioning in these roles are coded for in the mitochondrial genome. Thus, editing of the mitochondrial genome has diverse applications in both animals and plants. In humans, deleterious mitochondrial mutations are the source of a number of disorders for which gene editing therapies could be applied. Pathogenic mitochondrial DNA (mtDNA) mutations include large-scale rearrangements and point mutations in protein coding, transfer RNA (tRNA) or ribosomal RNA (rRNA) genes. Although the prevalence of mtDNA-related disease diagnosis is about 1 in 5,000, the population frequency of the ten most common pathogenic mtDNA mutations is much higher, approaching 1 in 200, implying that many “normal” individuals carry low levels of mutated genomes (Schon et al., Nat Rev Gen 13:878-890 (2012)). Mutated mtDNA, in most cases, co-exist with wild-type mtDNA in patients’ cells (mtDNA heteroplasmy). Several studies showed that the wild-type mtDNA has a strong protective effect, and biochemical abnormalities were observed only when the levels of the mutated mtDNA were higher than 80-90% (Schon et al., Nature Reviews Genetics 13:878-890 (2012)). It has been shown that muscle fibers develop an OXPHOS defect only when the mutation load is above 80% (Sciacco Attorney Docket No.: P893391190WO (01242) et al., Hum Mol Genet 3:13-19 (1994)). Therefore, any approach that could shift this balance by even a small percentage towards the wild-type would have strong therapeutic potential. However, mtDNA manipulation remains an underexplored area of science because of the inability to target mtDNA at high efficiencies and generate precise edits. The mitochondrial genome is difficult to edit because it requires predictable repair mechanisms and delivery of an editing technology to this organelle. In view of the difficulty and unpredictability associated with mitochondrial genome editing, there is an unmet need for precise editing of mtDNA, which would open up an entire field of inquiry and opportunity in life sciences. The ability to target and edit a defined region (preferably limited to just one gene) of the mitochondrial genome in a more predictable manner would be a clear benefit over currently available systems. SUMMARY OF THE INVENTION Provided herein are compositions and methods for precise editing of mutant mitochondrial DNA (mtDNA) comprising the 4977 basepair common deletion. The compositions and methods provided herein can be used for editing one specific mitochondrial gene without impacting surrounding regions. In one aspect, the invention provides an engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 in mitochondrial genomes of a eukaryotic cell, wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to a second recognition half- site of the recognition sequence and comprises a second hypervariable (HVR2) region. In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 7-10. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-10. In some embodiments, the HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-10. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of any one of SEQ ID NOs: 7-10. In some embodiments, the HVR1 region comprises residues 215-270 of any one of SEQ ID NOs: 7-10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 215-270 of any one of SEQ ID NOs: 7-10. Attorney Docket No.: P893391190WO (01242) In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of any one of SEQ ID NOs: 7-10. In some embodiments, the first subunit comprises a residue corresponding to residue 271 of any one of SEQ ID NOs: 7-10. In some embodiments, the first subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 9. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 210 of any one of SEQ ID NOs: 7-10. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 271 of any one of SEQ ID NOs: 7-10. In some embodiments, the first subunit comprises residues 198-344 of any one of SEQ ID NOs: 7-10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 198-344 of any one of SEQ ID NOs: 7-10. In some embodiments, the first subunit comprises residues 196-354 of any one of SEQ ID NOs: 7-10. In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs: 7-10. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-10. In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-10. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of any one of SEQ ID NOs: 7-10. In some embodiments, the HVR2 region comprises residues 24- 79 of any one of SEQ ID NOs: 7-10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 24-79 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of any one of SEQ ID NOs: 7-10. Attorney Docket No.: P893391190WO (01242) In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 9. In some embodiments, the second subunit comprises a residue corresponding to residue 80 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 19 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 80 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises residues 7- 153 of any one of SEQ ID NOs: 7-10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 7-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises residues 6-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises residues 5-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises residues 4-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises residues 3-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises residues 2-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of any one of SEQ ID NOs: 7-10. Attorney Docket No.: P893391190WO (01242) In some embodiments, the second subunit comprises residues 1-153 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit. In some embodiments, the N-terminus of the linker is fused to the residue (i.e., a D residue) corresponding to residue 153 of any one of SEQ ID NOs: 7-10, and the C-terminus of the linker is fused to the residue (i.e., a Y residue) corresponding to residue 196 of any one of SEQ ID NOs: 7- 10. In some embodiments, the linker comprises residues 154-195 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 4-343 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 3-343 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 2-343 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 7-10. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 19-22. In some Attorney Docket No.: P893391190WO (01242) embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 19-22. In some embodiments, the engineered meganuclease comprises a mitochondrial transit peptide (MTP). Such engineered meganucleases described herein comprising an MTP are mitochondria-targeted engineered meganucleases. In some embodiments, the MTP comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence set forth in any one of SEQ ID NOs: 23-25. In some embodiments, the MTP comprises an amino acid sequence set forth in any one of SEQ ID NOs: 23-25. In some embodiments, the MTP is attached to the C-terminus of the engineered meganuclease. In some embodiments, the MTP is attached to the N-terminus of the engineered meganuclease. In some embodiments, the MTP is fused to the engineered meganuclease. In some embodiments, the MTP is attached to the engineered meganuclease by a polypeptide linker. In some embodiments, the engineered meganuclease is attached to a first MTP and a second MTP. In some embodiments, the first MTP and/or the second MTP comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence set forth in any one of SEQ ID NOs: 23-25. In some embodiments, the first MTP and/or the second MTP comprises an amino acid sequence set forth in any one of SEQ ID NOs: 23-25. In some embodiments, the first MTP and the second MTP are identical. In some embodiments, the first MTP and the second MTP are not identical. In some embodiments, the first MTP and/or the second MTP is fused to the engineered meganuclease. In some embodiments, the first MTP and/or the second MTP is attached to the engineered meganuclease by a polypeptide linker. In some embodiments, the engineered meganuclease is attached to a nuclear export sequence (NES). In some embodiments, the NES comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence set forth in SEQ ID NO: 26 or 27. In some embodiments, the NES comprises an amino acid sequence set forth in SEQ ID NO: 26 or 27. In some embodiments, the NES is attached at the N-terminus of the engineered meganuclease. In some embodiments, the NES is attached at the C-terminus of the engineered meganuclease. In some embodiments, the NES is fused to the engineered meganuclease. In some embodiments, the NES is attached to the engineered meganuclease by a polypeptide linker. In some embodiments, the engineered meganuclease comprises a first NES and a second NES. In some embodiments, the first NES is attached at the N-terminus of the engineered meganuclease, and the second NES is attached at the C-terminus of the engineered meganuclease. In some embodiments, the first NES and/or the second NES comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, Attorney Docket No.: P893391190WO (01242) 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence set forth in SEQ ID NO: 26 or 27. In some embodiments, the first NES and/or the second NES comprises an amino acid sequence set forth in SEQ ID NO: 26 or 27. In some embodiments, the first NES and the second NES are identical. In some embodiments, the first NES and the second NES are not identical. In some embodiments, the first NES and/or the second NES is fused to the engineered meganuclease. In some embodiments, the first NES and/or the second NES is attached to the engineered meganuclease by a polypeptide linker. In another aspect, the invention provides a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein. In some embodiments, the polynucleotide is an mRNA. In another aspect, the invention provides a recombinant DNA construct comprising a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein. In some embodiments, the recombinant DNA construct encodes a recombinant virus genome comprising the polynucleotide. In some embodiments, the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adeno-associated virus (AAV). In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV is a muscle-tropic recombinant AAV or a central nervous system (CNS)-tropic recombinant AAV. In some embodiments, the recombinant AAV has an AAV9 capsid, a MyoAAV capsid, or an AAVMYO capsid. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the engineered meganuclease. In some embodiments, the promoter is a constitutive promoter. In some such embodiments, the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. In other embodiments, the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter. In some such embodiments, the promoter is an MHCK7 promoter, a truncated MCK (tMCK) promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter. In other embodiments, the promoter is a central nervous system (CNS) cell-specific promoter. In particular embodiments, the promoter is a stem cell-specific promoter, a CD34+ HSC-specific promoter, a muscle-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell-specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, a kidney cell-specific promoter, a bone marrow cell-specific promoter, or an ear hair cell-specific promoter. In some embodiments, the recombinant DNA construct is a plasmid DNA. Attorney Docket No.: P893391190WO (01242) In another aspect, the invention provides a recombinant virus comprising a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein. In some embodiments, the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant AAV. In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV is a muscle-tropic recombinant AAV or a central nervous system (CNS)-tropic recombinant AAV. In some embodiments, the recombinant AAV has an AAV9 capsid, a MyoAAV capsid, or an AAVMYO capsid. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the engineered meganuclease. In some embodiments, the promoter is a constitutive promoter. In some such embodiments, the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. In other embodiments, the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter. In some such embodiments, the promoter is an MHCK7 promoter, a truncated MCK (tMCK) promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter. In other embodiments, the promoter is a central nervous system (CNS) cell-specific promoter. In particular embodiments, the promoter is a stem cell-specific promoter, a CD34+ HSC-specific promoter, a muscle-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell-specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, a kidney cell-specific promoter, a bone marrow cell-specific promoter, or an ear hair cell-specific promoter. In another aspect, the invention provides a lipid nanoparticle composition comprising lipid nanoparticles comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence encoding an engineered meganuclease described herein. In some embodiments, the polynucleotide is an mRNA. In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an engineered meganuclease described herein. In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polynucleotide described herein. In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant DNA construct described herein. In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant virus described herein. Attorney Docket No.: P893391190WO (01242) In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid nanoparticle composition described herein. In another aspect, the invention provides a eukaryotic cell comprising any polynucleotide described herein. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the human cell is a human muscle cell, a human muscle stem cell, or a human CNS cell. In another aspect, the invention provides a method for producing a genetically-modified eukaryotic cell, the method comprising introducing into a eukaryotic cell: (a) a polynucleotide comprising a nucleic acid sequence encoding a mitochondria-targeted engineered meganuclease (e.g., comprising an MTP) described herein, wherein the engineered meganuclease is expressed in the eukaryotic cell; or (b) a mitochondria-targeted engineered meganuclease described herein; wherein the mitochondria-targeted engineered meganuclease produces a cleavage site at the recognition sequence comprising SEQ ID NO: 3 in mutant mitochondrial genomes of the eukaryotic cell. In some embodiments, the cleavage site is repaired by non-homologous end joining, such that the recognition sequence comprises an insertion or deletion. In some embodiments, the mutant mitochondrial genomes comprising the recognition sequence are degraded in the genetically-modified eukaryotic cell. In some embodiments, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of mutant mitochondrial genomes comprising the recognition sequence are degraded in the genetically-modified eukaryotic cell. In some embodiments, the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising the recognition sequence increases in the genetically-modified eukaryotic cell. In some embodiments, the ratio increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more. In some embodiments, the percentage of wild-type mitochondrial genomes in the genetically-modified eukaryotic cell is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, of the total mitochondrial genomes in the genetically-modified eukaryotic cell. In some embodiments, the percentage of mutant mitochondrial genomes comprising the recognition sequence in the genetically-modified eukaryotic cell decreases by about Attorney Docket No.: P893391190WO (01242) 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. In some embodiments, cellular respiration in the genetically-modified eukaryotic cell increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. In some embodiments, cellular respiration in the genetically-modified eukaryotic cell increases by about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more. In another aspect, the invention provides a method for producing a population of eukaryotic cells comprising a plurality of genetically-modified cells, the method comprising introducing into a plurality of eukaryotic cells in the population: (a) a polynucleotide comprising a nucleic acid sequence encoding a mitochondria-targeted engineered meganuclease (e.g., comprising an MTP) described herein, wherein the mitochondria-targeted engineered meganuclease is expressed in the plurality of eukaryotic cells; or (b) a mitochondria-targeted engineered meganuclease described herein; wherein the mitochondria-targeted engineered meganuclease produces a cleavage site at a recognition sequence comprising SEQ ID NO: 3 in mutant mitochondrial genomes of the plurality of eukaryotic cells. In some embodiments, the cleavage site is repaired by non-homologous end joining, such that the recognition sequence comprises an insertion or deletion. In some embodiments, the mutant mitochondrial genomes comprising the recognition sequence are degraded in the plurality of genetically-modified eukaryotic cells. In some embodiments, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of mutant mitochondrial genomes comprising the recognition sequence are degraded in the plurality of genetically-modified eukaryotic cells. In some embodiments, the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising the recognition sequence increases in the plurality of genetically-modified eukaryotic cells. In some embodiments, the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising the recognition sequence increases in the population of eukaryotic cells. In some embodiments, the ratio increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more. In some embodiments, the percentage of wild-type mitochondrial genomes in the plurality of Attorney Docket No.: P893391190WO (01242) genetically-modified eukaryotic cells increases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. In some embodiments, the percentage of wild-type mitochondrial genomes in the population of eukaryotic cells increases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. In some embodiments, the percentage of mutant mitochondrial genomes comprising the recognition sequence in the plurality of genetically-modified eukaryotic cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. In some embodiments, the percentage of mutant mitochondrial genomes comprising the recognition sequence in the population of eukaryotic cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. In some embodiments, cellular respiration in the plurality of genetically-modified eukaryotic cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. In some embodiments, cellular respiration in the plurality of genetically-modified eukaryotic cells increases by about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more. In some embodiments, cellular respiration in the population of eukaryotic cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. In some embodiments, cellular respiration in the population of eukaryotic cells increases by about 30-40%, about 40-50%, about 50-60%, about 60- 70%, about 70-80%, about 80-90%, about 90-100%, or more. In some embodiments, the recognition sequence is within a region of the mitochondrial DNA associated with a mitochondrial disorder. In some embodiments, the mitochondrial disorder is a mitochondrial DNA common deletion disorder. In some embodiments, the mutant mitochondrial genomes lack the nucleotides positioned between nucleotides 8,470 and 13,447 in a wild-type mitochondrial genome. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is any mRNA described herein. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is any recombinant DNA construct described herein. In some embodiments, the Attorney Docket No.: P893391190WO (01242) polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus. In some embodiments, the recombinant virus is any recombinant virus described herein. In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV is a muscle-tropic recombinant AAV or a CNS-tropic recombinant AAV. In some embodiments, the recombinant AAV has an AAV9 capsid, a MyoAAV capsid, or an AAVMYO capsid. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the mitochondria-targeted engineered meganuclease. In some embodiments, the promoter is a constitutive promoter. In some such embodiments, the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. In other embodiments, the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter. In some such embodiments, the promoter is an MHCK7 promoter, a truncated MCK (tMCK) promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter. In other embodiments, the promoter is a central nervous system (CNS) cell-specific promoter. In particular embodiments, the promoter is a stem cell-specific promoter, a CD34+ HSC-specific promoter, a muscle-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell- specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell- specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, a kidney cell-specific promoter, a bone marrow cell-specific promoter, or an ear hair cell-specific promoter. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the human cell is a human muscle cell, a human muscle stem cell, or a human CNS cell. In some embodiments, the eukaryotic cell is a stem cell, a CD34+ HSC, a muscle cell, a skeletal muscle cell, a myotube cell, a muscle satellite cell, a neuron, an astrocyte, a microglia cell, an eye cell, a retinal cell, a retinal ganglion cell, a retinal pigmentary epithelium cell, a pancreatic cell, a pancreatic beta cell, a kidney cell, a bone marrow cell, or an ear hair cell. In another aspect, the invention provides a genetically-modified eukaryotic cell, or a population of genetically-modified eukaryotic cells, produced by any method described herein for producing a genetically-modified eukaryotic cell or any method for producing a population of eukaryotic cells comprising a plurality of genetically-modified cells. In another aspect, the invention provides a method for degrading mutant mitochondrial genomes in a target cell in a subject, or in a population of target cells in a subject, the method comprising delivering to the target cell or the population of target cells: (a) a polynucleotide Attorney Docket No.: P893391190WO (01242) comprising a nucleic acid sequence encoding a mitochondria-targeted engineered meganuclease (e.g., comprising an MTP) described herein, wherein the mitochondria-targeted engineered meganuclease is expressed in the target cell or the population of target cells; or (b) a mitochondria- targeted engineered meganuclease described herein; wherein the mitochondria-targeted engineered meganuclease produces a cleavage site in the mutant mitochondrial genomes at a recognition sequence comprising SEQ ID NO: 3, and wherein the mutant mitochondrial genomes are degraded. In some embodiments, the mutant mitochondrial genomes comprise a mtDNA common deletion. In some embodiments, the mutant mitochondrial genomes lack the nucleotides positioned between nucleotides 8,470 and 13,447 in a wild-type mitochondrial genome. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the target cell or the population of target cells are human muscle cells, human muscle stem cells, or human CNS cells. In some embodiments, the target cell is a stem cell, a CD34+ HSC, a muscle cell, a skeletal muscle cell, a myotube cell, a muscle satellite cell, a neuron, an astrocyte, a microglia cell, an eye cell, a retinal cell, a retinal ganglion cell, a retinal pigmentary epithelium cell, a pancreatic cell, a pancreatic beta cell, a kidney cell, a bone marrow cell, or an ear hair cell, or the population of target cells is a population of stem cells, CD34+ HSCs, muscle cells, skeletal muscle cells, myotube cells, muscle satellite cells, neurons, astrocytes, microglia cells, eye cells, retinal cells, retinal ganglion cells, retinal pigmentary epithelium cells, pancreatic cells, pancreatic beta cells, kidney cells, bone marrow cells, or ear hair cells. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is any mRNA described herein. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is any recombinant DNA construct described herein. In some embodiments, the polynucleotide is introduced into the target cell, or the population of target cells, by a lipid nanoparticle. In some embodiments, the polynucleotide is introduced into the target cell, or the population of target cells, by a recombinant virus. In some embodiments, the recombinant virus is any recombinant virus described herein. In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV is a muscle-tropic recombinant AAV or a CNS-tropic recombinant AAV. In some embodiments, the recombinant AAV has an AAV9 capsid, a MyoAAV capsid, or an AAVMYO capsid. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the mitochondria-targeted engineered meganuclease. In some embodiments, the promoter is a constitutive promoter. In some such embodiments, the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. In other embodiments, the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter. In some such embodiments, the promoter is an MHCK7 promoter, a truncated MCK (tMCK) promoter, a CK8 Attorney Docket No.: P893391190WO (01242) promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter. In other embodiments, the promoter is a central nervous system (CNS) cell-specific promoter. In some embodiments, the promoter is a stem cell-specific promoter, a CD34+ HSC-specific promoter, a muscle-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell-specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, a kidney cell-specific promoter, a bone marrow cell-specific promoter, or an ear hair cell-specific promoter. In some embodiments, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of mutant mitochondrial genomes comprising the recognition sequence are degraded in the target cell or the population of the target cells. In some embodiments, the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising the recognition sequence increases in the target cell or the population of target cells. In some embodiments, the ratio increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more. In some embodiments, the percentage of wild-type mitochondrial genomes in the target cell or the population of target cells is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, of the total mitochondrial genomes in the target cell or the population of target cells. In some embodiments, the percentage of mutant mitochondrial genomes comprising the recognition sequence in the target cell or the population of target cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. In some embodiments, cellular respiration in the target cell or the population of target cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. In some embodiments, cellular respiration in Attorney Docket No.: P893391190WO (01242) the target cell or the population of target cells increases by about 30-40%, about 40-50%, about 50- 60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more. In another aspect, the invention provides a method for treating a condition associated with a mitochondrial mtDNA common deletion in a subject, the method comprising administering to the subject: (a) a therapeutically-effective amount of a polynucleotide comprising a nucleic acid sequence encoding a mitochondria-targeted engineered meganuclease (e.g., comprising an MTP) described herein, wherein the polynucleotide is delivered to a target cell, or a population of target cells, in the subject, wherein the mitochondria-targeted engineered meganuclease is expressed in the target cell or the population of target cells; or (b) a therapeutically-effective amount of a mitochondria-targeted engineered meganuclease described herein, wherein the engineered meganuclease is delivered to a target cell, or a population of target cells, in the subject; wherein the mitochondria-targeted engineered meganuclease produces a cleavage site in mutant mitochondrial genomes at a recognition sequence comprising SEQ ID NO: 3, and wherein the mutant mitochondrial genomes are degraded. In some embodiments, the mutant mitochondrial genomes comprise the mtDNA common deletion. In some embodiments, the mutant mitochondrial genomes lack the nucleotides positioned between nucleotides 8,470 and 13,447 in a wild-type mitochondrial genome. In some embodiments, the method reduces or ameliorates one or more symptoms associated with the mtDNA common deletion. In some embodiments, the method comprises administering any pharmaceutical composition described herein that comprises a mitochondria- targeted meganuclease described herein, or a polynucleotide described herein that encodes a mitochondria-targeted engineered meganuclease described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the target cell or population of target cells are human muscle cells, human muscle stem cells, or human CNS cells. In some embodiments, the target cell is a stem cell, a CD34+ HSC, a muscle cell, a skeletal muscle cell, a myotube cell, a muscle satellite cell, a neuron, an astrocyte, a microglia cell, an eye cell, a retinal cell, a retinal ganglion cell, a retinal pigmentary epithelium cell, a pancreatic cell, a pancreatic beta cell, a kidney cell, a bone marrow cell, or an ear hair cell, or the population of target cells is a population of stem cells, CD34+ HSCs, muscle cells, skeletal muscle cells, myotube cells, muscle satellite cells, neurons, astrocytes, microglia cells, eye cells, retinal cells, retinal ganglion cells, retinal pigmentary epithelium cells, pancreatic cells, pancreatic beta cells, kidney cells, bone marrow cells, or ear hair cells. In some embodiments, the condition is a condition of the bone marrow, the pancreas, muscle, skeletal muscle, central nervous system, the eye, or the ears. In some embodiments, the condition is Pearson Syndrome, Kearns-Sayre Syndrome (KSS), Progressive External Ophthalmoplegia (PEO), diabetes, or kidney dysfunction. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide Attorney Docket No.: P893391190WO (01242) is any mRNA described herein. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is any recombinant DNA construct described herein. In some embodiments, the polynucleotide is delivered to the target cell, or the population of target cells, by a lipid nanoparticle. In some embodiments, the polynucleotide is delivered to the target cell, or the population of target cells, by a recombinant virus. In some embodiments, the recombinant virus is any recombinant virus described herein. In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV is a muscle-tropic recombinant AAV or a CNS-tropic recombinant AAV. In some embodiments, the recombinant AAV has an AAV9 capsid, a MyoAAV capsid, or an AAVMYO capsid. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the mitochondria-targeted engineered meganuclease. In some embodiments, the promoter is a constitutive promoter. In some such embodiments, the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. In other embodiments, the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter. In some such embodiments, the promoter is an MHCK7 promoter, a truncated MCK (tMCK) promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter. In other embodiments, the promoter is a central nervous system (CNS) cell-specific promoter. In some embodiments, the promoter is a stem cell-specific promoter, a CD34+ HSC-specific promoter, a muscle-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell- specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, a kidney cell-specific promoter, a bone marrow cell-specific promoter, or an ear hair cell-specific promoter. In some embodiments, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of mutant mitochondrial genomes comprising the recognition sequence are degraded in the target cell or the population of the target cells. In some embodiments, the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising the recognition sequence increases in the target cell or the population of target cells. In some embodiments, the ratio increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about Attorney Docket No.: P893391190WO (01242) 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more. In some embodiments, the percentage of wild-type mitochondrial genomes in the target cell or the population of target cells is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, of the total mitochondrial genomes in the target cell or the population of target cells. In some embodiments, the percentage of mutant mitochondrial genomes comprising the recognition sequence in the target cell or population of target cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. In some embodiments, cellular respiration in the target cell or the population of target cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. In some embodiments, cellular respiration in the target cell or the population of target cells increases by about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more. The foregoing and other aspects and embodiments of the present disclosure can be more fully understood by reference to the following detailed description and claims. Certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All sub-combinations of features listed in the embodiments are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. Embodiments of each aspect of the present disclosure disclosed herein apply to each other aspect of the disclosure mutatis mutandis. BRIEF DESCRIPTION OF THE FIGURES Figure 1A shows the MIT 9-10 recognition sequence (SEQ ID NO: 3) present in mutant mitochondrial genomes comprising the common deletion. The MIT 9-10 recognition sequence targeted by engineered meganucleases of the invention comprises two recognition half-sites. Each recognition half-site comprises 9 base pairs, separated by a 4 base pair central sequence. The MIT Attorney Docket No.: P893391190WO (01242) 9-10 recognition sequence comprises two recognition half-sites referred to as MIT9 and MIT10. Figure 1B illustrates the deletion of nucleotides from human mitochondrial DNA that is referred to as the common deletion. The common deletion results in the removal of a 4977 basepair region of the mitochondrial DNA, reducing the size of the genome from 16,569 basepairs to 11,592 basepairs. Additionally, the MIT 9-10 recognition sequence (SEQ ID NO: 3) is not present in wild- type mitochondrial DNA but is generated following the common deletion. Figure 2 shows that the engineered meganucleases described herein comprise two subunits, wherein the first subunit comprising the HVR1 region binds to a first recognition half-site (e.g., MIT9) and the second subunit comprising the HVR2 region binds to a second recognition half-site (e.g., MIT10). In embodiments where the engineered meganuclease is a single-chain meganuclease, the first subunit comprising the HVR1 region can be positioned as either the N- terminal or C-terminal subunit. Likewise, the second subunit comprising the HVR2 region can be positioned as either the N-terminal or C-terminal subunit. Figure 3 shows an alignment of MIT 9-10 meganucleases exemplified herein, including MIT 9-10x.3 (SEQ ID NO: 7), MIT 9-10L.90 (SEQ ID NO: 8), MIT 9-10L.209 (SEQ ID NO: 9), and MIT 9-10L.210 (SEQ ID NO: 10). Figure 4 shows a schematic of reporter assay in CHO cells for evaluating engineered meganucleases targeting the MIT 9-10 recognition sequence (SEQ ID NO: 3) or a recognition sequence present in wild-type human mitochondrial DNA (SEQ ID NO: 5) that differs from MIT 9- 10 by only a single nucleotide. For the engineered meganucleases described herein, a CHO cell line was produced in which a reporter cassette was integrated stably into the genome of the cell. The reporter cassette comprised, in 5' to 3' order: an SV40 Early Promoter; the 5' 2/3 of the GFP gene; the recognition sequence for an engineered meganuclease described herein (e.g., the MIT 9- 10 recognition sequence or the corresponding wild-type sequence); the recognition sequence for the CHO-23/24 meganuclease (WO/2012/167192); and the 3' 2/3 of the GFP gene. Cells stably transfected with this cassette did not express GFP in the absence of a DNA break-inducing agent. Meganucleases were introduced by transduction of an mRNA encoding each meganuclease. When a DNA break was induced at either of the meganuclease recognition sequences, the duplicated regions of the GFP gene recombined with one another to produce a functional GFP gene. The percentage of GFP-expressing cells could then be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the meganucleases. Figure 5 shows flow cytometry results generated in CHO reporter cell lines, comprising either the MIT 9-10 recognition sequence (SEQ ID NO: 3) or the corresponding wild-type recognition sequence (SEQ ID NO: 5), that were transfected with mRNA encoding the MIT 9- Attorney Docket No.: P893391190WO (01242) 10x.3 meganuclease or a CHO 23-24 meganuclease (control) and assayed 48 hours post- transfection for the percentage of GFP+ cells. Figure 6 demonstrates mitochondrial targeting of a mitochondria-targeted MIT 9-10 engineered meganuclease. HELA cells were transfected with a plasmid coding for the MIT 9-10x.3 meganuclease that comprised a mitochondrial transit peptide (MTP) at its N-terminus. Immunocytological staining was achieved with anti-flag antibody (Meganucelase marker), and Mitotracker Red, which stains mitochondria. Cells were viewed under 40x magnification with Zeiss LSM710 confocal microscope. Figure 7 depicts the development of mitochondria-targeted engineered meganucleases specific for the human mtDNA common deletion, and a comparison to previously developed mitoTALENs. Figure 7A provides an illustration exemplifying the approach to specifically eliminate the common deletion mtDNA in living cells. The mitoTALEN monomers bind to regions that are too far apart in the wild-type mtDNA for FokI nuclease to dimerize and cleave the DNA. Only after the common deletion occurs are the mitoTALEN monomers close enough to allow the FokI nuclease to dimerize and cleave the genome. The mitochondria-targeted MIT 9-10 meganucleases described herein are monomeric and bind to the MIT 9-10 recognition sequence that spans the breakpoint after the common deletion occurs. The MIT 9-10 recognition sequence is not present until after the common deletion occurs. Figure 7B depicts the common deletion breakpoint region showing the DNA binding for mitoTALEN monomers and the MIT 9-10 meganuclease. The box in the center sequence illustrates the 13-bp direct repeat believed to mediate common deletion formation. The MIT 9-10 nuclease binding region avoids part of this direct repeat. Figure 7C shows relative levels of wild-type genomes were increased, using a 3-primer PCT, in sorted cells expressing both monomers (yellow) in the case of mitoTALEN and in “green” cells in the case of the mitochondria-targeted MIT 9-10 meganuclease. The arrows represent the amplifications depicted in panel A. Figure 7D provides results of experiments using the same 3-primer PCR technique in which sorted cells were analyzed after transfection with a plasmid coding for the MIT 9-10 meganuclease (co-transfected with a GFP-expressing plasmid). Sorted cells were “Black” (negative) and “Green” (positive). The lane marked “GFP” was transfected with GFP plasmid only. The lane marked “MitoMega” was transfected with the MIT 9-10 meganuclease but not sorted. Figure 8 shows the percentage (%) indel formation at either the mutant MIT 9-10 site or the corresponding wild-type site in cells transfected with either GFP mRNA at 500ng (negative control), or mRNA encoding the MIT 9-10x.3 or MIT 9-10L.90 engineered meganucleases at 500ng, 50ng, or 5ng, respectively. Each of the tested meganucleases were fused to a mitochondrial transit peptide (MTP) at their N-terminus. Attorney Docket No.: P893391190WO (01242) Figure 9 shows flow cytometry for GFP positive cells in a FlpIn CHO cell reporter system, wherein cells comprising either the MIT 9-10 recognition sequence or the corresponding wild-type sequence were transfected with mRNA encoding the indicated MIT 9-10 meganucleases or a mock negative control at low and high doses of mRNA. Figure 10 shows the immunocytochemistry imaging of COS-7 cells transfected with the MIT 9-10L.209, MIT 9-10L.210, or MIT 9-10L.90 meganucleases that each comprised an MTP at the N-terminus and were fused to a FLAG tag. Cells were stained with MitoTracker red to visualize the mitochondria and were stained with an anti-flag antibody. The left side of the figure (“merge”) shows cells where the meganucleases localized to the mitochondria. Figure 11 shows the shift in mitochondrial heteroplasmic DNA levels in BH10.9 cells clone 1-C-7-65% co-transfected with the MIT9-10 L.209, MIT9-10L.210 and MIT9-10L.90 meganucleases, each comprising an N-terminal MTP, and a GFP plasmid. Cells were sorted for GFP 48 hours after transfection and the percentage (%) change in levels of mutant mtDNA compared to cells not having been transfected with the mitochondria-targeted MIT9-10 meganucleases (“black cells”) was calculated. Figure 12 shows the shift in mitochondrial heteroplasmic DNA levels in BH10.9 cells clone 1-C-7-65% co-transfected with the MIT9-10 L.209, MIT9-10L.210 and MIT9-10L.90 meganucleases, each comprising an N-terminal MTP, and a GFP plasmid. Cells were sorted for GFP 48 hours after transfection and the percentage (%) change in levels of mutant mtDNA compared to untransfected control cells was calculated. Figure 13 provides semiquantitative PCR results of experiments where BH10.9 cells clone 1-C-7-65% were co-transfected with the MIT9-10 L.209, MIT9-10L.210, and MIT9-10L.90 meganucleases, each comprising an N-terminal MTP, and a GFP plasmid. Cells were sorted for GFP expression and a 3-primer semi-quantitative PCR technique was used to determine levels of WT or mutant mtDNA in untransfected “black cells” (Bl) and in transfected “green cells” (Gr). The upper band is the WT mtDNA sequence and the lower band is the mutant mtDNA sequence. Figure 14 shows PCR analysis of heteroplasmic shift after 7 days and 14 days in cybrid cells carrying approximately 80% mutant mtDNA, that were electroporated with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each comprising an MTP at their N-terminus, or a GFP mRNA control. Figure 15 shows a primer strategy for PCR analysis of wild-type mitochondrial DNA and mitochondrial DNA comprising the common deletion in cybrid cells, which carry approximately 80% mutant mtDNA. The cybrid cells were electroporated with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each comprising an MTP at their N-terminus, or a GFP mRNA control. Attorney Docket No.: P893391190WO (01242) Figure 16 shows a PCR analysis of wild-type mitochondrial DNA and mitochondrial DNA comprising the common deletion in cybrid cells, which carry approximately 80% mutant mtDNA. The cybrid cells were electroporated with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each comprising an MTP at their N-terminus, or a GFP mRNA control. PCR analysis was conducted on days 4, 7, and 14. Figure 17 shows a PCR analysis of mitochondrial DNA copy number in cybrid cells, which carry approximately 80% mutant mtDNA. The cybrid cells were electroporated with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each comprising an MTP at their N- terminus, or a GFP mRNA control. PCR analysis was conducted on days 4 and 14. Figure 18 shows cellular respiration of cybrids cells comprising the common deletion in mitochondrial DNA 21 days following electroporation with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each linked to an MTP at their N-terminus, or GFP mRNA as a control. Figure 18A shows changes in the normalized oxygen consumption rate (OCR). Figure 18B shows changes in the extracellular acidification rate (ECAR). Figure 19 shows basal and maximal cellular respiration rates of cybrids cells comprising the common deletion in mitochondrial DNA 21 days following electroporation with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each linked to an MTP at their N-terminus, or GFP mRNA as a control. BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 sets forth the amino acid sequence of the I-CreI meganuclease found in Chlamydomonas reinhardtii. SEQ ID NO: 2 sets forth the amino acid sequence of a LAGLIDADG motif. SEQ ID NO: 3 sets forth the nucleic acid sequence of the MIT 9-10 recognition sequence (sense). SEQ ID NO: 4 sets forth the nucleic acid sequence of the MIT 9-10 recognition sequence reverse complement. SEQ ID NO: 5 sets forth the nucleic acid sequence of nucleotides 13,445-13,466 of wild- type human mitochondrial DNA (sense). SEQ ID NO: 6 sets forth the nucleic acid sequence of the reverse complement of nucleotides 13,445-13,466 of wild-type human mitochondrial DNA. SEQ ID NO: 7 sets forth the nucleic acid sequence of the MIT 9-10x.3 engineered meganuclease. SEQ ID NO: 8 sets forth the amino acid sequence of the MIT 9-10.L90 engineered meganuclease. Attorney Docket No.: P893391190WO (01242) SEQ ID NO: 9 sets forth the amino acid sequence of the MIT 9-10.L209 engineered meganuclease. SEQ ID NO: 10 sets forth the amino acid sequence of the MIT 9-10.L210 engineered meganuclease. SEQ ID NO: 11 sets forth the amino acid sequence of the MIT 9-10x.3 MIT9-binding subunit. SEQ ID NO: 12 sets forth the amino acid sequence of the MIT 9-10.L90 MIT9- binding subunit. SEQ ID NO: 13 sets forth the amino acid sequence of the MIT 9-10.L209 MIT9- binding subunit. SEQ ID NO: 14 sets forth the amino acid sequence of the MIT 9-10.L210 MIT9- binding subunit. SEQ ID NO: 15 sets forth the amino acid sequence of the MIT 9-10x.3 MIT10- binding subunit. SEQ ID NO: 16 sets forth the amino acid sequence of the MIT 9-10.L90 MIT10- binding subunit. SEQ ID NO: 17 sets forth the amino acid sequence of the MIT 9-10.L209 MIT10- binding subunit. SEQ ID NO: 18 sets forth the amino acid sequence of the MIT 9-10.L210 MIT10- binding subunit. SEQ ID NO: 19 sets forth a nucleic acid sequence encoding the MIT 9-10x.3 engineered meganuclease. SEQ ID NO: 20 sets forth a nucleic acid sequence encoding the MIT 9-10.L90 engineered meganuclease. SEQ ID NO: 21 sets forth a nucleic acid sequence encoding the MIT 9-10.L209 engineered meganuclease. SEQ ID NO: 22 sets forth a nucleic acid sequence encoding the MIT 9-10.L210 engineered meganuclease. SEQ ID NO: 23 sets forth the amino acid sequence of the COX VIII MTP . SEQ ID NO: 24 sets forth the amino acid sequence of the SU9 MTP. SEQ ID NO: 25 sets forth the amino acid sequence of the COX VIII-SU9 MTP. SEQ ID NO: 26 sets forth the amino acid sequence of the MVMp NS2 NES sequence. SEQ ID NO: 27 sets forth the amino acid sequence of the NES sequence. SEQ ID NO: 28 sets forth the nucleic acid sequence of the PCR primer used to determine WT or mutant mtDNA frequency. Attorney Docket No.: P893391190WO (01242) SEQ ID NO: 29 sets forth the nucleic acid sequence of the PCR primer used to determine WT or mutant mtDNA frequency. SEQ ID NO: 30 sets forth the nucleic acid sequence of the PCR primer used to determine WT or mutant mtDNA frequency. SEQ ID NO: 31 sets forth the nucleic acid sequence of a ddPCR primer F1. SEQ ID NO: 32 sets forth the nucleic acid sequence of a ddPCR primer R1. SEQ ID NO: 33 sets forth the nucleic acid sequence of a ddPCR primer P1. SEQ ID NO: 34 sets forth the nucleic acid sequence of a ddPCR primer P2. SEQ ID NO: 35 sets forth the nucleic acid sequence of a ddPCR primer P3. SEQ ID NO: 36 sets forth the nucleic acid sequence of a ddPCR primer P3. SEQ ID NO: 37 sets forth the nucleic acid sequence of a ddPCR primer P3. SEQ ID NO: 38 sets forth the nucleic acid sequence of a ddPCR primer hND4-TAMRA. SEQ ID NO: 39 sets forth the nucleic acid sequence of a ddPCR primer PrF. SEQ ID NO: 40 sets forth the nucleic acid sequence of a ddPCR primer PrB. SEQ ID NO: 41 sets forth the nucleic acid sequence of a ddPCR primer hCOM deletion- FAM. SEQ ID NO: 42 sets forth the nucleic acid sequence of a ddPCR primer PrF. SEQ ID NO: 43 sets forth the nucleic acid sequence of a ddPCR primer PrB. SEQ ID NO: 44 sets forth the nucleic acid sequence of a ddPCR primer hND1-HEX. SEQ ID NO: 45 sets forth the nucleic acid sequence of a ddPCR primer PrF. SEQ ID NO: 46 sets forth the nucleic acid sequence of a ddPCR primer PrB. SEQ ID NO: 47 sets forth the nucleic acid sequence of a ddPCR primer mus18s-Cy5. SEQ ID NO: 48 sets forth the nucleic acid sequence of a ddPCR primer PrF. SEQ ID NO: 49 sets forth the nucleic acid sequence of a ddPCR primer PrB. DETAILED DESCRIPTION OF THE INVENTION 1.1 References and Definitions The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. The present invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this Attorney Docket No.: P893391190WO (01242) disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety. As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells. As used herein, the term "5’ cap" (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the “front” or 5’ end of a eukaryotic messenger RNA shortly after the start of transcription. The 5’ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5’ end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation. As used herein, the term “allele” refers to one of two or more variant forms of a gene. As used herein, the term “allogeneic” or, alternatively, “allogenic,” refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically. As used herein, the term “constitutive promoter" refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. Attorney Docket No.: P893391190WO (01242) As used herein, the term “a control” or “a control cell” refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a genetically-modified cell. A control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the genetically-modified cell; (b) a cell of the same genotype as the genetically-modified cell but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest); or, (c) a cell genetically identical to the genetically-modified cell but which is not exposed to conditions or stimuli or further genetic modifications that would induce expression of altered genotype or phenotype. For example, a control or control cell of the instant invention can be a cell or population of cells that does not comprise an engineered meganuclease or a polynucleotide having an amino acid sequence encoding an engineered meganuclease. As used herein, the term “corresponding to” with respect to modifications of two proteins or amino acid sequences is used to indicate that a specified modification in the first protein is a substitution of the same amino acid residue as in the modification in the second protein, and that the amino acid position of the modification in the first protein corresponds to or aligns with the amino acid position of the modification in the second protein when the two proteins are subjected to standard sequence alignments (e.g., using the BLASTp program) and aligned for maximum sequence identity across the entire subunit or protein. Thus, the modification of residue “X” to amino acid “A” in the first protein will correspond to the modification of residue “Y” to amino acid “A” in the second protein if residues X and Y correspond to each other in a sequence alignment and despite the fact that X and Y may be at different position relative to the N-terminus or C-terminus. As used herein, the term “disrupted” or “disrupts” or “disrupts expression” or “disrupting a target sequence” refers to the introduction of a mutation (e.g., frameshift mutation) that interferes with the gene function and prevents expression and/or function of the polypeptide/expression product encoded thereby. For example, nuclease-mediated disruption of a gene can result in the expression of a truncated protein and/or expression of a protein that does not retain its wild-type function. Additionally, introduction of a donor template into a gene can result in no expression of an encoded protein, expression of a truncated protein, and/or expression of a protein that does not retain its wild-type function. As used herein, the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or Attorney Docket No.: P893391190WO (01242) other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. As used herein, the term “endogenous” in reference to a nucleotide sequence or protein is intended to mean a sequence or protein that is naturally comprised within or expressed by a cell. As used herein, the terms “exogenous” or “heterologous” in reference to a nucleotide sequence or amino acid sequence are intended to mean a sequence that is purely synthetic, that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. As used herein, the term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter. As used herein, the term "expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. As used herein, the term “genetically-modified” refers to a cell or organism in which, or in an ancestor of which, a genomic DNA sequence has been deliberately modified by recombinant technology. As used herein, the term “genetically-modified” encompasses the term “transgenic.” For example, as used herein, a “genetically-modified” cell may refer to a cell wherein the mitochondrial DNA has been deliberately modified by recombinant technology. As used herein, the term “homologous recombination” or “HR” refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g., Cahill et al. (2006), Front. Biosci.11:1958-1976). The homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell. As used herein, the term “homology arms” or “sequences homologous to sequences flanking a nuclease cleavage site” refer to sequences flanking the 5' and 3' ends of a nucleic acid molecule, which promote insertion of the nucleic acid molecule into a cleavage site generated by a nuclease. In general, homology arms can have a length of at least 50 base pairs, preferably at least 100 base pairs, and up to 2000 base pairs or more, and can have at least 90%, preferably at least Attorney Docket No.: P893391190WO (01242) 95%, or more, sequence homology to their corresponding sequences in the genome. In some embodiments, the homology arms are about 500 base pairs. As used herein, the term “in vitro transcribed RNA” refers to RNA, preferably mRNA, which has been synthesized in vitro. Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA. As used herein, the term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. As used herein, the term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. As used herein, the term “lipid nanoparticle” refers to a lipid composition having a typically spherical structure with an average diameter between 10 and 1000 nanometers. In some formulations, lipid nanoparticles can comprise at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid. Lipid nanoparticles known in the art that are suitable for encapsulating nucleic acids, such as mRNA, are contemplated for use in the invention. As used herein, the term “modification” with respect to recombinant proteins means any insertion, deletion, or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., a wild-type or a native sequence). As used herein, the term “non-homologous end-joining” or “NHEJ” refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non-homologous DNA segments (see, e.g. Cahill et al. (2006), Front. Biosci.11:1958-1976). DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair. In some instances, cleavage at a target recognition sequence results in NHEJ at a target recognition site. Nuclease-induced cleavage of a target site in the coding sequence of a gene followed by DNA repair by NHEJ can introduce mutations into the coding sequence, such as frameshift mutations, that disrupt gene function. Thus, engineered nucleases can be used to effectively knock-out a gene in a population of cells. As used herein, the term “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino Attorney Docket No.: P893391190WO (01242) acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns. As used herein, the term “operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a nucleic acid sequence encoding a nuclease as disclosed herein and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the nucleic acid sequence encoding the nuclease. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and/or” and not the exclusive sense of “either/or. As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof. As used herein, the term “reduced” or “decreased” refers to a reduction in the percentage of cells or ratio of cells in a population of cells that comprise mutant mitochondrial genomes having the mtDNA common deletion when compared to a population of control cells. In some embodiments, “reduced” or “decreased” refers to a reduction in the percentage of mutant mitochondrial genomes or ratio of mutant mitochondrial genomes to wild-type mitochondrial genomes in a single cell or in a population of cells. Such a reduction is up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100%. Accordingly, the term “reduced” encompasses both a partial reduction and a complete reduction of mutant mtDNA. As used herein, the term with respect to both amino acid sequences and nucleic acid sequences, the terms “percent identity,” “sequence identity,” “percentage similarity,” “sequence similarity” and the like refer to a measure of the degree of similarity of two sequences based upon an alignment of the sequences that maximizes similarity between aligned amino acid residues or Attorney Docket No.: P893391190WO (01242) nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the number of total residues or nucleotides, and the presence and length of gaps in the sequence alignment. A variety of algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/), and are described in, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet.3:266-272; Madden et al. (1996), Meth. Enzymol.266:131-141; Altschul et al. (1997), Nucleic Acids Res.25:3389-3402); Zhang et al. (2000), J. Comput. Biol.7(1-2):203-14. As used herein, percent similarity of two amino acid sequences is the score based upon the following parameters for the BLASTp algorithm: word size=3; gap opening penalty=−11; gap extension penalty=−1; and scoring matrix=BLOSUM62. As used herein, percent similarity of two nucleic acid sequences is the score based upon the following parameters for the BLASTn algorithm: word size=11; gap opening penalty=−5; gap extension penalty=−2; match reward=1; and mismatch penalty=−3. As used herein, the term “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation. As used herein, the term “polyadenylation” refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3’ end. The 3’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3’ end at the cleavage site. Attorney Docket No.: P893391190WO (01242) As used herein, the term “promoter” or “regulatory sequence” refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. As used herein, the terms “recombinant” or “engineered,” with respect to a protein, means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein and cells or organisms that express the protein. With respect to a nucleic acid, the term “recombinant” or “engineered” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation, and other gene transfer technologies; homologous recombination; site- directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant or engineered. As used herein, the term “recombinant DNA construct,” “recombinant construct,” “expression cassette,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA fragment” are used interchangeably herein and are single or double-stranded polynucleotides. A recombinant construct comprises an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that are not found together in nature. For example, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. As used herein, the term “tissue-specific promoter" or “cell-specific promoter” refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter. As used herein, the terms “transfected” or “transformed” or “transduced” or “nucleofected” refer to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny. Attorney Docket No.: P893391190WO (01242) As used herein, the term “transfer vector” refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like. As used herein, the term “transient” refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell. As used herein, the term “vector” or “recombinant DNA vector” may be a construct that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. Vectors can include, without limitation, plasmid vectors and recombinant AAV vectors, or any other vector known in the art suitable for delivering a gene to a target cell. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleotides or nucleic acid sequences of the invention. In some embodiments, a “vector” also refers to a viral vector. Viral vectors can include, without limitation, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (AAV). As used herein, the term “wild-type” refers to the most common naturally occurring allele (i.e., polynucleotide sequence) in the allele population of the same type of gene, wherein a polypeptide encoded by the wild-type allele has its original functions. The term “wild-type” also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (i.e., polynucleotides) and polypeptides are distinguishable from mutant or variant alleles and polypeptides, which comprise one or more mutations and/or substitutions relative to the wild-type sequence(s). Whereas a wild- type allele or polypeptide can confer a normal phenotype in an organism, a mutant or variant allele or polypeptide can, in some instances, confer an altered phenotype. Wild-type nucleases are distinguishable from recombinant or non-naturally-occurring nucleases. The term “wild-type” can also refer to a cell, an organism, and/or a subject which possesses a wild-type allele of a particular gene, or a cell, an organism, and/or a subject used for comparative purposes. Attorney Docket No.: P893391190WO (01242) As used herein, the term “altered specificity,” when referencing to a nuclease, means that a nuclease binds to and cleaves a recognition sequence, which is not bound to and cleaved by a reference nuclease (e.g., a wild-type) under physiological conditions, or that the rate of cleavage of a recognition sequence is increased or decreased by a biologically significant amount (e.g., at least 2×, or 2×-10×) relative to a reference nuclease. As used herein, the term “center sequence” refers to the four base pairs separating half-sites in the meganuclease recognition sequence. These bases are numbered +1 through +4. The center sequence comprises the four bases that become the 3' single-strand overhangs following meganuclease cleavage. “Center sequence” can refer to the sequence of the sense strand or the antisense (opposite) strand. Meganucleases are symmetric and recognize bases equally on both the sense and antisense strand of the center sequence. For example, the sequence A+1A+2A+3A+4 on the sense strand is recognized by a meganuclease as T+1T+2T+3T+4 on the antisense strand and, thus, A+1A+2A+3A+4 and T+1T+2T+3T+4 are functionally equivalent (e.g., both can be cleaved by a given meganuclease). Thus, the sequence C+1T+2G+3C+4, is equivalent to its opposite strand sequence, G+1C+2A+3G+4 due to the fact that the meganuclease binds its recognition sequence as a symmetric homodimer. As used herein, the terms “cleave” or “cleavage” refer to the hydrolysis of phosphodiester bonds within the backbone of a recognition sequence within a target sequence that results in a double-stranded break within the target sequence, referred to herein as a “cleavage site”. As used herein, the terms “DNA-binding affinity” or “binding affinity” means the tendency of a nuclease to non-covalently associate with a reference DNA molecule (e.g., a recognition sequence or an arbitrary sequence). Binding affinity is measured by a dissociation constant, Kd. As used herein, a nuclease has “altered” binding affinity if the Kd of the nuclease for a reference recognition sequence is increased or decreased by a statistically significant percent change relative to a reference nuclease. As used herein, the term “hypervariable region” refers to a localized sequence within a meganuclease monomer or subunit that comprises amino acids with relatively high variability. A hypervariable region can comprise about 50-60 contiguous residues, about 53-57 contiguous residues, or preferably about 56 residues. In some embodiments, the residues of a hypervariable region may correspond to positions 24-79 or positions 215-270 of any one of SEQ ID NOs: 7-10. A hypervariable region can comprise one or more residues that contact DNA bases in a recognition sequence and can be modified to alter base preference of the monomer or subunit. A hypervariable region can also comprise one or more residues that bind to the DNA backbone when the meganuclease associates with a double-stranded DNA recognition sequence. Such residues can be modified to alter the binding affinity of the meganuclease for the DNA backbone and the target Attorney Docket No.: P893391190WO (01242) recognition sequence. In different embodiments of the invention, a hypervariable region may comprise between 1-20 residues that exhibit variability and can be modified to influence base preference and/or DNA-binding affinity. In particular embodiments, a hypervariable region comprises between about 15-20 residues that exhibit variability and can be modified to influence base preference and/or DNA-binding affinity. In some embodiments, variable residues within a hypervariable region correspond to one or more of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-10. In other embodiments, variable residues within a hypervariable region correspond to one or more of positions 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-10. As used herein, the term “linker” refers to an exogenous peptide sequence used to join two nuclease subunits into a single polypeptide. A linker may have a sequence that is found in natural proteins or may be an artificial sequence that is not found in any natural protein. A linker may be flexible and lacking in secondary structure or may have a propensity to form a specific three- dimensional structure under physiological conditions. A linker can include, without limitation, those encompassed by U.S. Patent Nos.8,445,251, 9,340,777, 9,434,931, and 10,041,053, each of which is incorporated by reference in its entirety. In some embodiments, a linker may have an amino acid sequence that sets forth residues 154-195 of any one of SEQ ID NOs: 7-10. As used herein, the term “meganuclease” refers to an endonuclease that binds double- stranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments, the recognition sequence for a meganuclease of the present disclosure is 22 base pairs. A meganuclease can be an endonuclease that is derived from I-CreI (SEQ ID NO: 1) and can refer to an engineered variant of I-CreI that has been modified relative to natural I-CreI with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties. Methods for producing such modified variants of I-CreI are known in the art (e.g., WO 2007/047859, incorporated by reference in its entirety). A meganuclease as used herein binds to double-stranded DNA as a heterodimer. A meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains is joined into a single polypeptide using a peptide linker. The term “homing endonuclease” is synonymous with the term “meganuclease.” Meganucleases of the present disclosure are substantially non-toxic when expressed in the targeted cells as described herein such that cells can be transfected and maintained at 37°C without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein. As used herein, the term “mitochondria-targeting engineered meganuclease” refers to an engineered meganuclease attached to a peptide, such as a mitochondrial transit peptide, or other molecule that is capable of directing the engineered meganuclease to the mitochondria such that the Attorney Docket No.: P893391190WO (01242) engineered meganuclease is capable of binding and cleaving mitochondrial DNA within the mitochondrial organelle. As used herein the term “mitochondrial transit peptide” or “MTP” refers to a peptide or fragment of amino acids that can be attached to a separate molecule in order to transport the molecule in the mitochondria. For example, an MTP can be attached to a nuclease, such as an engineered meganuclease, in order to transport the engineered meganuclease into the mitochondria. MTPs can consist of an alternating pattern of hydrophobic and positively charged amino acids to form what is called amphipathic helix. As used herein, the terms “nuclease” and “endonuclease” are used interchangeably to refer to naturally-occurring or engineered enzymes, which cleave a phosphodiester bond within a polynucleotide chain. As used herein, the term “recognition half-site,” “recognition sequence half-site,” or simply “half-site” means a nucleic acid sequence in a double-stranded DNA molecule that is recognized and bound by a monomer of a homodimeric or heterodimeric meganuclease or by one subunit of a single-chain meganuclease. As used herein, the terms “recognition sequence” or “recognition site” refers to a DNA sequence that is bound and cleaved by a nuclease. In the case of a meganuclease, a recognition sequence comprises a pair of inverted, 9 basepair “half sites” which are separated by four basepairs. In the case of a single-chain meganuclease, the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site. Cleavage by a meganuclease produces four basepair 3’ overhangs. “Overhangs,” or “sticky ends” are short, single-stranded DNA segments that can be produced by endonuclease cleavage of a double- stranded DNA sequence. In the case of meganucleases and single-chain meganucleases derived from I-CreI, the overhang comprises bases 10-13 of the 22 basepair recognition sequence. As used herein, the term “single-chain meganuclease” refers to a polypeptide comprising a pair of nuclease subunits joined by a linker. A single-chain meganuclease has the organization: N-terminal subunit – Linker – C-terminal subunit. The two meganuclease subunits will generally be non-identical in amino acid sequence and will bind non-identical DNA sequences. Thus, single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. A single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric. For clarity, unless otherwise specified, the term “meganuclease” can refer to a dimeric or single-chain meganuclease. As used herein, the term “specificity” means the ability of a nuclease to bind and cleave double-stranded DNA molecules only at a particular sequence of base pairs referred to as the recognition sequence, or only at a particular set of recognition sequences. The set of recognition Attorney Docket No.: P893391190WO (01242) sequences will share certain conserved positions or sequence motifs but may be degenerate at one or more positions. A highly-specific nuclease is capable of cleaving only one or a very few recognition sequences. Specificity can be determined by any method known in the art. As used herein, the terms “target site” or “target sequence” refers to a region of the chromosomal DNA of a cell comprising a recognition sequence for a nuclease. As used herein, a “vector” can also refer to a viral vector (i.e. a recombinant virus). Viral vectors can include, without limitation, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (AAV). As used herein, the term “serotype” or “capsid” refers to a distinct variant within a species of virus, such as recombinant adeno-associated viruses, that is determined based on the viral cell surface antigens. As used herein, a “control” or “control cell” refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a genetically-modified cell. A control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the genetically-modified cell; (b) a cell of the same genotype as the genetically-modified cell but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest); or, (c) a cell genetically identical to the genetically-modified cell but which is not exposed to conditions, stimuli, or further genetic modifications that would induce expression of altered genotype or phenotype. As used herein, the term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated. In some specific embodiments, an effective amount of the engineered meganuclease comprises about 1x1010 gc/kg to about 1x1014 gc/kg (e.g., 1x1010 gc/kg, 1x1011 gc/kg, 1x1012 gc/kg, 1x1013 gc/kg, or 1x1014 gc/kg) of a nucleic acid encoding the engineered meganuclease. In specific embodiments, an effective amount of a nucleic acid encoding an engineered meganuclease, or a pharmaceutical composition comprising a nucleic acid encoding an engineered meganuclease disclosed herein, reduces at least one symptom of a disease in a subject. As used herein, the term “effective dose”, “effective amount”, “therapeutically effective dose”, or “therapeutically effective amount,” as used herein, refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results. As used herein, the term “gc/kg” or “gene copies/kilogram” refers to the number of copies of a nucleic acid encoding an engineered meganuclease or the number of copies of a template Attorney Docket No.: P893391190WO (01242) nucleic acid described herein per weight in kilograms of a subject that is administered the nucleic acid encoding the engineered meganuclease. As used herein, the term “preventing” refers to the prevention of the disease or condition in the patient. As used herein, the term “prophylaxis” means the prevention of or protective treatment for a disease or disease state. As used herein, the term “reduced” refers to any reduction in the symptoms or severity of a disease. Such a reduction may be up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100%. Accordingly, the term “reduced” encompasses both a partial reduction and a complete reduction of a disease state. As used herein, the term “muscle stem cell” refers to a progenitor cell capable of developing into a muscle cell. Muscle stem cells can include, for example, muscle satellite cells. As used herein, the recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values ≧0 and ≦2 if the variable is inherently continuous. 2.1 Principle of the Invention Mitochondria regulate cellular energy and metabolism under normal growth and development, as well as in response to stress. Thus, editing of the mitochondrial genome has diverse applications in both animals and plants. In humans, deleterious mitochondrial mutations are the source of a number of disorders for which gene editing therapies could be applied. However, even in view of the potential of using mitochondrial genome editing for therapeutic applications, it still remains an underexplored area of science because of the inability to efficiently target mitochondrial DNA (mtDNA) and generate precise edits. The mitochondrial genome is difficult to edit as the editing technology needs to be delivered to this organelle. Moreover, the mitochondria lack predictable repair mechanisms. Previous attempts at editing the mitochondrial genome have resulted in large and unpredictable deletions/rearrangements. Hence, compositions and methods that would allow targeting and editing defined regions (preferably limited to just one gene) of the mitochondrial genome in a more predictable manner are desired. Attorney Docket No.: P893391190WO (01242) The present disclosure provides compositions and methods for binding and cleaving a recognition sequence on the mitochondrial genome without impacting the surrounding regions in the mitochondrial genome. Disclosed herein are engineered meganucleases, such as homing endonucleases, attached to MTPs such that DSBs can be generated in the mtDNA. The present invention demonstrates that engineered meganucleases can be directed into the mitochondria organelle and facilitate precise editing of mtDNA, thus opening up an entire field of prospects and opportunities in life sciences. 2.2 Engineered Meganucleases for Binding and Cleaving Recognition Sequences within the Human Mitochondrial DNA Engineered meganucleases described herein can be attached to a mitochondrial transit peptide (MTP) to generate a mitochondria-targeted engineered meganuclease that can effectively traffic from the cytoplasm of a eukaryotic cell into the mitochondria. Once inside the mitochondria organelle, the engineered meganuclease can bind and cleave a recognition sequence in the mitochondrial genome. It is known in the art that it is possible to use a site-specific nuclease to make a DNA break in the genome of a living cell, and that such a DNA break can result in permanent modification of the genome via mutagenic NHEJ repair or via homologous recombination with a transgenic DNA sequence. NHEJ can produce mutagenesis at the cleavage site, resulting in inactivation of the allele. NHEJ-associated mutagenesis may inactivate an allele via generation of early stop codons, frameshift mutations producing aberrant non-functional proteins, or could trigger mechanisms such as nonsense-mediated mRNA decay. The use of nucleases to induce mutagenesis via NHEJ can be used to target a specific mutation or a sequence present in a wild-type allele. Further, the use of nucleases to induce a double-strand break in a target locus is known to stimulate homologous recombination, particularly of transgenic DNA sequences flanked by sequences that are homologous to the genomic target. In this manner, exogenous nucleic acid sequences can be inserted into a target locus. Such exogenous nucleic acids can encode any sequence or polypeptide of interest. In some embodiments, a site-specific nuclease can cleave a recognition sequence in the mitochondrial genome that results in degradation of the mitochondrial genome from the cleaved ends created by the site-specific nuclease. The nucleases used to practice the invention are meganucleases. In particular embodiments, the meganucleases used to practice the invention are single-chain meganucleases. A single-chain meganuclease comprises an N-terminal subunit and a C-terminal subunit joined by a linker peptide. Each of the two domains recognizes and binds to half of the recognition sequence (i.e., a recognition half-site) and the site of DNA cleavage is at the middle of the recognition sequence Attorney Docket No.: P893391190WO (01242) near the interface of the two subunits. DNA strand breaks are offset by four base pairs such that DNA cleavage by a meganuclease generates a pair of four base pair, 3' single-strand overhangs. In some embodiments, an engineered meganuclease of the invention has been engineered to bind and cleave an MIT 9-10 recognition sequence (SEQ ID NO: 3). Such an engineered meganuclease is referred to herein as “MIT 9-10 meganuclease” or “MIT 9-10 nuclease.” In specific embodiments, the MIT 9-10 meganuclease is attached to an MTP to form a mitochondria- targeting engineered meganuclease that cleaves the MIT 9-10 recognition sequence of SEQ ID NO: 3. MIT 9-10 meganucleases comprising an MTP can further comprise an NES to further decrease localization to the nucleus and thus reduce any potential cleavage in the nuclear genome. Engineered meganucleases of the invention can comprise a first subunit, comprising a first hypervariable (HVR1) region, and a second subunit, comprising a second hypervariable (HVR2) region. Further, the first subunit can bind to a first recognition half-site in the recognition sequence (e.g., the MIT9 half-site), and the second subunit can bind to a second recognition half-site in the recognition sequence (e.g., the MIT10 half-site). In embodiments where the engineered meganuclease is a single-chain meganuclease, the first and second subunits can be oriented such that the first subunit, which comprises the HVR1 region and binds the first half-site, is positioned as the N-terminal subunit, and the second subunit, which comprises the HVR2 region and binds the second half-site, is positioned as the C-terminal subunit. In alternative embodiments, the first and second subunits can be oriented such that the first subunit, which comprises the HVR1 region and binds the first half-site, is positioned as the C-terminal subunit, and the second subunit, which comprises the HVR2 region and binds the second half-site, is positioned as the N-terminal subunit. Exemplary MIT 9-10 meganucleases described herein are provided in Tables 1 and 2 and are further described below.
Attorney Docket No.: P893391190WO (01242) Table 1. AA MIT9 MIT9 MIT10 MIT10 HVR1 *HVR1 HVR2 *HVR2 Meganuclease SEQ Subunit Subunit Subunit Subunit Residues % Residues % ID Residues SEQ ID Residues SEQ ID MIT9-10x.3 7 198-344 11 215-270 100 7-153 15 24-79 100 MIT9-10.L90 8 198-344 12 215-270 100 7-153 16 24-79 89.29 MIT9- 10.L209 9 198-344 13 215-270 100 7-153 17 24-79 89.29 MIT9- 10.L210 10 198-344 14 215-270 100 7-153 18 24-79 89.29 *“HVR1%” and “HVR2%” represent the amino acid sequence identity between the HVR1 and HVR2 regions of each meganuclease and the HVR1 and HVR2 regions, respectively, of the MIT9- 10x.3 meganuclease. Table 2. AA MIT9 MIT9 *MIT9 MIT10 MIT10 *MIT10 Meganuclease SEQ Subunit Subunit Subunit Subunit Subunit Subunit ID Residues SEQ ID % Residues SEQ ID % MIT9-10x.3 7 198-344 11 100 7-153 15 100 MIT9-10.L90 8 198-344 12 100 7-153 16 95.92 MIT9-10.L209 9 198-344 13 99.32 7-153 17 95.24 MIT9-10.L210 10 198-344 14 100 7-153 18 95.92 *“MIT9 Subunit %” and “MIT10 Subunit %” represent the amino acid sequence identity between the MIT9-binding and MIT10-binding subunit regions of each meganuclease and the MIT9-binding and MIT10-binding subunit regions, respectively, of the MIT9-10x.3 meganuclease. In certain embodiments of the invention, the engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 3 within mitochondrial genome, wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region. MIT 9-10x.3 (SEQ ID NO: 7) In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 215, Attorney Docket No.: P893391190WO (01242) 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises residues 215-270 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 215-270 of SEQ ID NO: 7. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 7. In some embodiments, the first subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 7. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 7. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 7. In some embodiments, the first subunit comprises residues 198-344 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 198-344 of SEQ ID NO: 7. In some embodiments, the first subunit comprises residues 196-354 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 7. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of SEQ ID NO: 7. In Attorney Docket No.: P893391190WO (01242) some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 7. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 7. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 7. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 6-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 5-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 4-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 3-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 2-153 of SEQ ID NO: 7. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 1-153 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit. In some embodiments, the N-terminus of the linker is fused to the residue (i.e., a D residue) corresponding to residue 153 of SEQ ID NO: 7, and the C-terminus of the linker is fused to the residue (i.e., a Y residue) corresponding to residue 196 of SEQ ID NO: 7. In some embodiments, the linker comprises residues 154-195 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of SEQ ID NO: 7. In some embodiments, the engineered Attorney Docket No.: P893391190WO (01242) meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 4-343 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 3-343 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 2-343 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of SEQ ID NO: 19. MIT 9-10L.90 (SEQ ID NO: 8) In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises residues 215-270 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 215-270 of SEQ ID NO: 8. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 8. In some embodiments, the first subunit comprises a residue Attorney Docket No.: P893391190WO (01242) corresponding to residue 271 of SEQ ID NO: 8. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 8. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 8. In some embodiments, the first subunit comprises residues 198-344 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 198-344 of SEQ ID NO: 8. In some embodiments, the first subunit comprises residues 196-354 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, Attorney Docket No.: P893391190WO (01242) 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 8. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 8. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 6-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 5-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 4-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 3-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 2-153 of SEQ ID NO: 8. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 1-153 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit. In some embodiments, the N-terminus of the linker is fused to the residue (i.e., a D residue) corresponding to residue 153 of SEQ ID NO: 8, and the C-terminus of the linker is fused to the residue (i.e., a Y residue) corresponding to residue 196 of SEQ ID NO: 8. In some embodiments, the linker comprises residues 154-195 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 4-343 of SEQ ID NO: 8. In some embodiments, the engineered Attorney Docket No.: P893391190WO (01242) meganuclease comprises an amino acid sequence of residues 3-343 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 2-343 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of SEQ ID NO: 20. MIT 9-10L.209 (SEQ ID NO: 9) In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises residues 215-270 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 215-270 of SEQ ID NO: 9. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 9. In some embodiments, the first subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 9. In some embodiments, the first subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 9. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 9. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 9. In some embodiments, the first subunit comprises residues 198-344 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 198-344 of SEQ ID NO: 9. In some embodiments, the first subunit comprises residues 196-354 of SEQ ID NO: 9. Attorney Docket No.: P893391190WO (01242) In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 9. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 9. In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 9. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 9. In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 9. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 9. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 9. In some embodiments, the second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 9. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 9. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 9. In some embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino Attorney Docket No.: P893391190WO (01242) acid substitutions. In some embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises residues 6-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises residues 5-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises residues 4-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises residues 3-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises residues 2-153 of SEQ ID NO: 9. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 9. In some embodiments, the second subunit comprises residues 1-153 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit. In some embodiments, the N-terminus of the linker is fused to the residue (i.e., a D residue) corresponding to residue 153 of SEQ ID NO: 9, and the C-terminus of the linker is fused to the residue (i.e., a Y residue) corresponding to residue 196 of SEQ ID NO: 9. In some embodiments, the linker comprises residues 154-195 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 4-343 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 3-343 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 2-343 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more Attorney Docket No.: P893391190WO (01242) sequence identity to a nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of SEQ ID NO: 21. MIT 9-10L.210 (SEQ ID NO: 10) In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises residues 215-270 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 215-270 of SEQ ID NO: 10. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 10. In some embodiments, the first subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 10. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 10. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 10. In some embodiments, the first subunit comprises residues 198-344 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 198-344 of SEQ ID NO: 10. In some embodiments, the first subunit comprises residues 196-354 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or Attorney Docket No.: P893391190WO (01242) 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 24-79 of SEQ ID NO: 10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 10. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 10. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 7-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 6-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 5-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 4-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 3-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 2-153 of SEQ ID NO: 10. In some embodiments, the second subunit comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 1-153 of SEQ ID NO: 10. Attorney Docket No.: P893391190WO (01242) In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit. In some embodiments, the N-terminus of the linker is fused to the residue (i.e., a D residue) corresponding to residue 153 of SEQ ID NO: 10, and the C-terminus of the linker is fused to the residue (i.e., a Y residue) corresponding to residue 196 of SEQ ID NO: 10. In some embodiments, the linker comprises residues 154-195 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 4-343 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 3-343 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of residues 2-343 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises a residue other than M at a position corresponding to position 1 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of SEQ ID NO: 22. MTPs for directing the engineered meganuclease into the mitochondria can be from 10-100 amino acids in length. In specific embodiments, the MTP is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, or more amino acids long. MTPs can contain additional signals that subsequently target Attorney Docket No.: P893391190WO (01242) the protein to different regions of the mitochondria, such as the mitochondrial matrix. Non limiting examples of MTPs for use in the compositions and methods disclose herein include, Neurospora crassa F0 ATPase subunit 9 (SU9) MTP, human cytochrome c oxidase subunit VIII (CoxVIII or Cox8) MTP, the P1 isoform of subunit c of human ATP synthase MTP, aldehyde dehydrogenase targeting sequence MTP, Glutaredoxin 5 MTP, Pyruvate dehydrogenase MTP, Peptidyl-prolyl isomerase MTP, Acetyltransferase MTP, Isocitrate dehydrogenase MTP, cytochrome oxidase MTP, and the subunits of the FA portion of ATP synthase MTP, CPN60/No GGlinker MTP, Superoxide dismutase (SOD) MTP, Superoxide dismutase doubled(2SOD) MTP, Superoxide dismutase modified(SODmod) MTP, Superoxide dismutase modified (2SODmod) doubled MTP, L29 MTP, gATPase gamma subunit (FAγ51) MTP, CoxIV twin strep (ABM97483) MTP, and CoxIV 10xHis MTP. In specific embodiments, the MTP comprises a combination of at least two MTPs. The combination of MTPs can be a combination of identical MTPs or a combination of different MTPs. In specific embodiments, the MTP comprises the Cox VIII MTP (SEQ ID NO: 23) and the SU9 MTP (SEQ ID NO: 24) combined into a single MTP represented by SEQ ID NO: 25. In order to form a mitochondria-targeted engineered meganuclease, an MTP can be attached by any appropriate means to an engineered meganuclease disclosed herein. In specific embodiments, the MTP can be attached to the N-terminus of the engineered meganuclease. In other embodiments the MTP can be attached to the C-terminus of the engineered meganuclease. In some embodiments multiple MTPs can be attached to a single engineered meganuclease. For example, a first MTP can be attached to the N-terminus of the engineered meganuclease and a second MTP can be attached to the C-terminus of the engineered meganuclease. In some embodiments, the first and second MTP are identical and in other embodiments, the first and second MTP not identical. The MTP(s) can be attached by any means that allows for transport of the engineered meganuclease into the mitochondria of a cell. In specific embodiments, the MTP is attached by fusing the MTP to the N- or C-terminus of the engineered meganuclease. The MTP can also be attached to the engineered meganuclease by a peptide linker. The linker can be, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20 amino acids. In specific embodiments the MTP is attached to a peptide linker at the N- or C-terminus of the engineered meganuclease. In some embodiments, a mitochondria-targeted engineered meganuclease described herein is attached to a nuclear export sequence (NES) in order to help prevent the engineered meganuclease from cleaving the nuclear genome. In some such embodiments, the NES comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 26 or 27. For example, the NES may Attorney Docket No.: P893391190WO (01242) comprise the amino acid sequence of SEQ ID NO: 26 or 27. In certain embodiments, the NES is attached at the N-terminus of the engineered meganuclease. In other embodiments, the NES is attached at the C-terminus of the engineered meganuclease. In certain embodiments, the NES is fused to the engineered meganuclease. In certain embodiments, the NES is attached to the engineered meganuclease by a polypeptide linker. In specific embodiments, more than one NES is attached to the engineered meganuclease. For example, an engineered meganuclease disclosed herein can comprise a first NES and a second NES. In some such embodiments, the first NES is attached at the N-terminus of the engineered meganuclease, and the second NES is attached at the C-terminus of the engineered meganuclease. In some such embodiments, the first NES and/or the second NES comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26 or 27. For example, the first NES and/or the second NES may comprise the amino acid sequence set forth in SEQ ID NO: 26 or 27. In some embodiments, the first NES and the second NES are identical. In other embodiments, the first NES and the second NES are not identical. The NES can be attached to the engineered meganuclease by any appropriate means known in the art. For example, the first NES and/or the second NES can be fused to the engineered meganuclease. In some embodiments, the first NES and/or the second NES is attached to the engineered meganuclease by a polypeptide linker. An engineered meganuclease with an NES may have reduced or decreased transport to the nucleus of a target cell or target cell population (e.g., a eukaryotic cell or eukaryotic cell population), compared to an engineered meganuclease without an NES. For example, nuclear transport of an engineered meganuclease with an NES may be less than that of an engineered meganuclease without an NES, by about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60- 70%, 70-80%, 80-90%, 90-100%, or more (e.g., by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more). In some embodiments, an engineered meganuclease with an NES may induce fewer nuclear indels (i.e., less cleavage and resulting deletion in nuclear genome of a target cell or target cell population) compared to an engineered meganuclease without an NES. For example, nuclear indels induced by an engineered meganuclease with an NES may be less than that induced by an engineered meganuclease without an NES, by about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, or more. In certain embodiments of the engineered meganucleases described herein, the first subunit (i.e., comprising HVR1) can be positioned as the C-terminal subunit, and the second subunit (i.e., comprising HVR2) can be positioned as the N-terminal subunit. In some embodiments of such a Attorney Docket No.: P893391190WO (01242) configuration, such as those exemplified in SEQ ID NOs: 7-10, the first subunit (i.e., the C-terminal subunit) can lack residues at its N-terminus that correspond to residues 1-4 of wild-type I-CreI because the binding site of the polypeptide linker is at the Y residue corresponding to position 5 of wild-type I-CreI. The first subunit can further comprise residues at its C-terminus that correspond to residues 154-163 of wild-type I-CreI. Also, in some embodiments of such a configuration, the second subunit (i.e., the N-terminal subunit) can lack residues at its C-terminus that correspond to residues 154-163 of wild-type I-CreI because the binding site of the polypeptide linker is at the D residue corresponding to position 153 of wild-type I-CreI. The second subunit can further comprise one or more residues at its N-terminus that correspond to one or more of residues 1-6 of wild-type I-CreI (e.g., residues 1-6, 2-6, 3-6, 4-6, or 5-6). In other embodiments of the engineered meganucleases described herein, the first subunit (i.e., comprising HVR1) can be positioned as the N-terminal subunit, and the second subunit (i.e., comprising HVR2) can be positioned as the C-terminal subunit. In some embodiments of such a configuration, the first subunit (i.e., the N-terminal subunit) can lack residues at its C-terminus that correspond to residues 154-163 of wild-type I-CreI because the binding site of the polypeptide linker is at the D residue corresponding to position 153 of wild-type I-CreI. The first subunit can further comprise one or more residues at its N-terminus that correspond to one or more of residues 1-6 of wild-type I-CreI (e.g., residues 1-6, 2-6, 3-6, 4-6, or 5-6). Also, in some embodiments of such a configuration, the second subunit (i.e., the C-terminal subunit) can lack residues at its N- terminus that correspond to residues 1-4 of wild-type I-CreI because the binding site of the polypeptide linker is at the Y residue corresponding to position 5 of wild-type I-CreI. The second subunit can further comprise residues at its C-terminus that correspond to residues 154-163 of wild- type I-CreI. In some embodiments, the disclosed engineered meganucleases comprise (i) an inactivating amino acid in the N-terminal subunit that reduces or abolishes cleavage activity; (ii) an inactivating amino acid in the C-terminal subunit that reduces or abolishes cleavage activity; or (iii) an inactivating amino acid in the N-terminal subunit that reduces or abolishes cleavage activity and an inactivating amino acid in the C-terminal subunit that reduces or abolishes cleavage activity. As used here, an inactivating amino acid that “reduces” cleavage activity of an engineered meganuclease inactivates only the subunit comprising that amino acid, while not affecting the ability of the other subunit to cleave its DNA strand. For example, in cases where only one subunit comprises an inactivating amino acid that reduces cleavage activity, the other subunit remains active and the engineered meganuclease becomes a nickase that remains capable of cleaving one strand of the double-stranded DNA. In other cases where both subunits comprise an inactivating amino acid that reduces cleavage activity, neither subunit is active, the engineered meganuclease Attorney Docket No.: P893391190WO (01242) does not comprise any cleavage activity, and it cannot generate a single-strand or double-strand break in the DNA. By comparison, an inactivating amino acid that “abolishes” cleavage activity of an engineered meganuclease can be present in only one subunit but will inactivate both subunits of the engineered meganuclease, such that it does not comprise any cleavage activity and cannot generate a single-strand or double-strand break in the DNA. In some embodiments, the inactivating amino acid is an A at a position corresponding to position 20 (i.e., D20A) or position 211 (i.e., D211A) of SEQ ID NOs: 7-10. In some embodiments, the inactivating amino acid is an E at a position corresponding to position 47 (i.e., Q47E) or position 238 (i.e., Q238E) of SEQ ID NOs: 7-10. In some embodiments, the N-terminal subunit comprises an E at a position corresponding to position 47 (i.e., Q47E) of SEQ ID NOs: 7-10, and the C-terminal subunit comprises an E at a position corresponding to position 238 (Q238E) of SEQ ID NOs: 7-10, wherein the engineered meganuclease does not comprise cleavage activity (i.e., activity is abolished). In some embodiments, the N-terminal subunit comprises an A at a position corresponding to position 20 (i.e., D20A) of SEQ ID NOs: 7-10, and/or the C-terminal subunit comprises an A at a position corresponding to position 211 (i.e., D211A) of SEQ ID NOs: 7-10, wherein the engineered meganuclease does not comprise cleavage activity (i.e., activity is abolished). In some embodiments, the N-terminal subunit comprises an E at a position corresponding to position 47 (i.e., Q47E) of SEQ ID NOs: 7-10 and the C-terminal subunit does not comprise an inactivating amino acid, wherein the engineered meganuclease is a nickase that is only capable of cleaving the antisense strand of a dsDNA target site. In some embodiments, the C-terminal subunit comprises an E at a position corresponding to position 238 (i.e., Q238E) of SEQ ID NOs: 7-10 and the N-terminal subunit does not comprise an inactivating amino acid, wherein the engineered meganuclease is a nickase that is only capable of cleaving the sense strand of a dsDNA target site. In those embodiments wherein the engineered meganuclease does not comprise cleavage activity (i.e., activity is abolished) due to one or more inactivating amino acid modifications, such engineered meganucleases are capable of binding to a double-stranded DNA comprising the recognition sequence of SEQ ID NO: 3 (i.e., MIT 9-10) without cleaving the double-stranded DNA. In those embodiments wherein the engineered meganuclease comprises an inactivating amino acid modification such that only one subunit has cleavage activity, and the engineered meganuclease is a nickase, such engineered meganucleases are capable of binding to a double- stranded DNA comprising the recognition sequence of SEQ ID NO: 3 (i.e., MIT 9-10) and cleaving either the sense or antisense strand of the DNA. Attorney Docket No.: P893391190WO (01242) 2.3 Pharmaceutical Compositions In some embodiments, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an engineered meganuclease described herein, or a pharmaceutically acceptable carrier and a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein. In particular, pharmaceutical compositions are provided that comprise a pharmaceutically acceptable carrier and a therapeutically effective amount of a nucleic acid encoding an engineered meganuclease described herein. Such pharmaceutical compositions can be prepared in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21st ed., Philadelphia, Lippincott, Williams & Wilkins, 2005). In the manufacture of a pharmaceutical formulation according to the invention, nuclease polypeptides (or DNA/RNA encoding the same or cells expressing the same) are typically admixed with a pharmaceutically acceptable carrier, and the resulting composition is administered to a subject. The carrier must be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. In some embodiments, pharmaceutical compositions of the invention can further comprise one or more additional agents or biological molecules useful in the treatment of a disease in the subject. Likewise, the additional agent(s) and/or biological molecule(s) can be co-administered as a separate composition. In particular embodiments of the invention, the pharmaceutical composition comprises a recombinant virus (i.e., a viral vector) comprising a polynucleotide (e.g., a viral genome) comprising a nucleic acid sequence encoding an engineered meganuclease described herein. Such recombinant viruses are known in the art and include recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, and recombinant adeno-associated viruses (AAV) (reviewed in Vannucci, et al. (2013 New Microbiol.36:1-22). Recombinant AAVs useful in the invention can have any capsid or serotype that allows for transduction of the virus into a target cell type and expression of the engineered meganuclease by the target cell. For example, in some embodiments, recombinant AAV has a capsid of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVHSC, or other AAVs known in the art. In some embodiments, the recombinant virus is injected directly into target tissues. In alternative embodiments, the recombinant virus is delivered systemically via the circulatory system. It is known in the art that different AAVs tend to localize to different tissues, and one could select an appropriate AAV capsid/serotype for preferential delivery to a particular tissue. Accordingly, in some embodiments, the recombinant AAV has an AAV9 capsid, a MyoAAV capsid (e.g., Attorney Docket No.: P893391190WO (01242) Tabebordbar, et al. (2021) Cell.184: 4919-4938), or an AAVMYO capsid (e.g., Weinmann et al., (2020) Nature Communications.11:5432; Andari et al., (2022) Science Advances.8(38)). AAVs can also be self-complementary such that they do not require second-strand DNA synthesis in the host cell (McCarty, et al. (2001) Gene Ther.8:1248-54). Nucleic acids delivered by recombinant AAVs can include left (5') and right (3') inverted terminal repeats. In particular embodiments of the invention, the pharmaceutical composition comprises one or more mRNAs described herein (e.g., mRNAs encoding an engineered meganuclease) formulated within lipid nanoparticles. The selection of cationic lipids, non-cationic lipids and/or lipid conjugates which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, and the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios of each individual component may be adjusted accordingly. The lipid nanoparticles for use in the method of the invention can be prepared by various techniques which are presently known in the art. Nucleic acid-lipid particles and their method of preparation are disclosed in, for example, U.S. Patent Publication Nos.20040142025 and 20070042031, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Selection of the appropriate size of lipid nanoparticles must take into consideration the site of the target cell and the application for which the lipid nanoparticles is being made. Generally, the lipid nanoparticles will have a size within the range of about 25 to about 500 nm. In some embodiments, the lipid nanoparticles have a size from about 50 nm to about 300 nm or from about 60 nm to about 120 nm. The size of the lipid nanoparticles may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421^150 (1981), incorporated herein by reference. A variety of methods are known in the art for producing a population of lipid nanoparticles of particular size ranges, for example, sonication or homogenization. One such method is described in U.S. Pat. No.4,737,323, incorporated herein by reference. Some lipid nanoparticles contemplated for use in the invention comprise at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid. In more particular examples, lipid nanoparticles can comprise from about 50 mol % to about 85 mol % of a cationic lipid, from about 13 mol % to about 49.5 mol % of a non-cationic lipid, and from about 0.5 mol % to about 10 mol % of a lipid conjugate, and are produced in such a manner as to have a non- lamellar (i.e., non-bilayer) morphology. In other particular examples, lipid nanoparticles can Attorney Docket No.: P893391190WO (01242) comprise from about 40 mol % to about 85 mol % of a cationic lipid, from about 13 mol % to about 49.5 mol % of a non-cationic lipid, and from about 0.5 mol % to about 10 mol % of a lipid conjugate, and are produced in such a manner as to have a non-lamellar (i.e., non-bilayer) morphology. Cationic lipids can include, for example, one or more of the following: palmitoyi-oleoyl- nor-arginine (PONA), MPDACA, GUADACA, ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate) (MC3), LenMC3, CP-LenMC3, γ-LenMC3, CP-γ- LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide, Pan-MC3, Pan-MC4 and Pan MC5, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; “XTC2”), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K- C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2- dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N- methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]- dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3- morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2- dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2- propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2- N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N- dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N′,N′- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4- oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-beta-oxy)-3′- oxapentoxy)-3-dimethy-1-(cis,cis-9′,1-2′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl- 3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or mixtures Attorney Docket No.: P893391190WO (01242) thereof. The cationic lipid can also be DLinDMA, DLin-K-C2-DMA (“XTC2”), MC3, LenMC3, CP-LenMC3, γ-LenMC3, CP-γ-LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide, Pan-MC3, Pan-MC4, Pan MC5, or mixtures thereof. In various embodiments, the cationic lipid comprises from about 50 mol % to about 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, or from about 50 mol % to about 60 mol % of the total lipid present in the particle. In other embodiments, the cationic lipid comprises from about 40 mol % to about 90 mol %, from about 40 mol % to about 85 mol %, from about 40 mol % to about 80 mol %, from about 40 mol % to about 75 mol %, from about 40 mol % to about 70 mol %, from about 40 mol % to about 65 mol %, or from about 40 mol % to about 60 mol % of the total lipid present in the particle. The non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids. In particular embodiments, the non-cationic lipid comprises one of the following neutral lipid components: (1) cholesterol or a derivative thereof; (2) a phospholipid; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2′- hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, and mixtures thereof. The phospholipid may be a neutral lipid including, but not limited to, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), and mixtures thereof. In certain particular embodiments, the phospholipid is DPPC, DSPC, or mixtures thereof. In some embodiments, the non-cationic lipid (e.g., one or more phospholipids and/or cholesterol) comprises from about 10 mol % to about 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol % to about 60 mol %, from about 25 mol % to about 60 mol %, from about 30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, from about 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %, from about 25 mol % to about 55 mol %, from about 30 mol % to about 55 mol %, from about 13 mol % to about 50 mol %, from about 15 mol % to about 50 mol % or from about 20 mol % to about 50 mol % of the total lipid present in the particle. When the non-cationic lipid is a mixture of a phospholipid and cholesterol or a Attorney Docket No.: P893391190WO (01242) cholesterol derivative, the mixture may comprise up to about 40, 50, or 60 mol % of the total lipid present in the particle. The conjugated lipid that inhibits aggregation of particles may comprise, e.g., one or more of the following: a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, a cationic-polymer-lipid conjugates (CPLs), or mixtures thereof. In one particular embodiment, the nucleic acid-lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate. In certain embodiments, the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL. The conjugated lipid that inhibits aggregation of particles may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG- di lauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18), or mixtures thereof. Additional PEG-lipid conjugates suitable for use in the invention include, but are not limited to, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT Application No. PCT/US08/88676. Yet additional PEG-lipid conjugates suitable for use in the invention include, without limitation, 1-[8′-(1,2-dimyristoyl-3- propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethylene glycol) (2KPEG- DMG). The synthesis of 2KPEG-DMG is described in U.S. Pat. No.7,404,969. In some cases, the conjugated lipid that inhibits aggregation of particles (e.g., PEG-lipid conjugate) may comprise from about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7 mol %, from about 1 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. Typically, in such instances, the PEG moiety has an average molecular weight of about 2,000 Daltons. In other cases, the conjugated lipid that inhibits aggregation of particles (e.g., PEG-lipid conjugate) may comprise from about 5.0 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. Typically, in such instances, the PEG moiety has an average molecular weight of about 750 Daltons. In other embodiments, the composition comprises amphoteric liposomes, which contain at least one positive and at least one negative charge carrier, which differs from the positive one, the Attorney Docket No.: P893391190WO (01242) isoelectric point of the liposomes being between 4 and 8. This objective is accomplished owing to the fact that liposomes are prepared with a pH-dependent, changing charge. Liposomal structures with the desired properties are formed, for example, when the amount of membrane-forming or membrane-based cationic charge carriers exceeds that of the anionic charge carriers at a low pH and the ratio is reversed at a higher pH. This is always the case when the ionizable components have a pKa value between 4 and 9. As the pH of the medium drops, all cationic charge carriers are charged more and all anionic charge carriers lose their charge. Cationic compounds useful for amphoteric liposomes include those cationic compounds previously described herein above. Without limitation, strongly cationic compounds can include, for example: DC-Chol 3-β-[N-(N′,N′-dimethylmethane) carbamoyl] cholesterol, TC-Chol 3-β-[N- (N′, N′, N′-trimethylaminoethane) carbamoyl cholesterol, BGSC bisguanidinium-spermidine- cholesterol, BGTC bis-guadinium-tren-cholesterol, DOTAP (1,2-dioleoyloxypropyl)-N,N,N- trimethylammonium chloride, DOSPER (1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylarnide, DOTMA (1,2-dioleoyloxypropyl)-N,N,N-trimethylamronium chloride) (Lipofectin®), DORIE 1,2-dioleoyloxypropyl)-3-dimethylhydroxyethylammonium bromide, DOSC (1,2-dioleoyl-3- succinyl-sn-glyceryl choline ester), DOGSDSO (1,2-dioleoyl-sn-glycero-3-succinyl-2- hydroxyethyl disulfide omithine), DDAB dimethyldioctadecylammonium bromide, DOGS ((C18)2GlySper3+) N,N-dioctadecylamido-glycol-spermin (Transfectam®) (C18)2Gly+ N,N- dioctadecylamido-glycine, CTAB cetyltrimethylarnmonium bromide, CpyC cetylpyridinium chloride, DOEPC 1,2-dioleoly-sn-glycero-3-ethylphosphocholine or other O-alkyl- phosphatidylcholine or ethanolamines, amides from lysine, arginine or ornithine and phosphatidyl ethanolamine. Examples of weakly cationic compounds include, without limitation: His-Chol (histaminyl- cholesterol hemisuccinate), Mo-Chol (morpholine-N-ethylamino-cholesterol hemisuccinate), or histidinyl-PE. Examples of neutral compounds include, without limitation: cholesterol, ceramides, phosphatidyl cholines, phosphatidyl ethanolamines, tetraether lipids, or diacyl glycerols. Anionic compounds useful for amphoteric liposomes include those non-cationic compounds previously described herein. Without limitation, examples of weakly anionic compounds can include: CHEMS (cholesterol hemisuccinate), alkyl carboxylic acids with 8 to 25 carbon atoms, or diacyl glycerol hemisuccinate. Additional weakly anionic compounds can include the amides of aspartic acid, or glutamic acid and PE as well as PS and its amides with glycine, alanine, glutamine, asparagine, serine, cysteine, threonine, tyrosine, glutamic acid, aspartic acid or other amino acids or aminodicarboxylic acids. According to the same principle, the esters of hydroxycarboxylic acids or hydroxydicarboxylic acids and PS are also weakly anionic compounds. Attorney Docket No.: P893391190WO (01242) In some embodiments, amphoteric liposomes contain a conjugated lipid, such as those described herein above. Particular examples of useful conjugated lipids include, without limitation, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3-amines. Some particular examples are PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, the neutral lipids comprise from about 10 mol % to about 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol % to about 60 mol %, from about 25 mol % to about 60 mol %, from about 30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, from about 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %, from about 25 mol % to about 55 mol %, from about 30 mol % to about 55 mol %, from about 13 mol % to about 50 mol %, from about 15 mol % to about 50 mol % or from about 20 mol % to about 50 mol % of the total lipid present in the particle. In some cases, the conjugated lipid that inhibits aggregation of particles (e.g., PEG-lipid conjugate) comprises from about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7 mol %, from about 1 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. Typically, in such instances, the PEG moiety has an average molecular weight of about 2,000 Daltons. In other cases, the conjugated lipid that inhibits aggregation of particles (e.g., PEG-lipid conjugate) may comprise from about 5.0 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle. Typically, in such instances, the PEG moiety has an average molecular weight of about 750 Daltons. Considering the total amount of neutral and conjugated lipids, the remaining balance of the amphoteric liposome can comprise a mixture of cationic compounds and anionic compounds formulated at various ratios. The ratio of cationic to anionic lipid may selected in order to achieve the desired properties of nucleic acid encapsulation, zeta potential, pKa, or other physicochemical property that is at least in part dependent on the presence of charged lipid components. In some embodiments, the lipid nanoparticles have a composition that specifically enhances delivery and uptake in a eukaryotic cell, such as a mammalian cell (e.g., a human cell). In certain Attorney Docket No.: P893391190WO (01242) embodiments, the lipid nanoparticles have a composition that specifically enhances delivery and uptake in the liver or specifically within hepatocytes. In certain embodiments, the lipid nanoparticles have a composition that specifically enhances delivery and uptake in a nerve cell. 2.4 Methods for Producing Recombinant Viruses In some embodiments, the invention provides recombinant viruses (e.g., recombinant AAVs) for use in the methods of the invention. Recombinant AAVs are typically produced in mammalian cell lines such as HEK-293. Because the viral cap and rep genes are removed from the vector to prevent its self-replication to make room for the therapeutic gene(s) to be delivered (e.g. the nuclease gene), it is necessary to provide these in trans in the packaging cell line. In addition, it is necessary to provide the “helper” (e.g., adenoviral) components necessary to support replication (Cots et al. (2013), Curr. Gene Ther.13(5): 370-81). Frequently, recombinant AAVs are produced using a triple-transfection in which a cell line is transfected with a first plasmid encoding the “helper” components, a second plasmid comprising the cap and rep genes, and a third plasmid comprising the viral ITRs containing the intervening DNA sequence to be packaged into the virus. Viral particles comprising a genome (ITRs and intervening gene(s) of interest) encased in a capsid are then isolated from cells by freeze-thaw cycles, sonication, detergent, or other means known in the art. Particles are then purified using cesium-chloride density gradient centrifugation or affinity chromatography and subsequently delivered to the gene(s) of interest to cells, tissues, or an organism such as a human patient. Because recombinant AAVs are typically produced (manufactured) in cells, precautions must be taken in practicing the current invention to ensure that the engineered meganuclease is not expressed in the packaging cells. Because the viral genomes of the invention may comprise a recognition sequence for the nuclease, any nuclease expressed in the packaging cell line may be capable of cleaving the viral genome before it can be packaged into viral particles. This will result in reduced packaging efficiency and/or the packaging of fragmented genomes. Several approaches can be used to prevent nuclease expression in the packaging cells. Engineered meganucleases described herein can be placed under the control of any promoter suitable for expression of the engineered meganuclease. In some embodiments, the promoter is a constitutive promoter. In some such embodiments, the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. In other embodiments, the promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter. In other embodiments, the promoter is a central nervous system (CNS) cell-specific promoter. Attorney Docket No.: P893391190WO (01242) In some embodiments, the promoter is a constitutive promoter, or the promoter is a tissue- specific promoter such as, for example, a muscle cell-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte-specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell-specific promoter, a retinal ganglion cell-specific promoter, a retinal pigmentary epithelium-specific promoter, a pancreatic cell-specific promoter, or a pancreatic beta cell-specific promoter. In some embodiments, the constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. In specific embodiments, the engineered meganuclease can be placed under control of a tissue-specific promoter that is not active in the packaging cells. For example, if a viral vector is developed for delivery of a nuclease gene(s) to muscle tissue, a muscle cell-specific promoter can be used. Examples of muscle cell-specific promoters include C5-12 (Liu, et al. (2004) Hum Gene Ther.15:783-92), the muscle-specific creatine kinase (MCK) promoter (Yuasa, et al. (2002) Gene Ther.9:1576-88), or the smooth muscle 22 (SM22) promoter (Haase, et al. (2013) BMC Biotechnol.13:49-54). Others include, for example, a truncated MCK (tMCK) promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter. Examples of CNS (neuron)-specific promoters include the NSE, Synapsin, and MeCP2 promoters (Lentz, et al. (2012) Neurobiol Dis.48:179-88). Examples of liver-specific promoters include albumin promoters (such as Palb), human α1-antitrypsin (such as Pa1AT), and hemopexin (such as Phpx) (Kramer et al., (2003) Mol. Therapy 7:375-85), hybrid liver-specific promoter (hepatic locus control region from ApoE gene (ApoE-HCR) and a liver- specific alpha1-antitrypsin promoter), human thyroxine binding globulin (TBG) promoter, and apolipoprotein A-II promoter. Examples of eye-specific promoters include opsin, and corneal epithelium-specific K12 promoters (Martin et al. (2002) Methods (28): 267-75) (Tong et al., (2007) J Gene Med, 9:956-66). These promoters, or other tissue-specific promoters known in the art, are not highly-active in HEK-293 cells and, thus, will not be expected to yield significant levels of nuclease gene expression in packaging cells when incorporated into viral vectors of the present invention. Similarly, the viral vectors of the present invention contemplate the use of other cell lines with the use of incompatible tissue specific promoters (i.e., the well-known HeLa cell line (human epithelial cell) and using the liver-specific hemopexin promoter). Other examples of tissue-specific promoters include: synovial sarcomas PDZD4 (cerebellum), C6 (liver), ASB5 (muscle), PPP1R12B (heart), SLC5A12 (kidney), cholesterol regulation APOM (liver), ADPRHL1 (heart), and monogenic malformation syndromes TP73L (muscle). (Jacox et al., (2010), PLoS One v.5(8):e12274). Alternatively, the recombinant virus can be packaged in cells from a different species in Attorney Docket No.: P893391190WO (01242) which the nuclease is not likely to be expressed. For example, viral particles can be produced in microbial, insect, or plant cells using mammalian promoters, such as the well-known cytomegalovirus- or SV40 virus-early promoters, which are not active in the non-mammalian packaging cells. In a particular embodiment, viral particles are produced in insect cells using the baculovirus system as described by Gao, et al. (Gao et al. (2007), J. Biotechnol.131(2):138-43). A nuclease under the control of a mammalian promoter is unlikely to be expressed in these cells (Airenne et al. (2013), Mol. Ther.21(4):739-49). Moreover, insect cells utilize different mRNA splicing motifs than mammalian cells. Thus, it is possible to incorporate a mammalian intron, such as the human growth hormone (HGH) intron or the SV40 large T antigen intron, into the coding sequence of a nuclease. Because these introns are not spliced efficiently from pre-mRNA transcripts in insect cells, insect cells will not express a functional nuclease and will package the full-length genome. In contrast, mammalian cells to which the resulting recombinant AAV particles are delivered will properly splice the pre-mRNA and will express functional nuclease protein. Haifeng Chen has reported the use of the HGH and SV40 large T antigen introns to attenuate expression of the toxic proteins barnase and diphtheria toxin fragment A in insect packaging cells, enabling the production of recombinant AAV vectors carrying these toxin genes (Chen, H (2012) Mol Ther Nucleic Acids.1(11): e57). The engineered meganuclease gene can be operably linked to an inducible promoter such that a small-molecule inducer is required for nuclease expression. Examples of inducible promoters include the Tet-On system (Clontech; Chen et al. (2015), BMC Biotechnol.15(1):4)) and the RheoSwitch system (Intrexon; Sowa et al. (2011), Spine, 36(10): E623-8). Both systems, as well as similar systems known in the art, rely on ligand-inducible transcription factors (variants of the Tet Repressor and Ecdysone receptor, respectively) that activate transcription in response to a small-molecule activator (Doxycycline or Ecdysone, respectively). Practicing the current invention using such ligand-inducible transcription activators includes: 1) placing the nuclease gene under the control of a promoter that responds to the corresponding transcription factor, the nuclease gene having (a) binding site(s) for the transcription factor; and 2) including the gene encoding the transcription factor in the packaged viral genome. The latter step is necessary because the nuclease will not be expressed in the target cells or tissues following recombinant AAV delivery if the transcription activator is not also provided to the same cells. The transcription activator then induces nuclease gene expression only in cells or tissues that are treated with the cognate small- molecule activator. This approach is advantageous because it enables nuclease gene expression to be regulated in a spatio-temporal manner by selecting when and to which tissues the small- molecule inducer is delivered. However, the requirement to include the inducer in the viral genome, which has significantly limited carrying capacity, creates a drawback to this approach. Attorney Docket No.: P893391190WO (01242) In another particular embodiment, recombinant AAVs are produced in a mammalian cell line that expresses a transcription repressor that prevents expression of the nuclease. Transcription repressors are known in the art and include the Tet-Repressor, the Lac-Repressor, the Cro repressor, and the Lambda-repressor. Many nuclear hormone receptors such as the ecdysone receptor also act as transcription repressors in the absence of their cognate hormone ligand. To practice the current invention, packaging cells are transfected/transduced with a vector encoding a transcription repressor and the nuclease gene in the viral genome (packaging vector) is operably linked to a promoter that is modified to comprise binding sites for the repressor such that the repressor silences the promoter. The gene encoding the transcription repressor can be placed in a variety of positions. It can be encoded on a separate vector; it can be incorporated into the packaging vector outside of the ITR sequences; it can be incorporated into the cap/rep vector or the adenoviral helper vector; or it can be stably integrated into the genome of the packaging cell such that it is expressed constitutively. Methods to modify common mammalian promoters to incorporate transcription repressor sites are known in the art. For example, Chang and Roninson modified the strong, constitutive CMV and RSV promoters to comprise operators for the Lac repressor and showed that gene expression from the modified promoters was greatly attenuated in cells expressing the repressor (Chang and Roninson (1996), Gene 183:137-42). The use of a non- human transcription repressor ensures that transcription of the nuclease gene will be repressed only in the packaging cells expressing the repressor and not in target cells or tissues transduced with the resulting recombinant AAV. 2.5 Methods for Producing Genetically-Modified Cells The invention provides methods for producing genetically-modified cells, both in vitro and in vivo, using engineered meganucleases comprising an MTP (i.e., mitochondria-targeted engineered meganucleases) that bind and cleave recognition sequences found within mtDNA, such as human mtDNA. Cleavage at such recognition sequences can allow for NHEJ at the cleavage site, insertion of an exogenous sequence via homologous recombination, or degradation of the mtDNA. The invention includes that an engineered meganuclease described herein, or a nucleic acid encoding an engineered meganuclease described herein, can be delivered (i.e., introduced) into cells, such as eukaryotic cells (e.g., human cells). Engineered meganucleases described herein can be delivered into a cell in the form of protein or, preferably, as a nucleic acid encoding the engineered meganuclease. Such nucleic acids can be DNA (e.g., circular or linearized plasmid DNA or PCR products) or RNA (e.g., mRNA). Attorney Docket No.: P893391190WO (01242) Accordingly, polynucleotides are provided herein that comprise a nucleic acid sequence encoding an engineered meganuclease disclosed herein. In specific embodiments, the polynucleotide is an mRNA. The polynucleotides encoding an engineered meganuclease disclosed herein can be operably linked to a promoter. In specific embodiments, expression cassettes are provided that comprise a promoter operably linked to a polynucleotide having a nucleic acid sequence encoding a engineered meganuclease disclosed herein. For embodiments in which the engineered meganuclease coding sequence is delivered in DNA form, it should be operably linked to a promoter to facilitate transcription of the engineered meganuclease-encoding sequence. Mammalian promoters suitable for the invention include constitutive promoters such as the cytomegalovirus early (CMV) promoter (Thomsen et al. (1984), Proc Natl Acad Sci USA.81(3):659-63), the SV40 early promoter (Benoist and Chambon (1981), Nature.290(5804):304-10), a CAG promoter, an EF1 alpha promoter, or a UbC promoter, as well as inducible promoters such as the tetracycline-inducible promoter (Dingermann et al. (1992), Mol Cell Biol.12(9):4038-45). An engineered meganuclease described herein can also be operably linked to a synthetic promoter. Synthetic promoters can include, without limitation, the JeT promoter (WO 2002/012514). In specific embodiments, a nucleic acid sequence encoding an engineered meganuclease described hereinis operably linked to a tissue or cell-specific promoter, such as a muscle cell-specific promoter, a skeletal muscle-specific promoter, a myotube-specific promoter, a muscle satellite cell-specific promoter, a neuron-specific promoter, an astrocyte- specific promoter, a microglia-specific promoter, an eye cell-specific promoter, a retinal cell- specific promoter, a retinal ganglion cell-specific promoter, a retinal pigmentary epithelium- specific promoter, a pancreatic cell-specific promoter, a pancreatic beta cell-specific promoter, or other promoters described herein. In specific embodiments, a nucleic acid sequence encoding an engineered meganuclease is delivered on a recombinant DNA construct or expression cassette. For example, the recombinant DNA construct can comprise an expression cassette (i.e., “cassette”) comprising a promoter and a polynucleotide having a nucleic acid sequence encoding an engineered meganuclease described herein. The polynucleotides provided herein can be mRNA or DNA. In particular embodiments, the polynucleotides further comprise a sequence encoding a selectable marker. The selectable marker can be any marker that allows selection of cells or organisms (e.g., bacteria, eukaryotic cells, mammalian cells, plant cells, plants, and/or plant parts) that contain a polynucleotide disclosed herein. In specific embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, mRNA encoding the engineered meganuclease is delivered to a cell because this reduces the likelihood that the gene encoding the engineered meganuclease will integrate into the genome of the cell. Attorney Docket No.: P893391190WO (01242) Such mRNA encoding an engineered meganuclease can be produced using methods known in the art such as in vitro transcription. In some embodiments, the mRNA is 5' capped using 7- methyl-guanosine, anti-reverse cap analogs (ARCA) (US 7,074,596), CLEANCAP® analogs such as Cap 1 analogs (Trilink, San Diego, CA), or enzymatically capped using vaccinia capping enzyme or similar. In some embodiments, the mRNA may be polyadenylated. The mRNA may contain various 5’ and 3’ untranslated sequence elements to enhance expression the encoded engineered meganuclease and/or stability of the mRNA itself. Such elements can include, for example, posttranslational regulatory elements such as a woodchuck hepatitis virus posttranslational regulatory element. The mRNA may contain nucleoside analogs or naturally- occurring nucleosides, such as pseudouridine, 5-methylcytidine, N6-methyladenosine, 5- methyluridine, or 2-thiouridine. Additional nucleoside analogs include, for example, those described in US 8,278,036. Purified engineered meganucleases can be delivered into cells to cleave mitochondrial DNA by a variety of different mechanisms known in the art, including those further detailed herein. In another particular embodiment, a nucleic acid encoding an engineered meganuclease described herein is introduced into the cell using a single-stranded DNA template. The single- stranded DNA can further comprise a 5' and/or a 3' AAV inverted terminal repeat (ITR) upstream and/or downstream of the sequence encoding the engineered meganuclease. The single-stranded DNA can further comprise a 5' and/or a 3' homology arm upstream and/or downstream of the sequence encoding the engineered meganuclease. In another particular embodiment, genes encoding an engineered meganuclease described herein are introduced into a cell using a linearized DNA template. Such linearized DNA templates can be produced by methods known in the art. For example, a plasmid DNA encoding a nuclease can be digested by one or more restriction enzymes such that the circular plasmid DNA is linearized prior to being introduced into a cell. Purified engineered meganucleases, or nucleic acids encoding engineered meganucleases, can be delivered into cells to cleave mitochondrial DNA by a variety of different mechanisms known in the art, including those further detailed herein below. In some embodiments, engineered meganucleases, DNA/mRNA encoding engineered meganucleases, or cells expressing engineered meganucleases are formulated for systemic administration, or administration to target tissues, in a pharmaceutically acceptable carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21st ed., Philadelphia, Lippincott, Williams & Wilkins, 2005). In the manufacture of a pharmaceutical formulation according to the invention, proteins/RNA/mRNA/cells are typically admixed with a pharmaceutically acceptable carrier. The carrier must, of course, be acceptable in Attorney Docket No.: P893391190WO (01242) the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier can be a solid or a liquid, or both, and can be formulated with the compound as a unit-dose formulation. In some embodiments, the engineered meganucleases, or DNA/mRNA encoding the engineered meganucleases, are coupled to a cell penetrating peptide or targeting ligand to facilitate cellular uptake. Examples of cell penetrating peptides known in the art include poly-arginine (Jearawiriyapaisarn, et al. (2008) Mol Ther.16:1624-9), TAT peptide from the HIV virus (Hudecz et al. (2005), Med. Res. Rev.25: 679-736), MPG (Simeoni, et al. (2003) Nucleic Acids Res. 31:2717–2724), Pep-1 (Deshayes et al. (2004) Biochemistry 43: 7698–7706, and HSV-1 VP-22 (Deshayes et al. (2005) Cell Mol Life Sci.62:1839-49. In an alternative embodiment, engineered meganucleases, or DNA/mRNA encoding engineered meganucleases, are coupled covalently or non-covalently to an antibody that recognizes a specific cell-surface receptor expressed on target cells such that the engineered meganuclease protein/DNA/mRNA binds to and is internalized by the target cells. Alternatively, engineered meganuclease protein/DNA/mRNA can be coupled covalently or non-covalently to the natural ligand (or a portion of the natural ligand) for such a cell- surface receptor. (McCall, et al. (2014) Tissue Barriers.2(4):e944449; Dinda, et al. (2013) Curr Pharm Biotechnol.14:1264-74; Kang, et al. (2014) Curr Pharm Biotechnol.15(3):220-30; Qian et al. (2014) Expert Opin Drug Metab Toxicol.10(11):1491-508). In some embodiments, engineered meganucleases, or DNA/mRNA encoding engineered meganucleases, are encapsulated within biodegradable hydrogels. Hydrogels can provide sustained and tunable release of the therapeutic payload to the desired region of the target tissue without the need for frequent injections, and stimuli-responsive materials (e.g., temperature- and pH-responsive hydrogels) can be designed to release the payload in response to environmental or externally applied cues (Kang Derwent et al. (2008) Trans Am Ophthalmol Soc.106:206-214). In some embodiments, engineered meganuclease, or DNA/mRNA encoding engineered meganucleases, are coupled covalently or, preferably, non-covalently to a nanoparticle or encapsulated within such a nanoparticle using methods known in the art (Sharma, et al. (2014) Biomed Res Int.2014). A nanoparticle is a nanoscale delivery system whose length scale is <1 µm, preferably <100 nm. Such nanoparticles may be designed using a core composed of metal, lipid, polymer, or biological macromolecule, and multiple copies of the nuclease proteins, mRNA, or DNA can be attached to or encapsulated with the nanoparticle core. This increases the copy number of the protein/mRNA/DNA that is delivered to each cell and, so, increases the intracellular expression of each engineered meganuclease to maximize the likelihood that the target recognition sequences will be cut. The surface of such nanoparticles may be further modified with polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) to form a core-shell nanoparticle whose Attorney Docket No.: P893391190WO (01242) surface confers additional functionalities to enhance cellular delivery and uptake of the payload (Jian et al. (2012) Biomaterials.33(30): 7621-30). Nanoparticles may additionally be advantageously coupled to targeting molecules to direct the nanoparticle to the appropriate cell type and/or increase the likelihood of cellular uptake. Examples of such targeting molecules include antibodies specific for cell-surface receptors and the natural ligands (or portions of the natural ligands) for cell surface receptors. In some embodiments, the engineered meganucleases or DNA/mRNA encoding the engineered meganucleases are encapsulated within liposomes or complexed using cationic lipids (see, e.g., LIPOFECTAMINE™, Life Technologies Corp., Carlsbad, CA; Zuris et al. (2015) Nat Biotechnol.33: 73-80; Mishra et al. (2011) J Drug Deliv.2011:863734). The liposome and lipoplex formulations can protect the payload from degradation, enhance accumulation and retention at the target site, and facilitate cellular uptake and delivery efficiency through fusion with and/or disruption of the cellular membranes of the target cells. In some embodiments, engineered meganucleases, or DNA/mRNA encoding engineered meganucleases, are encapsulated within polymeric scaffolds (e.g., PLGA) or complexed using cationic polymers (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv.2(4): 523-536). Polymeric carriers can be designed to provide tunable drug release rates through control of polymer erosion and drug diffusion, and high drug encapsulation efficiencies can offer protection of the therapeutic payload until intracellular delivery to the desired target cell population. In some embodiments, engineered meganucleases, or DNA/mRNA encoding engineered meganucleases, are combined with amphiphilic molecules that self-assemble into micelles (Tong et al. (2007) J Gene Med.9(11): 956-66). Polymeric micelles may include a micellar shell formed with a hydrophilic polymer (e.g., polyethyleneglycol) that can prevent aggregation, mask charge interactions, and reduce nonspecific interactions. In some embodiments, engineered meganucleases, or DNA/mRNA encoding engineered meganucleases, are formulated into an emulsion or a nanoemulsion (i.e., having an average particle diameter of < 1nm) for administration and/or delivery to the target cell. The term “emulsion” refers to, without limitation, any oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersions or droplets, including lipid structures that can form as a result of hydrophobic forces that drive apolar residues (e.g., long hydrocarbon chains) away from water and polar head groups toward water, when a water immiscible phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. Emulsions are composed of an aqueous phase and a lipophilic phase (typically containing an oil and an organic solvent). Emulsions also frequently contain one or more surfactants. Nanoemulsion formulations are well known, e.g., as described in Attorney Docket No.: P893391190WO (01242) US Pat. Nos.6,015,832, 6,506,803, 6,635,676, 6,559,189, and 7,767,216, each of which is incorporated herein by reference in its entirety. In some embodiments, engineered meganucleases, or DNA/mRNA encoding engineered meganucleases, are covalently attached to, or non-covalently associated with, multifunctional polymer conjugates, DNA dendrimers, and polymeric dendrimers (Mastorakos et al. (2015) Nanoscale.7(9): 3845-56; Cheng et al. (2008) J Pharm Sci.97(1): 123-43). The dendrimer generation can control the payload capacity and size, and can provide a high payload capacity. Moreover, display of multiple surface groups can be leveraged to improve stability, reduce nonspecific interactions, and enhance cell-specific targeting and drug release. In some embodiments, polynucleotides having nucleic acid sequences encoding an engineered meganuclease are introduced into a cell using a recombinant virus. Such recombinant viruses are known in the art and include recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, and recombinant AAVs (reviewed in Vannucci, et al. (2013 New Microbiol.36:1-22). Recombinant AAVs useful in the invention can have any capsid or serotype that allows for transduction of the virus into a target cell type and expression of the engineered meganuclease by the target cell. For example, in some embodiments, a recombinant AAV has a capsid of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVHSC, or other capsids known in the art. In some embodiments, the recombinant virus is injected directly into target tissues. In alternative embodiments, the recombinant virus is delivered systemically via the circulatory system. It is known in the art that different AAVs tend to localize to different tissues, and one could select an appropriate AAV capsid/serotype for preferential delivery to a particular tissue. Accordingly, in some embodiments, the recombinant AAV has an AAV9 capsid, a MyoAAV capsid (e.g., Tabebordbar, et al. (2021) Cell.184: 4919- 4938), or an AAVMYO capsid (e.g., Weinmann et al., (2020) Nature Communications.11:5432; Andari et al., (2022) Science Advances.8(38)). AAVs can also be self-complementary such that they do not require second-strand DNA synthesis in the host cell (McCarty, et al. (2001) Gene Ther.8:1248-54). Nucleic acids delivered by recombinant AAVs can include left (5') and right (3') inverted terminal repeats. In one embodiment, a recombinant virus used for delivery of a polynucleotide having nucleic acid sequences encoding an engineered meganuclease is a self-limiting recombinant virus. A self-limiting recombinant virus can have limited persistence time in a cell or organism due to the presence of a recognition sequence for an engineered meganuclease within the viral genome. Thus, a self-limiting recombinant virus can be engineered to provide coding for a promoter, an engineered meganuclease described herein, and a meganuclease recognition site within the ITRs. The self-limiting recombinant virus delivers the meganuclease gene to a cell, tissue, or organism, Attorney Docket No.: P893391190WO (01242) such that the engineered meganuclease is expressed and able to cut the genome of the cell at an endogenous recognition sequence within the genome. The delivered engineered meganuclease will also find its target site within the self-limiting recombinant virus itself, and cut the viral genome at this target site. Once cut, the 5' and 3' ends of the viral genome will be exposed and degraded by exonucleases, thus killing the virus and ceasing production of the engineered meganuclease. If the polynucleotides having nucleic acid sequences encoding an engineered meganuclease are delivered in DNA form (e.g. plasmid) and/or via a viral vector (e.g. AAV) they must be operably linked to a promoter, such as those promoters described elsewhere herein. In some embodiments, this can be a viral promoter such as endogenous promoters from the viral vector (e.g. the LTR of a lentiviral vector) or constitutive or tissue-specific promoters described elsewhere herein. In a particular embodiment, polynucleotides having nucleic acid sequences encoding an engineered meganuclease are operably linked to a promoter that drives gene expression preferentially in the target cells or tissues. In some embodiments, provided herein are methods for producing a genetically-modified eukaryotic cell or a genetically-modified eukaryotic cell population by introducing into the eukaryotic cell or eukaryotic cell population a polynucleotide of the present disclosure, such as a polynucleotide containing a nucleic acid sequence that encodes an engineered meganuclease described herein. Upon expression in the eukaryotic cell or eukaryotic cell population, the engineered meganuclease localizes to the mitochondria, binds a recognition sequence in the mitochondrial genome, and generates a cleavage site. The cleavage site generated by the engineered meganuclease can be repaired by NHEJ repair pathway which may result in a nucleic acid insertion or deletion at the cleavage site. Additionally or alternatively, the cleavage site generated by the engineered meganuclease in the mitochondrial genome of the eukaryotic cell or eukaryotic cell population can be repaired by alternative nonhomologous end-joining (Alt-NHEJ) or microhomology-mediated end joining (MMEJ). The NHEJ or Alt-NHEJ/MMEJ can result in insertion and/or deletion of a nucleic acid at the cleavage site. In particular, the NHEJ or Alt- NHEJ/MMEJ can result in insertion and/or deletion of 1-1000 (e.g., 1-10, 10-100, 100-200, 200- 300, 300-400, 400-500, 500-600, 600-700, 700-80, 800-900, or 900-1000) nucleotides, such as about 1, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 nucleotides at the cleavage site. In some embodiments, mitochondrial genomes in a genetically-modified eukaryotic cell disclosed herein or a genetically-modified eukaryotic cell population disclosed herein can be degraded. In some such embodiments, the percentage of mitochondrial genomes comprising the recognition sequence is decreased by about 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Attorney Docket No.: P893391190WO (01242) 99%, or 100%, or can be degraded by about can be degraded by about 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, or more, compared to a control cell. In specific embodiments, mutant mitochondrial genomes comprising the recognition sequence of SEQ ID NO: 3 are degraded. By degrading mutant mitochondrial genomes having the recognition sequence of SEQ ID NO: 3, the overall ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes will increase following administration or expression of an engineered meganuclease disclosed herein. In some embodiments, the ratio of wild-type to mutant mitochondrial genomes in a single genetically-modified eukaryotic cell disclosed herein or a population genetically-modified eukaryotic cells increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more. In particular embodiments, the percentage of wild-type genomes in a single genetically- modified eukaryotic cell disclosed herein, or a population of genetically-modified eukaryotic cells disclosed herein, can increase as mutant mitochondrial genomes comprising SEQ ID NO: 3 are recognized, cleaved, and degraded by the engineered meganuclease. The percentage of wild-type mitochondrial genomes in a genetically-modified eukaryotic cell or genetically modified cell population disclosed herein can be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, of the total mitochondrial genomes in the genetically-modified eukaryotic cell or genetically modified cell population when compared to a eukaryotic cell or eukaryotic cell population that does not express an engineered meganuclease disclosed herein. Likewise the percentage of mutant mitochondrial genomes comprising the recognition sequence of SEQ ID NO: 3 in the genetically-modified eukaryotic cell or genetically-modified cell population can decrease by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more when compared to a eukaryotic cell or eukaryotic cell population that does not express an engineered meganuclease disclosed herein. In some embodiments, mitochondrial respiration in a genetically-modified eukaryotic cell or a genetically-modified eukaryotic cell population disclosed herein can increase by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, Attorney Docket No.: P893391190WO (01242) about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more when compared to a eukaryotic cell that does not express an engineered meganuclease disclosed herein. Mitochondrial respiration in a genetically-modified eukaryotic cell or a genetically-modified eukaryotic cell population disclosed herein can be increased by about 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, or more when compared to a eukaryotic cell or eukaryotic cell population that does not express an engineered meganuclease disclosed herein. In certain instances, the recognition sequence is within a region of the mitochondrial genome associated with a mitochondrial disorder. For example, the recognition sequence can be within a region of the mitochondrial genome associated with human mtDNA common deletion. This mutation removes all or part of the genes encoding four complex I subunits, one complex IV subunit, two complex V subunits and five tRNA genes, which are indispensable for maintaining normal mitochondrial function. The mitochondrial common deletion has attracted tremendous interest as it is associated with several sporadic diseases including myopathies, Alzheimer disease, Pearson’ s syndrome, photoaging of the skin, Kearns-Sayre syndrome (KSS) and chronic progressive external ophthalmoplegia (CPEO). Furthermore, this deletion also accumulates in many tissues during aging, and has been used as an indication of mtDNA oxidative damage. The mitochondrial common deletion is a deletion of 4,977bp between nucleotides 8,470 and 13,447 of the mitochondrial genome. Both normal and mutated mtDNA can exist in the same cell, a situation known as heteroplasmy. The number of defective mitochondria may be out-numbered by the number of normal mitochondria. Symptoms may not appear in any given generation until the mutation affects a significant proportion of mtDNA. The uneven distribution of normal and mutant mtDNA in different tissues can affect different organs in members of the same family. This can result in a variety of symptoms in affected family members. In specific embodiments, the recognition sequence disclosed herein in the mitochondrial genome of the eukaryotic cell or eukaryotic cell population is positioned between nucleotides 8,470 and 13,447 of the mitochondrial genome. In particular embodiments, the recognition sequence in the mitochondrial genome of the eukaryotic cell or eukaryotic cell population is positioned between nucleotides 8,460 and 8,580 or between nucleotides 13,437 and 13,457. In particular embodiments, the recognition sequence of SEQ ID NO: 3 is located only on mutant mitochondrial genomes. Upon expression in the genetically-modified eukaryotic cell or genetically-modified eukaryotic cell population, the engineered meganuclease can localize to the mitochondria, bind the recognition sequence in the mitochondrial genome, and generate a cleavage site. Thus, by targeting a recognition sequence only located on mutant genomes, the genomes can be cleaved and subsequently degraded. This specific degradation of mutant mitochondrial genomes can be used to Attorney Docket No.: P893391190WO (01242) help treat or alleviate the symptoms of the mitochondrial common deletion (i.e., mitochondrial common deletion disorder). Accordingly, methods are provided herein for degrading mutant mitochondrial genomes in a target cell or a population of target cells by delivering to the target cell or population a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease or an engineered meganuclease disclosed herein. In specific embodiments, the target cell or population of target cells comprise mutant mitochondrial genomes having the mitochondrial common deletion, and the engineered meganuclease recognizes and cleaves the recognition sequence of SEQ ID NO: 3. The target cell or target cell population can be in a mammalian subject, such as a human subject. In some embodiments, the target cell is a muscle cell, a skeletal muscle cell, a myotube cell, a muscle stem cell or satellite cell, a neuron, an astrocyte, a microglia cell, an eye cell, a retinal cell, a retinal ganglion cell, a retinal pigmentary epithelium cell, a pancreatic cell, or a pancreatic beta cell, or wherein said population of target cells is a population of muscle cells, skeletal muscle cells, myotube cells, muscle stem cells or satellite cells, neurons, astrocytes, microglia cells, eye cells, retinal cells, retinal ganglion cells, retinal pigmentary epithelium cells, pancreatic cells, or pancreatic beta cells. Methods of treating a condition associated with a mitochondrial mtDNA common deletion in a subject are disclosed herein. Such methods include administering to a subject a therapeutically- effective amount of a polynucleotide having a nucleic acid sequence encoding an engineered meganuclease described herein, or a therapeutically-effective amount of an engineered meganuclease described herein, wherein the engineered meganuclease produces a cleavage site at the recognition sequence of SEQ ID NO: 3 in mutant mitochondrial genomes having the common deletion. The cleavage site produced in mutant mitochondrial genomes can lead to degradation of the mutant mitochondrial genomes. In specific embodiments, treating comprises reducing or alleviating at least one symptom of a condition associated with the mtDNA common deletion. Symptoms of the mtDNA common deletion include but are not limited to any symptom of myopathies, Alzheimer disease, Pearson’ s syndrome, photoaging of the skin, Kearns-Sayre syndrome (KSS), or chronic progressive external ophthalmoplegia (CPEO). Specifically, symptoms of the mtDNA common deletion can include pigmentary retinopathy, and PEO, cerebellar ataxia, impaired intellect (intellectual disability, dementia, or both), sensorineural hearing loss, ptosis, oropharyngeal and esophageal dysfunction, exercise intolerance, muscle weakness, cardiac conduction block, endocrinopathy, sideroblastic anemia and exocrine pancreas dysfunction, ptosis, impaired eye movements due to paralysis of the extraocular muscles (ophthalmoplegia), oropharyngeal weakness, or variably severe proximal limb weakness with exercise intolerance. In some embodiments, the condition is a condition of the bone marrow, the pancreas, muscle, skeletal Attorney Docket No.: P893391190WO (01242) muscle, central nervous system, the eye, or the ears. In some embodiments, the condition is Pearson Syndrome, Kearns-Sayre Syndrome (KSS), Progressive External Ophthalmoplegia (PEO), diabetes, or kidney dysfunction. In specific embodiments, the methods of treating a condition associated with a mtDNA common deletion in a subject involve administration of a pharmaceutical composition disclosed herein. In some embodiments, a subject is administered a pharmaceutical composition disclosed herein at a dose of about 1x1010 gc/kg to about 1x1014 gc/kg (e.g., 1x1010 gc/kg, 1x1011 gc/kg, 1x1012 gc/kg, 1x1013 gc/kg, or 1x1014 gc/kg) of a nucleic acid encoding an engineered meganuclease. In some embodiments, a subject is administered a pharmaceutical composition at a dose of at least about 1x1010 gc/kg, at least about 1x1011 gc/kg, at least about 1x1012 gc/kg, at least about 1x1013 gc/kg, or at least about 1x1014 gc/kg of a nucleic acid encoding an engineered meganuclease. In some embodiments, a subject is administered a pharmaceutical composition at a dose of about 1x1010 gc/kg to about 1x1011 gc/kg, about 1x1011 gc/kg to about 1x1012 gc/kg, about 1x1012 gc/kg to about 1x1013 gc/kg, or about 1x1013 gc/kg to about 1x1014 gc/kg of a nucleic acid encoding an engineered meganuclease. In certain embodiments, a subject is administered a pharmaceutical composition at a dose of about 1x1012 gc/kg to about 9x1013 gc/kg (e.g., about 1x1012 gc/kg, about 2x1012 gc/kg, about 3x1012 gc/kg, about 4x1012 gc/kg, about 5x1012 gc/kg, about 6x1012 gc/kg, about 7x1012 gc/kg, about 8x1012 gc/kg, about 9x1012 gc/kg, about 1x1013 gc/kg, about 2x1013 gc/kg, about 3x1013 gc/kg, about 4x1013 gc/kg, about 5x1013 gc/kg, about 6x1013 gc/kg, about 7x1013 gc/kg, about 8x1013 gc/kg, or about 9x1013 gc/kg) of a nucleic acid encoding an engineered meganuclease. In some embodiments, a subject is administered a lipid nanoparticle formulation at a dose of about 0.1 mg/kg to about 3 mg/kg of mRNA encoding an engineered meganuclease. In some embodiments, the subject is administered a lipid nanoparticle formulation at a dose of at least about 0.1 mg/kg, at least about 0.25 mg/kg, at least about 0.5 mg/kg, at least about 0.75 mg/kg, at least about 1.0 mg/kg, at least about 1.5 mg/kg, at least about 2.0 mg/kg, at least about 2.5 mg/kg, or at least about 3.0 mg/kg of mRNA encoding an engineered meganuclease. In some embodiments, the subject is administered a lipid nanoparticle formulation at a dose of within about 0.1 mg/kg to about 0.25 mg/kg, about 0.25 mg/kg to about 0.5 mg/kg, about 0.5 mg/kg to about 0.75 mg/kg, about 0.75 mg/kg to about 1.0 mg/kg, about 1.0 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 2.0 mg/kg, about 2.0 mg/kg to about 2.5 mg/kg, or about 2.5 mg/kg to about 3.0 mg/kg of mRNA encoding an engineered meganuclease. The target tissue(s) for delivery of engineered meganucleases disclosed herein, or nucleic acids encoding engineered meganucleases disclosed herein, include without limitation, nerve tissue, muscle tissue, neuromuscular tissue, pancreatic tissue, and ocular/retinal tissue. In some Attorney Docket No.: P893391190WO (01242) embodiments, the target cell for delivery is a muscle cell, a skeletal muscle cell, a myotube cell, a muscle satellite cell, a neuron, an astrocyte, a microglia cell, an eye cell, a retinal cell, a retinal ganglion cell, a retinal pigmentary epithelium cell, a pancreatic cell, or a pancreatic beta cell, or wherein said population of target cells is a population of muscle cells, skeletal muscle cells, myotube cells, muscle stem cells or satellite cells, neurons, astrocytes, microglia cells, eye cells, retinal cells, retinal ganglion cells, retinal pigmentary epithelium cells, pancreatic cells, or pancreatic beta cells or the population of target cells is a population of muscle cells, skeletal muscle cells, myotube cells, muscle stem cells or satellite cells, neurons, astrocytes, microglia cells, eye cells, retinal cells, retinal ganglion cells, retinal pigmentary epithelium cells, pancreatic cells, or pancreatic beta cells. In various embodiments of the methods described herein, the one or more engineered meganucleases, polynucleotides encoding such engineered meganucleases, or recombinant viruses comprising one or more polynucleotides encoding such engineered meganucleases, as described herein, can be administered via any suitable route of administration known in the art. Accordingly, the one or more engineered meganucleases, polynucleotides encoding such engineered meganucleases, or recombinant viruses comprising one or more polynucleotides encoding such engineered meganucleases, as described herein may be administered by an administration route comprising intravenous, intramuscular, intraperitoneal, subcutaneous, intrahepatic, transmucosal, transdermal, intraarterial, and sublingual. In some embodiments, engineered meganucleases, or mRNA, or DNA vectors encoding such engineered meganucleases, are supplied to target cells (e.g., nerve cells, muscle cells, pancreatic cells, ocular cells, etc.) via injection directly to the target tissue. Other suitable routes of administration of the engineered meganucleases, polynucleotides encoding such engineered meganucleases, or recombinant viruses comprising one or more polynucleotides encoding such engineered meganucleases may be readily determined by the treating physician as necessary. In some embodiments, a therapeutically effective amount of an engineered meganuclease described herein is administered to a subject in need thereof. As appropriate, the dosage or dosing frequency of the engineered meganuclease may be adjusted over the course of the treatment, based on the judgment of the administering physician. Appropriate doses will depend, among other factors, on the specifics of any AAV chosen (e.g., serotype, etc.), on the route of administration, on the subject being treated (i.e., age, weight, sex, and general condition of the subject), and the mode of administration. Thus, the appropriate dosage may vary from patient to patient. An appropriate effective amount can be readily determined by one of skill in the art. Dosage treatment may be a single dose schedule or a multiple dose schedule. Moreover, the subject may be administered as many doses as appropriate. One of skill in the art can readily determine an appropriate number of Attorney Docket No.: P893391190WO (01242) doses. The dosage may need to be adjusted to take into consideration an alternative route of administration or balance the therapeutic benefit against any side effects. Exogenous nucleic acid molecules of the invention may be introduced into a cell and/or delivered to a subject by any of the means previously discussed. In a particular embodiment, exogenous nucleic acid molecules are introduced by way of a recombinant virus, such as a lentivirus, retrovirus, adenovirus, or a recombinant AAV. Recombinant AAVs useful for introducing an exogenous nucleic acid molecule can have any serotype that allows for transduction of the virus into the cell and insertion of the exogenous nucleic acid molecule sequence into the cell genome, including those serotypes/capsids previously described herein. The recombinant AAVs can also be self-complementary such that they do not require second-strand DNA synthesis in the host cell. Exogenous nucleic acid molecules introduced using a recombinant AAV can be flanked by a 5' (left) and 3' (right) inverted terminal repeat. In another particular embodiment, an exogenous nucleic acid molecule can be introduced into a cell using a single-stranded DNA template. The single-stranded DNA can comprise the exogenous nucleic acid molecule and, in particular embodiments, can comprise 5' and 3' homology arms to promote insertion of the nucleic acid sequence into the nuclease cleavage site by homologous recombination. The single-stranded DNA can further comprise a 5' AAV inverted terminal repeat (ITR) sequence 5' upstream of the 5' homology arm, and a 3' AAV ITR sequence 3' downstream of the 3' homology arm. In another particular embodiment, polynucleotides comprising nucleic acid sequences encoding engineered meganucleases of the invention and/or an exogenous nucleic acid molecule of the invention can be introduced into a cell by transfection with a linearized DNA template. A plasmid DNA encoding an engineered meganuclease and/or an exogenous nucleic acid molecule can, for example, be digested by one or more restriction enzymes such that the circular plasmid DNA is linearized prior to transfection into the cell. When delivered to a cell, an exogenous nucleic acid of the invention can be operably linked to any promoter suitable for expression of the encoded polypeptide in the cell, including those mammalian promoters and inducible promoters previously discussed. An exogenous nucleic acid of the invention can also be operably linked to a synthetic promoter. 2.6 Variants The present invention encompasses variants of the polypeptide and polynucleotide sequences described herein. Attorney Docket No.: P893391190WO (01242) As used herein, “variants” is intended to mean substantially similar sequences. A “variant” polypeptide is intended to mean a polypeptide derived from the “native” polypeptide by deletion or addition of one or more amino acids at one or more internal sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native polypeptide. As used herein, a “native” polynucleotide or polypeptide comprises a parental sequence from which variants are derived. Variant polypeptides encompassed by the embodiments are biologically active. That is, they continue to possess the desired biological activity of the native protein; for example, the ability to bind and cleave recognition sequences found in mtDNA (e.g., human mtDNA), such as MIT 9-10 recognition sequence (SEQ ID NO: 3). Such variants may result, for example, from human manipulation. In some embodiments, biologically active variants of a native polypeptide of the embodiments (e.g., any one of SEQ ID NOs: 7-10), or biologically active variants of the recognition half-site binding subunits described herein, will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, sequence identity to the amino acid sequence of the native polypeptide, native subunit, native HVR1, or native HVR2 as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a polypeptide or subunit of the embodiments may differ from that polypeptide or subunit by as few as about 1-40 amino acid residues, as few as about 1-20, as few as about 1-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acid residue. The polypeptides of the embodiments may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol.154:367-382; U.S. Pat. No.4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal. In some embodiments, engineered meganucleases of the invention can comprise variants of the HVR1 and HVR2 regions disclosed herein. Parental HVR regions can comprise, for example, residues 24-79 or residues 215-270 of the exemplified engineered meganucleases. Thus, variant Attorney Docket No.: P893391190WO (01242) HVRs can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to an amino acid sequence corresponding to residues 24-79 or residues 215-270 of the engineered meganucleases exemplified herein, such that the variant HVR regions maintain the biological activity of the engineered meganuclease (i.e., binding to and cleaving the recognition sequence). Further, in some embodiments of the invention, a variant HVR1 region or variant HVR2 region can comprise residues corresponding to the amino acid residues found at specific positions within the parental HVR. In this context, “corresponding to” means that an amino acid residue in the variant HVR is the same amino acid residue (i.e., a separate identical residue) present in the parental HVR sequence in the same relative position (i.e., in relation to the remaining amino acids in the parent sequence). By way of example, if a parental HVR sequence comprises a serine residue at position 26, a variant HVR that “comprises a residue corresponding to” residue 26 will also comprise a serine at a position that is relative (i.e., corresponding) to parental position 26. In particular embodiments, engineered meganucleases of the invention comprise an HVR1 that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 7-10. In certain embodiments, engineered meganucleases of the invention comprise an HVR2 that has 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7-10. A substantial number of amino acid modifications to the DNA recognition domain of the wild-type I-CreI meganuclease have previously been identified (e.g., U.S.8,021,867) which, singly or in combination, result in engineered meganucleases with specificities altered at individual bases within the DNA recognition sequence half-site, such that the resulting rationally-designed meganucleases have half-site specificities different from the wild-type enzyme. Table 3 provides potential substitutions that can be made in an engineered meganuclease monomer or subunit to enhance specificity based on the base present at each half-site position (-1 through -9) of a recognition half-site. Such substitutions are incorporated into variants of the meganucleases disclosed herein. Attorney Docket No.: P893391190WO (01242) TABLE 3. Potential substitutions in engineered meganuclease variants Favored Sense-Strand Base Posn. A C G T A/T A/C A/G C/T G/T A/G/T A/C/G/T -1 Y75 R70* K70 Q70* T46* G70 L75* H75* E70* C70 A70 C75* R75* E75* L70 S70 Y139* H46* E46* Y75* G46* C46* K46* D46* Q75* A46* R46* H75* H139 Q46* H46* -2 Q70 E70 H70 Q44* C44* T44* D70 D44* A44* K44* E44* V44* R44* I44* L44* N44* -3 Q68 E68 R68 M68 H68 Y68 K68 C24* F68 C68 I24* K24* L68 R24* F68 -4 A26* E77 R77 S77 S26* Q77 K26* E26* Q26* -5 E42 R42 K28* C28* M66 Q42 K66 -6 Q40 E40 R40 C40 A40 S40 C28* R28* I40 A79 S28* V40 A28* C79 H28* I79 V79 Q28* -7 N30* E38 K38 I38 C38 H38 Q38 K30* R38 L38 N38 R30* E30* Q30* -8 F33 E33 F33 L33 R32* R33 Y33 D33 H33 V33 I33 F33 C33 -9 E32 R32 L32 D32 S32 K32 V32 I32 N32 A32 H32 Attorney Docket No.: P893391190WO (01242) Favored Sense-Strand Base Posn. A C G T A/T A/C A/G C/T G/T A/G/T A/C/G/T C32 Q32 T32 Bold entries are wild-type contact residues and do not constitute “modifications” as used herein. An asterisk indicates that the residue contacts the base on the antisense strand. Certain modifications can be made in an engineered meganuclease monomer or subunit to modulate DNA-binding affinity and/or activity. For example, an engineered meganuclease monomer or subunit described herein can comprise a G, S, or A at a residue corresponding to position 19 of I-CreI (WO 2009001159), a Y, R, K, or D at a residue corresponding to position 66 of I-CreI and/or an E, Q, or K at a residue corresponding to position 80 of I-CreI (US8021867). For polynucleotides, a “variant” comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide. One of skill in the art will recognize that variants of the nucleic acids of the embodiments will be constructed such that the open reading frame is maintained. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the embodiments. Variant polynucleotides include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode an engineered meganuclease, or an exogenous nucleic acid molecule, or template nucleic acid of the embodiments. Generally, variants of a particular polynucleotide of the embodiments will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein. Variants of a particular polynucleotide of the embodiments (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by screening the polypeptide for its ability to preferentially bind and cleave recognition sequences found within human mtDNA, such as the MIT 9-10 recognition sequence (SEQ ID NO: 3). Attorney Docket No.: P893391190WO (01242) EXAMPLES This disclosure is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are intended to be encompassed in the scope of the claims that follow the examples below. EXAMPLE 1 Reporter Assay for MIT 9-10 Meganuclease Activity The purpose of this experiment was to determine whether a MIT 9-10 meganuclease could bind and cleave the human MIT 9-10 recognition sequence (SEQ ID NO: 3) in mammalian cells, and to determine whether the MIT 9-10 meganuclease could discriminate against the corresponding sequence found on wild-type mitochondrial DNA (mtDNA) (SEQ ID NO: 5) that spans nucleotides 13,445-13,466. To do this, a meganuclease engineered to bind and cleave the MIT 9-10 recognition sequence, referred to as MIT 9-10x.3 (SEQ ID NO: 7), was evaluated using the CHO cell reporter assay previously described (see, WO/2012/167192) and depicted in Figure 4. Briefly, if the CHO cell gene reporter gets cleaved, it restores GFP. To perform the assays, two CHO cell reporter lines were produced, which carried a non-functional Green Fluorescent Protein (GFP) gene expression cassette integrated into the genome of the cells. The GFP gene in each cell line contains a direct sequence duplication separated by a pair of recognition sequences such that intracellular cleavage of either recognition sequence by the meganuclease would stimulate a homologous recombination event resulting in a functional GFP gene. In the CHO reporter cell lines developed for this study, two recognition sequences were inserted into the GFP gene. One recognition sequence was for the human MIT 9-10 recognition sequence – either the mutant (SEQ ID NO: 3) or wild-type (SEQ ID NO: 5) sequence, which only differ by one base. Cell line number 1 (mutant) contained the mutant allele, while cell line number 2 (wild-type) contained the wild-type allele. The second recognition sequence inserted into both lines was a CHO-23/24 recognition sequence, which is recognized and cleaved by a control meganuclease called “CHO-23/24.” The CHO-23/24 recognition sequence is used as a positive control and standard measure of activity. The CHO reporter cells detailed above were transfected with mRNA encoding the MIT 9- 10x.3 meganuclease. A control sample of CHO reporter cells were transfected with mRNA encoding the CHO-23/24 meganuclease. In each assay, 5e4 CHO reporter cells were transfected with 2.5ng (low dose) of mRNA in a 96-well plate using LIPOFECTAMINE® Attorney Docket No.: P893391190WO (01242) MESSENGERMAX (ThermoFisher) according to the manufacturer’s instructions. The transfected CHO cells were evaluated by image cytometry at 2 days post transfection to determine the percentage of GFP-positive cells compared to an untransfected negative control. This was done for both the wild-type and mutant cell lines to determine specificity of the nuclease for the mutant sequence. The MIT 9-10x.3 meganuclease was able to bind and cleave the MIT 9-10 recognition sequence in the mutant reporter line (Figure 5). Additionally, the MIT 9-10x.3 nuclease was highly specific for the mutant site versus the corresponding wild-type site. Specifically, the MIT 9-10 nuclease yielded ~80% GFP+ cells when tested against the intended (deletion breakpoint) target site but <5% GFP+ cells when tested against the wild-type target sites (Figure 5). In summary, these studies demonstrated that the early-generation MIT 9-10x.3 meganuclease could efficiently and selectively bind and cleave the MIT 9-10 recognition sequence in cells. EXAMPLE 2 Localization of Mitochondria-Targeted MIT 9-10 Meganucleases The purpose of this experiment was to visualize MIT 9-10x.3 localization when a mitochondrial transit peptide (MTP) was fused to the N-terminus. The MIT 9-10 protein was further modified to comprise a FLAG tag on its C-terminus. In this study, 6e5 HELA cells were nucleofected with 600ng MTP-MIT 9-10x.3-FLAG mRNA using the Lonza 4D-NucleofectorTM (SE buffer, condition CM-150). At 24 hours post- nucleofection, the cells were stained with 50nM MitoTrackerTM Deep Red FM (ThermoFisher Scientific, M22426) for 30 minutes and then washed with PBS. Cells were then fixed with 4% PFA with HIER for 15 minutes and stained with an anti-FLAG antibody. Cells were imaged using a Zeiss LSM710 confocal microscope using 40x Z-stack images. When fused with a mitochondrial targeting sequence, MIT 9-10x.3 staining appears punctate and overlays with MitoTracker staining (Figure 6). There does not appear to be any nuclear localization of the MIT 9-10x.3 nuclease when attached to the MTP. In summary, this study demonstrates that fusion of an MTP to MIT 9-10 meganucleases effectively localizes the nuclease to mitochondria of cells. EXAMPLE 3 MIT 9-10 Meganuclease Efficacy and Function in Cybrid Cells Attorney Docket No.: P893391190WO (01242) The purpose of this experiment was to show efficacy of the mitochondria-targeting engineered meganuclease MIT 9-10x.3 in a cell line that harbors heteroplasmic levels of the mtDNA common deletion, and to compare its efficacy with a previously characterized mitoTALEN that binds to regions that are far too apart in the wild-type mtDNA for FokI nuclease to dimerize and cleave the DNA (Figure 7A) (Bacman et al.2013. Nat Med 19:1111-1113). The cell line used is a cybrid (i.e., cytoplasmic hybrid) that contains both wild-type and mutant mtDNA comprising the common deletion. The cell line is 91% mutant – that is, 91% of the mtDNA population contains the mutant allele and 9% contains the wildtype allele. In this study, 8e5 cybrid cells were nucleofected with MTP-MIT 9-10x.3 mRNA across a dose titration using the Lonza 4D-NucleofectorTM (SF buffer, condition CA-137). The MTP-MIT 9-10x.3 mRNA doses started at 1e5 RNA copies/cell; this translates to 8e10 RNA copies total, or 94.8ng of RNA. The mRNA was then serially diluted 1:10 down to 1e2 RNA copies/cell. Cells were collected at one day post-nucleofection for gDNA extraction and evaluated for transfection efficiency using a Beckman Coulter CytoFlex S cytometer. Transfection efficiency exceeded 95%. gDNA was isolated using the Macherey Nagel NucleoSpin Blood QuickPure kit. mitoTALEN are expressed from two plasmids, each coding for a mitoTALEN monomer. The plasmids also express either eGFP or mCherry. Transfected cells were sorted 48 hours after transfection and populations expressing eGFP and mCherry (yellow cells), only eGFP (green) or neither fluorescent marker (Blafigck cells) was isolated and had their DNA extracted and analyzed by the 3-primer PCR as illustrated in Figure 7A (see arrows) with one forward-primer and two backward primers of the mtDNA Primer B1 corresponds to a mtDNA region inside the common deletion, whereas primers F1 and B2 flank the deleted region. Primers F1 and B1 only amplify WT–mtDNAs, and primers F and B2 amplify Δ– mtDNAs. F1-(8273-8289)-5’-CCCCTCTACCCCCTCTA-3’ (SEQ ID NO: 28) B1-(9020-9008)-5’-GCCTGCAGTAATGTTAGCGG-3’ (SEQ ID NO: 29) B2-(13720-13705)-5’-GGCTTCCGGCTGCCAG-3’ (SEQ ID NO: 30) Since the vector expressing the MTP-MIT 9-10x.3 meganuclease lacks a fluorescent marker, cells were co-transfected with the MIT 9-10x.3 meganuclease and a plasmid coding for a cytosolic eGFP (ratio of 3:1 of MIT 9-10:eGFP). As shown in Figures 7C and 7D, transfected “green” cells co- expressing the MIT 9-10 nuclease and eGFP had a marked reduction in mutant mtDNA (Figure 7D), which was similar to the reduction obtained with mitoTALEN in the yellow cells carrying both mitoTALENs (Figure 7C). Attorney Docket No.: P893391190WO (01242) Therefore, it was observed in this study that heteroplasmy was very effectively shifted in cybrid cells using a mitochondria-targeted MIT 9-10 meganuclease described herein. EXAMPLE 4 Evaluation of Mitochondria-Targeted MIT 9-10 Meganucleases in FlpIn CHO Cells The purpose of this experiment was to evaluate several MIT 9-10 meganucleases for (1) activity against the mutant target site and (2) specificity against the corresponding wild-type sequence in an in vitro model. This was done using two FlpIn CHO cell lines that contain a portion of the human mitochondrial genome integrated onto the nuclear chromosome. The integrated sequence contains either the wild-type recognition sequence (SEQ ID NO: 5) or the mutant recognition sequence (i.e., MIT 9-10; SEQ ID NO: 3), as well as surrounding mtDNA sequence. The mutant and wild-type binding sites only differ by one nucleotide and therefore meganuclease specificity is paramount, as the objective is to generate an engineered meganuclease that can cleave the mutant sequence at high efficiency while not cleaving the wild-type sequence. Specificity and potency were evaluated by droplet digital PCR (ddPCR) by calculating insertion/deletion (indel) formation at each site. The engineered meganucleases compared in this study were MIT 9-10x.3 (SEQ ID NO: 7) and MIT 9-10L.90 (SEQ ID NO: 8), an engineered meganuclease that was further optimized from the MIT 9-10x.3 sequence. FlpIn CHO lines were made using the Flp-InTM system from ThermoFisher Scientific. The integration cassette contained either the MIT 9-10 recognition sequence or the wild-type sequence, as well as surrounding mtDNA sequence. To compare specificity and potency of the two tested MIT 9-10 meganucleases, 6e5 FlpIn CHO cells were nucleofected using the Lonza 4D- NucleofectorTM at MIT 9-10 meganuclease mRNA doses of either 500ng, 50ng, or 5ng (SF buffer, condition EN-138). Cells were collected at two days post-nucleofection for gDNA extraction and evaluated for transfection efficiency using a Beckman Coulter CytoFlex S cytometer. Transfection efficiency exceeded 95% for both cell lines. gDNA was isolated using the Macherey Nagel NucleoSpin Blood QuickPure kit. Droplet digital PCR (ddPCR) was utilized to determine indel frequency at both the MIT 9- 10 mutant and wild-type sites using P1/P2, F1, and R1 to generate an amplicon surrounding the binding site, as well as P3, F2, R2 to generate a reference amplicon that acts as a genomic counter. The ratio of the two amplicons should be equal in an un-treated population and drop relative to indel formation at the binding site in treated samples. Amplifications were multiplexed in a 24µL reaction containing 1x ddPCR Supermix for Probes (no dUTP, BioRad), 250nM of each probe, Attorney Docket No.: P893391190WO (01242) 900nM of each primer, 20 U/µL Kpn-I HF (NEB), and 150ng cellular gDNA. Droplets were generated using a QX100 droplet generator (BioRad). Cycling conditions were as follows: 1 cycle of 95˚C (2˚C/s ramp) for 10 minutes, 45 cycles of 94˚C (2˚C/s ramp) for 10 seconds, 59.2˚C (2˚C/s ramp) for 30 seconds, 72C (0.2˚C/s ramp) for 1 minute 30 seconds, 1 cycle of 98˚C for 10 minutes, 4˚C hold. Droplets were analyzed using a QX200 droplet reader (BioRad) and QuantaSoft analysis software (BioRad) was used to acquire and analyze data. The following ddPCR primers were used in the assay: F1: CGTATGGCCCACCATAAT (SEQ ID NO: 31) R1: AGTAGAAACCTGTGAGGAAAG (SEQ ID NO: 32) P1 (mutant): AATGGTGAGGGAGGTAGGTGGTA (SEQ ID NO: 33) P2 (WT): TACCACCAACCTCCCT (SEQ ID NO: 34) P3: AGCAGTTCTACCGTACAACCCTAACA (SEQ ID NO: 35) F2: GGCAGTTGAGGTGGATTA (SEQ ID NO: 36) R2: GGAATGCGGTAGTAGTTAGG (SEQ ID NO: 37) The two MIT 9-10 meganucleases were evaluated for indel formation in both the mutant and wild- type sequences at three mRNA doses. Both of the MIT 9-10 meganucleases exhibited activity at the wild-type site at a very high mRNA dose (500ng) (Figure 8). At a dose of 50ng of mRNA, MIT 9-10x.3 generated 78.7% indels in the mutant cell line and only 2.7% indels in the wild-type cell line. At the same dose, MIT 9-10L.90 generated 91.1% indels in the mutant line and 16.2% indels in the wild-type line. At a dose of 5ng of mRNA, MIT 9-10x.3 generated 29.2% indels in the mutant line and 1.9% indels in the wild-type line. At the same dose, MIT 9-10L.90 generated 69.6% indels in the mutant line and 3.2% indels in the wild-type line. Together, these data suggest that while MIT 9-10L.90 is a more potent nuclease, MIT 9- 10x.3 appears to be more specific for the mutant recognition sequence found with the common deletion. EXAMPLE 5 Optimization and Evaluation of Additional MIT 9-10 Meganucleases The purpose of this experiment was to determine whether further-optimized MIT 9-10 meganucleases, referred to as MIT 9-10L.209 (SEQ ID NO: 9) and MIT 9-10L.210 (SEQ ID NO: Attorney Docket No.: P893391190WO (01242) 10), could bind and cleave the MIT 9-10 recognition sequence (SEQ ID NO: 3) in mammalian cells, and to determine how well they discriminate between the MIT 9-10 recognition sequence and the corresponding wild-type sequence (SEQ ID NO: 5). MIT 9-10L.209 and MIT 9-10L.210 were each optimized from the MIT 9-10L.90 meganuclease. To do this, each of the engineered meganucleases was evaluated using the CHO cell reporter assay depicted in Figure 3 and described in Example 1. The CHO reporter cells were transfected with mRNA encoding each of the MIT 9-10L.90, MIT 9-10L.209, and MIT 9-10L.210 meganucleases. A control sample of CHO reporter cells were transfected with mRNA encoding the CHO-23/24 meganuclease. In each assay, 5e4 CHO reporter cells comprising the MIT 9-10 recognition sequence (SEQ ID NO: 3) were transfected with 2.5ng (low dose) or 90 ng (high dose) of mRNA in a 96-well plate using LIPOFECTAMINE® MESSENGERMAX (ThermoFisher) according to the manufacturer’s instructions. CHO reporter cells comprising the corresponding wild-type sequence (SEQ ID NO: 5) (shown in Figure 9 as “9A-10”) were transfected with 90 ng (high dose) of mRNA only. The transfected CHO cells were evaluated by image cytometry at 2 days post transfection to determine the percentage of GFP- positive cells compared to an untransfected negative control. This percentage was then normalized to the activity of the positive control CHO 23-24 meganuclease to compare data between experiments. This was done for both the wild-type and mutant cell lines to determine specificity of the nuclease for the mutant sequence. Each of the tested MIT 9-10 meganucleases were able to bind and cleave the MIT 9-10 recognition sequence in the mutant reporter line with a low dose of mRNA as indicated by the light gray bars in Figure 9. Additionally, the MIT 9-10 nucleases were significantly impaired in binding and cleaving the wild-type MIT 9-10 sequence with a high dose of mRNA, as indicated by the dark gray bars in Figure 9, indicating a high level of specificity for the mutant sequence. All of the tested meganucleases had increased activity against the mutant site and/or decreased activity against the wild-type site, and none had increased activity against the wild-type site. As shown, the MIT 9- 10L.209 meganuclease exhibited similar activity against the MIT 9-10 recognition sequence as the earlier-generation MIT 9-10L.90 meganuclease but exhibited substantially lower activity against the wild-type sequence. The MIT 9-10L.210 meganuclease exhibited much greater activity against the MIT 9-10 recognition sequence than the MIT 9-10L.90 meganuclease, and while it also exhibited lower activity against the wild-type sequence, the MIT 9-10L.209 meganuclease exhibited the lowest activity of the three meganucleases evaluated. These studies demonstrated that the further-optimized engineered MIT 9-10 meganucleases could efficiently and selectively bind and cleave the MIT 9-10 recognition sequence in cells, and that both binding and selectivity could be improved relative to earlier-generation meganucleases. Attorney Docket No.: P893391190WO (01242) EXAMPLE 6 Evaluation of MIT 9-10 Meganucleases For Shifting mtDNA Heteroplasmy In this study, MIT 9-10 meganucleases were tested to determine if they could shift mtDNA heteroplasmy in human cells. Initially, to determine if these meganucleases could appropriately localize to the mitochondria where the heteroplasmic mtDNA is located, Cos-7 cells were transfected with C-terminal FLAG-tagged MIT 9-10L.209, MIT 9-10L.210, and MIT 9-10L.90 meganucleases that each comprised an N-terminal MTP. Immunocytochemistry was performed after 24h with an Anti –FLAG antibody and mitotracker red. The results of the immunofluorescent staining showed a strict mitochondrial localization of all three of the tested MIT 9-10 meganucleases (Figure 10). Next, each of the MIT 9-10 meganucleases were tested in human osteosarcoma cells heteroplasmic for the mtDNA common deletion (BH10.9 cells clone 1-C-7-65% heteroplasmy mutant) to determine whether they could shift mtDNA heteroplasmy. Approximately two million BH10.9 cells were transfected with two independent plasmids: 25 ug of plasmids encoding the MIT 9-10L.209, MIT 9-10L.210, or MIT 9-10L.90 meganucleases each together with 5 ug of a plasmid carrying GFP (pLenti-GFP). Transfection was carried out with the GenJet DNA in vitro transfection reagent version II (#SL100489; SignaGen Laboratories. Each of the MIT 9-10 meganucleases in this experiment comprised an N-terminal MTP (Cox8Su9; SEQ ID NO: 25) and a C-terminal FLAG Tag. After 48 hours, cells were FACS sorted for eGFP expression using a FACSAria IIu by gating on single cell fluorescence using 561 nm laser and 600LP with a 530/30 filter set for eGFP. Cells expressing the green marker were labeled as “green” because they were expected to co-express the MIT 9-10 meganucleases together with the plasmid carrying GFP. Cells were also isolated that did not express the fluorescent markers, which were labeled as “black” and therefore would not be expected to have any meganuclease expression. Cells were expanded and DNA was extracted at day 0 (day of the sorting) and days 4, 8 and 12 days after sorting from both green and black populations. Figure 11 shows the summary of three independent experiments demonstrating a shift in mtDNA heteroplasmy, which is indicated by the percentage of change in the mutant in green cells normalized to black cells (non-transfected). The reduction in heteroplasmy was approximately 40% in cells treated with the MIT 9-10L.209 meganuclease, which persisted over days 4, 8, and 12. The MIT 9-10L.210 and MIT 9-10L.90 meganucleases also showed a decrease in the mutant mtDNA but to a lesser extent. Figure 12 shows a corresponding shift in heteroplasmy in the green cells normalized to control heteroplasmic cells prior to transfection. Attorney Docket No.: P893391190WO (01242) DNA was also extracted from some of these cells using the NucleoSpin Tissue XS kit (#740901.50; Macherey–Nagel, Clontech) to perform semi-quantitative PCR analysis. DNA from cells transfected and sorted at day 0 (day of the sorting) and after 4, 8 and 12 days after sorting were analyzed for heteroplasmy with a 3-primer PCR technique using the following primers of mtDNA: F–m.8273-8289, B1–m.9028-9008 and B2–m.13720-13705 (see Example 3 above). Analysis of the semi quantitative PCR with Image J program showed that all meganucleases were effective in reducing the mtDNA deleted molecules and changing mtDNA heteroplasmy towards a predominance of wild-type mtDNA as shown in Figure 13. In conclusion, each of the MIT 9-10 meganucleases evaluated in this study produced a shift in heteroplasmy levels towards wild-type. Of the meganucleases tested, the MIT 9-10L.209 meganuclease showed the most rapid and persistent shift in heteroplasmy, which was still observed 12 weeks after transfection. EXAMPLE 7 Evaluation of MIT 9-10 Meganucleases For Shifting mtDNA Heteroplasmy after mRNA Electroporation of Cybrid Cells The MIT 9-10x.3 and MIT 9-10L.209 meganucleases, each linked to an MTP (SEQ ID NO: 25) at their N-terminus, were cloned into a plasmid vector containing a T7 promoter, 5' and 3' untranslated regions, and a polyT repeat to serve as a template for a >100 bp polyA tail. Cybrid cells carrying approximately 80% mutant mtDNA (Clone CD-H11) were electroporated with mRNA coding for 2 different meganucleases, MIT 9-10x.3 or MIT 9-10L.209, or GFP-mRNA as a control. Two different concentrations of mRNA were used: 1e4 and 1e5 RNA copies/cell. Electroporation was done at 1250V, 30sec, 1 pulse with Neon transfection system (Invitrogen). As shown in Figure 14, a shift in heteroplasmy analyzed by 3-primer PCR (as described previously in Figure 7) was observed after 4 days of electroporation with both mRNA- MIT 9- 10x.3 and MIT 9-10 L.209, but not with mRNA-GFP. The decrease in mutant mtDNA was persistent after 7 (not shown) and 14 days after electroporation. Similar analysis was performed by dPCR after 4, 7 and 14 days after electroporation. Deleted molecules and WT molecules were evaluated using 3 sets of primer/probes describe below) (Figure 15). The following dPCR primers were used in the assay: Probe hND4-TAMRA (inside the deletion): CCCTTCCTTGTACTATCCCTA (11567-11587) (SEQ ID NO: 38) PrF=CGCCTCACACTCATTCTCAA (11522-11541) (SEQ ID NO: 39) Attorney Docket No.: P893391190WO (01242) PrB=GTAGGCAGATGGAGCTTGTTA (11621-11600) (SEQ ID NO: 40) Probe hCom deletion-FAM (deleted molecules): TGGCAGCCTAGCATTAGC (13491-13608) (SEQ ID NO: 41) PrF: CACTATTCCTCATCACCCAACTAA (8439-8462) (SEQ ID NO: 42) PrB: TGTGGTCTTTGGAGTAGAAACC (13549-13528) (SEQ ID NO: 43) Probe hND1-HEX (Ref mitochondria DNA): AAGGGTGGAGAGGTTAAAG (3795-3813) (SEQ ID NO: 44) PrF: GAAGTCACCCTAGCCATCATTC (3745-3766) (SEQ ID NO: 45) PrB: GCAGGAGTAATCAGAGGTGTTC (3847-3826) (SEQ ID NO: 46) Probe mus18s-Cy5 (Ref nuclear DNA): AGAAACGGCTACCACATCC (SEQ ID NO: 47) PrF: CGTCTGCCCTATCAACTTT (SEQ ID NO: 48) PrR: CCTCGAAAGAGTCCTGTATTG (SEQ ID NO: 49) Deleted molecules were decreased when using MIT 9-10x.3 or MIT 9-10L.209 but not GFP mRNA while WT molecules were increased (Figure 16). Copy number was also evaluated showing the high mRNA meganuclease mRNA dose generated a transient depletion in mtDNA at 4 days, that was recovered after 14 days (Figure 17). EXAMPLE 8 Evaluation of MIT 9-10 Meganucleases For Respiration Heteroplasmy after mRNA electroporation of cybrid cells Cellular respiration of cybrids cells carrying the “common deletion” (clone CD-H11) was evaluated after electroporation with mRNA encoding the MIT 9-10x.3 or MIT 9-10L.209 meganucleases, each linked to an MTP (SEQ ID NO: 25) at their N-terminus, or mRNA-GFP used as a control after 21 days. Oxygen consumption rates were measured using the Seahorse XFe96 Analyzer (Agilent Technologies). OCR values were normalized to proteins levels and data analysis was conducted using Wave software (Agilent Technologies). A clear recovery was observed in the cells treated with mRNA encoding the mitochondria- targeted MIT 9-10x.3 or MIT 9-10L.209 meganucleases compared to mRNA GFP (1e4 copies/cell) (Figure 18A). Changes in the extracellular acidification rate (ECAR) over time are shown in Figure Attorney Docket No.: P893391190WO (01242) 18B. Figure 19 shows that both basal and maximal respiration values were significantly increased in cells treated with the mitochondria-targeted MIT 9-10 meganucleases.
Attorney Docket No.: P893391190WO (01242) Sequence Listing SEQ ID NO: 1 MNTKYNKEFLLYLAGFVDGDGSIIAQIKPNQSYKFKHQLSLAFQVTQKTQRRWFLDKLVD EIGVGYVRDRGSVSDYILSEIKPLHNFLTQLQPFLKLKQKQANLVLKIIWRLPSAKESPDKF LEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKKKSSP SEQ ID NO: 2 LAGLIDADG SEQ ID NO: 3 CTACCTCCCTCACCATTGGCAG SEQ ID NO: 4 CTGCCAATGGTGAGGGAGGTAG SEQ ID NO: 5 CAACCTCCCTCACCATTGGCAG SEQ ID NO: 6 CTGCCAATGGTGAGGGAGGTTG SEQ ID NO: 7 MNTKYNKEFLLYLAGFVDGDGSICASIRPSQKTKFKHVLRLIFAVSQKTQRRWFLDKLVDE IGVGYVTDCGSVSQYTLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKFL EVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSAASSASSSPGSGISEA LRAGAGSGTGYNKEFLLYLAGFVDGDGSIFASIHPQQDLKFKHDLRLSFRVHQKTQRRWF LDKLVDEIGVGYVYDSGSVSFYSLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAK ESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKKKSSP Attorney Docket No.: P893391190WO (01242) SEQ ID NO: 8 MNTKYNKEFLLYLAGFVDGDGSICASIRPRQDLKFKHGLRLIFAVTQKTQRRWFLDKLVD EIGVGYVTDGGSVSQYTLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKF LEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSAASSASSSPGSGISE ALRAGAGSGTGYNKEFLLYLAGFVDGDGSIFASIHPQQDLKFKHDLRLSFRVHQKTQRRW FLDKLVDEIGVGYVYDSGSVSFYSLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSA KESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKKKSSP SEQ ID NO: 9 MNTKYNKEFLLYLAGFVDADGSICASIRPMQDFKFKHQLRLIFAVTQKTQRRWFLDKLVD EIGVGYVTDGGSVSQYTLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKF LEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSAASSASSSPGSGISE ALRAGAGSGTGYNKEFLLYLAGFVDGDGSIFASIHPQQDLKFKHDLRLSFRVHQKTQRRW FLDKLVDEIGVGYVYDSGSVSFYSLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSA KESPDKFLEVCTWVDQIAALNDSRTRKTTSETVRAVLDSLSEKKKSSP SEQ ID NO: 10 MNTKYNKEFLLYLAGFVDGDGSICASIRPRQDLKFKHGLRLIFAVTQKTQRRWFLDKLVD EIGVGYVTDGGSVSQYTLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKF LEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLPGSVGGLSPSQASSAASSASSSPGSGISE ALRAGAGSGTGYNKEFLLYLAGFVDGDGSIFASIHPQQDLKFKHDLRLSFRVHQKTQRRW FLDKLVDEIGVGYVYDSGSVSFYSLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSA KESPDKFLEVCTWVDQIAALNDSKTRKTTSETVRAVLDSLSEKKKSSP SEQ ID NO: 11 KEFLLYLAGFVDGDGSIFASIHPQQDLKFKHDLRLSFRVHQKTQRRWFLDKLVDEIGVGYV YDSGSVSFYSLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKFLEVCTWV DQIAALNDSKTRKTTSETVRAVLD SEQ ID NO: 12 KEFLLYLAGFVDGDGSIFASIHPQQDLKFKHDLRLSFRVHQKTQRRWFLDKLVDEIGVGYV YDSGSVSFYSLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKFLEVCTWV DQIAALNDSKTRKTTSETVRAVLD SEQ ID NO: 13 Attorney Docket No.: P893391190WO (01242) KEFLLYLAGFVDGDGSIFASIHPQQDLKFKHDLRLSFRVHQKTQRRWFLDKLVDEIGVGYV YDSGSVSFYSLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKFLEVCTWV DQIAALNDSRTRKTTSETVRAVLD SEQ ID NO: 14 KEFLLYLAGFVDGDGSIFASIHPQQDLKFKHDLRLSFRVHQKTQRRWFLDKLVDEIGVGYV YDSGSVSFYSLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKFLEVCTWV DQIAALNDSKTRKTTSETVRAVLD SEQ ID NO: 15 KEFLLYLAGFVDGDGSICASIRPSQKTKFKHVLRLIFAVSQKTQRRWFLDKLVDEIGVGYV TDCGSVSQYTLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKFLEVCTWV DQIAALNDSKTRKTTSETVRAVLD SEQ ID NO: 16 KEFLLYLAGFVDGDGSICASIRPRQDLKFKHGLRLIFAVTQKTQRRWFLDKLVDEIGVGYV TDGGSVSQYTLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKFLEVCTWV DQIAALNDSKTRKTTSETVRAVLD SEQ ID NO: 17 KEFLLYLAGFVDADGSICASIRPMQDFKFKHQLRLIFAVTQKTQRRWFLDKLVDEIGVGYV TDGGSVSQYTLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKFLEVCTWV DQIAALNDSKTRKTTSETVRAVLD SEQ ID NO: 18 KEFLLYLAGFVDGDGSICASIRPRQDLKFKHGLRLIFAVTQKTQRRWFLDKLVDEIGVGYV TDGGSVSQYTLSQIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKFLEVCTWV DQIAALNDSKTRKTTSETVRAVLD SEQ ID NO: 19 ATGAATACAAAATATAATAAAGAGTTCTTACTCTACTTAGCAGGGTTTGTAGACGGTG ACGGTTCCATCTGTGCAAGTATCAGGCCTAGTCAAAAGACGAAGTTCAAGCACGTGCT GAGGCTCATTTTTGCTGTCTCTCAAAAGACACAGCGCCGTTGGTTCCTCGACAAGCTGG TGGACGAGATCGGTGTGGGTTACGTGACGGACTGTGGCAGCGTCTCCCAGTACACGCT GAGCCAGATCAAGCCTTTGCATAATTTTTTAACACAACTACAACCTTTTCTAAAACTAA Attorney Docket No.: P893391190WO (01242) AACAAAAACAAGCAAATTTAGTTTTAAAAATTATTGAACAACTTCCGTCAGCAAAAGA ATCCCCGGACAAATTCTTAGAAGTTTGTACATGGGTGGATCAAATTGCAGCTCTGAAT GATTCGAAGACGCGTAAAACAACTTCTGAAACCGTTCGTGCTGTGCTAGACAGTTTAC CAGGATCCGTGGGAGGTCTATCGCCATCTCAGGCATCCAGCGCCGCATCCTCGGCTTC CTCAAGCCCGGGTTCAGGGATCTCCGAAGCACTCAGAGCTGGAGCAGGTTCCGGCACT GGATACAACAAGGAATTCCTGCTCTACCTGGCGGGCTTCGTCGACGGGGACGGCTCCA TCTTTGCCTCGATCCATCCTCAGCAAGATCTTAAGTTCAAGCACGATCTGCGTCTCTCT TTCCGTGTCCATCAGAAGACACAGCGCCGTTGGTTCCTCGACAAGCTGGTGGACGAGA TCGGTGTGGGTTACGTGTATGACTCGGGCAGCGTCTCCTTTTACTCTCTGTCCCAAATC AAGCCTCTGCACAACTTCCTGACCCAGCTCCAGCCCTTCCTGAAGCTCAAGCAGAAGC AGGCCAACCTCGTGCTGAAGATCATCGAGCAGCTGCCCTCCGCCAAGGAATCCCCGGA CAAGTTCCTGGAGGTGTGCACCTGGGTGGACCAGATCGCCGCTCTGAACGACTCCAAG ACCCGCAAGACCACTTCCGAAACCGTCCGCGCCGTTCTAGACAGTCTCTCCGAGAAGA AGAAGTCGTCCCCC SEQ ID NO: 20 ATGGCACCGAAGAAGAAGCGCAAGGTGCATATGAATACAAAATATAATAAAGAGTTC TTACTCTACTTAGCAGGGTTTGTAGACGGTGACGGTTCCATCTGTGCCAGTATCAGACC TCGTCAAGATCTTAAGTTCAAGCACGGGCTGAGGCTCATCTTCGCTGTCACCCAAAAG ACACAGCGCCGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGA CGGACGGTGGCAGCGTCTCCCAGTACACGCTGTCCCAGATCAAGCCTTTGCATAATTTT TTAACACAACTACAACCTTTTCTAAAACTAAAACAAAAACAAGCAAATTTAGTTTTAA AAATTATTGAACAACTTCCGTCAGCAAAAGAATCCCCGGACAAATTCTTAGAAGTTTG TACATGGGTGGATCAAATTGCAGCTCTGAATGATTCGAAGACGCGTAAAACAACTTCT GAAACCGTTCGTGCTGTGCTAGACAGTTTACCAGGATCCGTGGGAGGTCTATCGCCAT CTCAGGCATCCAGCGCCGCATCCTCGGCTTCCTCAAGCCCGGGTTCAGGGATCTCCGA AGCACTCAGAGCTGGAGCAGGTTCCGGCACTGGATACAACAAGGAATTCCTGCTCTAC CTGGCGGGCTTCGTCGACGGGGACGGCTCCATCTTTGCCTCGATCCATCCTCAGCAAG ATCTTAAGTTCAAGCACGATCTGCGTCTCTCTTTCCGTGTCCATCAGAAGACACAGCGC CGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGTATGACTCGG GCAGCGTCTCCTTTTACTCTCTGTCCCAAATCAAGCCTCTGCACAACTTCCTGACCCAG CTCCAGCCCTTCCTGAAGCTCAAGCAGAAGCAGGCCAACCTCGTGCTGAAGATCATCG AGCAGCTGCCCTCCGCCAAGGAATCCCCGGACAAGTTCCTGGAGGTGTGCACCTGGGT GGACCAGATCGCCGCTCTGAACGACTCCAAGACCCGCAAGACCACTTCCGAAACCGTC CGCGCCGTTCTAGACAGTCTCTCCGAGAAGAAGAAGTCGTCCCCC Attorney Docket No.: P893391190WO (01242) SEQ ID NO: 21 ATGGCACCGAAGAAGAAGCGCAAGGTGCATATGAATACAAAATATAATAAAGAGTTC TTACTCTACTTAGCAGGGTTTGTAGACGCTGACGGTTCCATCTGTGCCTCCATCAGACC TATGCAAGATTTTAAGTTCAAGCACCAGCTGCGTCTCATCTTCGCTGTCACCCAAAAGA CACAGCGCCGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGAC GGACGGTGGCAGCGTCTCCCAGTACACGCTGTCCCAGATCAAGCCTTTGCATAATTTTT TAACACAACTACAACCTTTTCTAAAACTAAAACAAAAACAAGCAAATTTAGTTTTAAA AATTATTGAACAACTTCCGTCAGCAAAAGAATCCCCGGACAAATTCTTAGAAGTTTGT ACATGGGTGGATCAAATTGCAGCTCTGAATGATTCGAAGACGCGTAAAACAACTTCTG AAACCGTTCGTGCTGTGCTAGACAGTTTACCAGGATCCGTGGGAGGTCTATCGCCATCT CAGGCATCCAGCGCCGCATCCTCGGCTTCCTCAAGCCCGGGTTCAGGGATCTCCGAAG CACTCAGAGCTGGAGCAGGTTCCGGCACTGGATACAACAAGGAATTCCTGCTCTACCT GGCGGGCTTCGTCGACGGGGACGGCTCCATCTTTGCCTCGATCCATCCTCAGCAAGAT CTTAAGTTCAAGCACGATCTGCGTCTCTCTTTCCGTGTCCATCAGAAGACACAGCGCCG TTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGTATGACTCGGGC AGCGTCTCCTTTTACTCTCTGTCCCAGATCAAGCCTCTGCACAACTTCCTGACCCAGCT CCAGCCCTTCCTGAAGCTCAAGCAGAAGCAGGCCAACCTCGTGCTGAAGATCATCGAG CAGCTGCCCTCCGCCAAGGAATCCCCGGACAAGTTCCTGGAGGTGTGCACCTGGGTGG ACCAGATCGCCGCTCTGAACGACTCCAGGACCCGCAAGACCACTTCCGAAACCGTCCG CGCCGTTCTAGACAGTCTCTCCGAGAAGAAGAAGTCGTCCCCC SEQ ID NO: 22 ATGGCACCGAAGAAGAAGCGCAAGGTGCATATGAATACAAAATATAATAAAGAGTTC TTACTCTACTTAGCAGGGTTTGTAGACGGTGACGGTTCCATCTGTGCCAGTATCAGACC TCGTCAAGATCTTAAGTTCAAGCACGGGCTGAGGCTCATCTTCGCTGTCACCCAAAAG ACACAGCGCCGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGA CGGACGGTGGCAGCGTCTCCCAGTACACGCTGTCCCAGATCAAGCCTTTGCATAATTTT TTAACACAACTACAACCTTTTCTAAAACTAAAACAAAAACAAGCAAATTTAGTTTTAA AAATTATTGAACAACTTCCGTCAGCAAAAGAATCCCCGGACAAATTCTTAGAAGTTTG TACATGGGTGGATCAAATTGCAGCTCTGAATGATTCGAAGACGCGTAAAACAACTTCT GAAACCGTTCGTGCTGTGCTAGACAGTTTACCAGGATCCGTGGGAGGTCTATCGCCAT CTCAGGCATCCAGCGCCGCATCCTCGGCTTCCTCAAGCCCGGGTTCAGGGATCTCCGA AGCACTCAGAGCTGGAGCAGGTTCCGGCACTGGATACAACAAGGAATTCCTGCTCTAC CTGGCGGGCTTCGTCGACGGGGACGGCTCCATCTTTGCCTCGATCCATCCTCAGCAAG Attorney Docket No.: P893391190WO (01242) ATCTTAAGTTCAAGCACGATCTGCGTCTCTCTTTCCGTGTCCATCAGAAGACACAGCGC CGTTGGTTCCTCGACAAGCTGGTGGACGAGATCGGTGTGGGTTACGTGTATGACTCGG GCAGCGTCTCCTTTTACTCTCTGTCCCAGATCAAGCCTCTGCACAACTTCCTGACCCAG CTCCAGCCCTTCCTGAAGCTCAAGCAGAAGCAGGCCAACCTCGTGCTGAAGATCATCG AGCAGCTGCCCTCCGCCAAGGAATCCCCGGACAAGTTCCTGGAGGTGTGCACCTGGGT GGACCAGATCGCCGCTCTGAACGACTCCAAGACCCGCAAGACCACTTCCGAAACCGTC CGCGCCGTTCTAGACAGTCTCTCCGAGAAGAAGAAGTCGTCCCCC SEQ ID NO: 23 MSVLTPLLLRGLTGSARRLPVPRAKIHSLPPEGKL SEQ ID NO: 24 MASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQ AFQ SEQ ID NO: 25 MSVLTPLLLRGLTGSARRLPVPRAKIHSLPPEGKLMASTRVLASRLASQMAASAKVA RPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQ SEQ ID NO: 26 VDEMTKKFGTLTIHDTEK SEQ ID NO: 27 LGAGLGALGL SEQ ID NO: 28 CCCCTCTACCCCCTCTA SEQ ID NO: 29 GCCTGCAGTAATGTTAGCGG SEQ ID NO: 30 GGCTTCCGGCTGCCAG SEQ ID NO: 31 Attorney Docket No.: P893391190WO (01242) CGTATGGCCCACCATAAT SEQ ID NO: 32 AGTAGAAACCTGTGAGGAAAG SEQ ID NO: 33 AATGGTGAGGGAGGTAGGTGGTA SEQ ID NO: 34 TACCACCAACCTCCCT SEQ ID NO: 35 AGCAGTTCTACCGTACAACCCTAACA SEQ ID NO: 36 GGCAGTTGAGGTGGATTA SEQ ID NO: 37 GGAATGCGGTAGTAGTTAGG SEQ ID NO: 38 CCCTTCCTTGTACTATCCCTA SEQ ID NO: 39 CGCCTCACACTCATTCTCAA SEQ ID NO: 40 GTAGGCAGATGGAGCTTGTTA SEQ ID NO: 41 TGGCAGCCTAGCATTAGC SEQ ID NO: 42 CACTATTCCTCATCACCCAACTAA Attorney Docket No.: P893391190WO (01242) SEQ ID NO: 43 TGTGGTCTTTGGAGTAGAAACC SEQ ID NO: 44 AAGGGTGGAGAGGTTAAAG SEQ ID NO: 45 GAAGTCACCCTAGCCATCATTC SEQ ID NO: 46 GCAGGAGTAATCAGAGGTGTTC SEQ ID NO: 47 AGAAACGGCTACCACATCC SEQ ID NO: 48 CGTCTGCCCTATCAACTTT SEQ ID NO: 49 CCTCGAAAGAGTCCTGTATTG

Claims

Attorney Docket No.: P893391190WO (01242) CLAIMS 1. An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 in mitochondrial genomes of a eukaryotic cell, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region. 2. The engineered meganuclease of claim 1, wherein said HVR1 region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 7-10. 3. The engineered meganuclease of claim 1 or claim 2, wherein said HVR1 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-10. 4. The engineered meganuclease of any one of claims 1-3, wherein said HVR1 region comprises residues 215-270 of any one of SEQ ID NOs: 7-10. 5. The engineered meganuclease of any one of claims 1-4, wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 198-344 of any one of SEQ ID NOs: 7-10. 6. The engineered meganuclease of any one of claims 1-5, wherein said first subunit comprises a residue corresponding to residue 271 of any one of SEQ ID NOs: 7-10. 7. The engineered meganuclease of any one of claims 1-6, wherein said first subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 9. 8. The engineered meganuclease of any one of claims 1-7, wherein said first subunit comprises residues 198-344 of any one of SEQ ID NOs: 7-10. 9. The engineered meganuclease of any one of claims 1-8, wherein said HVR2 region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs: 7-10. Attorney Docket No.: P893391190WO (01242) 10. The engineered meganuclease of any one of claims 1-9, wherein said HVR2 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-10. 11. The engineered meganuclease of any one of claims 1-10, wherein said HVR2 region comprises residues 24-79 of any one of SEQ ID NOs: 7-10. 12. The engineered meganuclease of any one of claims 1-11, wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 7-153 of any one of SEQ ID NOs: 7-10. 13. The engineered meganuclease of any one of claims 1-12, wherein said second subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 9. 14. The engineered meganuclease of any one of claims 1-13, wherein said second subunit comprises a residue corresponding to residue 80 of any one of SEQ ID NOs: 7-10. 15. The engineered meganuclease of any one of claims 1-14, wherein said second subunit comprises residues 7-153 of any one of SEQ ID NOs: 7-10. 16. The engineered meganuclease of any one of claims 1-15, wherein said engineered meganuclease is a single-chain meganuclease comprising a linker, wherein said linker covalently joins said first subunit and said second subunit. 17. The engineered meganuclease of any one of claims 1-16, wherein said engineered meganuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 7-10. 18. The engineered meganuclease of any one of claims 1-17, wherein said engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 7-10. 19. An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 in mitochondrial genomes of a eukaryotic cell, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a Attorney Docket No.: P893391190WO (01242) first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region, and wherein said engineered meganucleases comprises an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. 20. An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 in mitochondrial genomes of a eukaryotic cell, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region, and wherein said engineered meganucleases comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 9 or SEQ ID NO: 10. 21. The engineered meganuclease of any one of claims 1-20, wherein said engineered meganuclease is encoded by a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 19-22. 22. The engineered meganuclease of any one of claims 1-21, wherein said engineered meganuclease is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 19-22. 23. The engineered meganuclease of any one of claims 1-22, wherein said engineered meganuclease comprises a mitochondrial transit peptide (MTP). 24. The engineered meganuclease of claim 23, wherein said MTP comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in any one of SEQ ID NOs: 23-25. 25. The engineered meganuclease of claim 23 or claim 24, wherein said MTP comprises an amino acid sequence set forth in any one of SEQ ID NOs: 23-25. 26. The engineered meganuclease of any one of claims 23-25, wherein said MTP is attached to the C-terminus of said engineered meganuclease. Attorney Docket No.: P893391190WO (01242) 27. The engineered meganuclease of any one of claims 23-25, wherein said MTP is attached to the N-terminus of said engineered meganuclease. 28. The engineered meganuclease of any one of claims 23-27, wherein said MTP is fused to said engineered meganuclease. 29. The engineered meganuclease of any one of claims 23-27, wherein said MTP is attached to said engineered meganuclease by a polypeptide linker. 30. The engineered meganuclease of any one of claims 23-29, wherein said engineered meganuclease is attached to a first MTP and a second MTP. 31. The engineered meganuclease of claim 28, wherein said first MTP and/or said second MTP comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in any one of SEQ ID NOs: 23-25. 32. The engineered meganuclease of claim 30 or claim 31, wherein said first MTP and/or said second MTP comprises an amino acid sequence set forth in any one of SEQ ID NOs: 23-25. 33. The engineered meganuclease of any one of claims 30-32, wherein said first MTP and said second MTP are identical. 34. The engineered meganuclease of any one of claims 30-32, wherein said first MTP and said second MTP are not identical. 35. The engineered meganuclease of any one of claims 30-34, wherein said first MTP and/or said second MTP is fused to said engineered meganuclease. 36. The engineered meganuclease of any one of claims 30-34, wherein said first MTP and/or said second MTP is attached to said engineered meganuclease by a polypeptide linker. 37. The engineered meganuclease of any one of claims 1-36, wherein said engineered meganuclease comprises a nuclear export sequence (NES). Attorney Docket No.: P893391190WO (01242) 38. The engineered meganuclease of claim 37, wherein said NES comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 26 or 27. 39. The engineered meganuclease of claim 37 or claim 38, wherein said NES comprises an amino acid sequence set forth in SEQ ID NO: 26 or 27. 40. The engineered meganuclease of any one of claims 37-39, wherein said NES is attached at the N-terminus of said engineered meganuclease. 41. The engineered meganuclease of any one of claims 37-39, wherein said NES is attached at the C-terminus of said engineered meganuclease. 42. The engineered meganuclease of any one of claims 37-41, wherein said NES is fused to said engineered meganuclease. 43. The engineered meganuclease of any one of claims 37-41, wherein said NES is attached to said engineered meganuclease by a polypeptide linker. 44. The engineered meganuclease of any one of claims 1-36, wherein said engineered meganuclease comprises a first NES and a second NES. 45. The engineered meganuclease of claim 44, wherein said first NES is attached at the N-terminus of said engineered meganuclease, and wherein said second NES is attached at the C- terminus of said engineered meganuclease. 46. The engineered meganuclease of claim 44 or claim 45, wherein said first NES and/or said second NES comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 26 or 27. 47. The engineered meganuclease of any one of claims 44-46, wherein said first NES and/or said second NES comprises an amino acid sequence set forth in SEQ ID NO: 26 or 27. 48. The engineered meganuclease of any one of claims 44-47, wherein said first NES and said second NES are identical. Attorney Docket No.: P893391190WO (01242) 49. The engineered meganuclease of any one of claims 44-47, wherein said first NES and said second NES are not identical. 50. The engineered meganuclease of any one of claims 44-49, wherein said first NES and/or said second NES is fused to said engineered meganuclease. 51. The engineered meganuclease of any one of claims 44-49, wherein said first NES and/or said second NES is attached to said engineered meganuclease by a polypeptide linker. 52. A polynucleotide comprising a nucleic acid sequence encoding said engineered meganuclease of any one of claims 1-51. 53. The polynucleotide of claim 52, wherein said polynucleotide is an mRNA. 54. A recombinant DNA construct comprising a polynucleotide comprising a nucleic acid sequence encoding said engineered meganuclease of any one of claims 1-51. 55. The recombinant DNA construct of claim 54, wherein said recombinant DNA construct encodes a recombinant virus genome comprising said polynucleotide. 56. The recombinant DNA construct of claim 54, wherein said recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adeno-associated virus (AAV). 57. The recombinant DNA construct of claim 55 or claim 56, wherein said recombinant virus is a recombinant AAV. 58. The recombinant DNA construct of claim 56 or claim 57, wherein said recombinant AAV is a muscle-tropic recombinant AAV or a central nervous system (CNS)-tropic recombinant AAV. 59. The recombinant DNA construct of any one of claims 56-58, wherein said recombinant AAV has an AAV9 capsid, a MyoAAV capsid or an AAVMYO capsid. Attorney Docket No.: P893391190WO (01242) 60. The recombinant DNA construct of any one of claims 54-59, wherein said polynucleotide comprises a promoter operably linked to said nucleic acid sequence encoding said engineered meganuclease. 61. The recombinant DNA construct of claim 60, wherein said promoter is a constitutive promoter. 62. The recombinant DNA construct of claim 61, wherein said constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. 63. The recombinant DNA construct of claim 60, wherein said promoter is a cell- specific promoter. 64. The recombinant DNA construct of claim 63, wherein said promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter. 65. The recombinant DNA construct of claim 63 or claim 64, wherein said promoter is an MHCK7 promoter, a truncated MCK (tMCK) promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter. 66. A recombinant virus comprising a polynucleotide comprising a nucleic acid sequence encoding said engineered meganuclease of any one of claims 1-51. 67. The recombinant virus of claim 66, wherein said recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant AAV. 68. The recombinant virus of claim 66 or claim 67, wherein said recombinant virus is a recombinant AAV. 69. The recombinant virus of claim 67 or claim 68, wherein said recombinant AAV is a muscle-tropic recombinant AAV or a CNS-tropic recombinant AAV. 70. The recombinant virus of any one of claims 67-69, wherein said recombinant AAV has an AAV9 capsid, a MyoAAV capsid or an AAVMYO capsid. Attorney Docket No.: P893391190WO (01242) 71. The recombinant virus of any one of claims 66-70, wherein said polynucleotide comprises a promoter operably linked to said nucleic acid sequence encoding said engineered meganuclease. 72. The recombinant virus of claim 71, wherein said promoter is a constitutive promoter. 73. The recombinant virus of claim 72, wherein said constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. 74. The recombinant virus of claim 71, wherein said promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter. 75. The recombinant virus of claim 74, wherein said promoter is an MHCK7 promoter, a tMCK promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter. 76. The recombinant virus of claim 71, wherein said promoter is a CNS cell-specific promoter. 77. A lipid nanoparticle composition comprising lipid nanoparticles comprising a polynucleotide, wherein said polynucleotide comprises a nucleic acid sequence encoding said engineered meganuclease of any one of claims 1-51. 78. The lipid nanoparticle composition of claim 77, wherein said polynucleotide is an mRNA. 79. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and said engineered meganuclease of any one of claims 1-51. 80. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and said polynucleotide of claim 52 or claim 53. 81. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and said recombinant DNA construct of any one of claims 54-65. Attorney Docket No.: P893391190WO (01242) 82. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and said recombinant virus of any one of claims 66-76. 83. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and said lipid nanoparticle composition of claim 77 or claim 78. 84. A eukaryotic cell comprising said polynucleotide of claim 52 or claim 53. 85. The eukaryotic cell of claim 84, wherein said eukaryotic cell is a mammalian cell. 86. The eukaryotic cell of claim 84 or claim 85, wherein said eukaryotic cell is a human cell. 87. The eukaryotic cell of claim 86, wherein said human cell is a human muscle cell, a human muscle stem cell, or a human CNS cell. 88. A method for producing a genetically-modified eukaryotic cell, said method comprising introducing into a eukaryotic cell: (a) a polynucleotide comprising a nucleic acid sequence encoding said engineered meganuclease of any one of claims 23-51, wherein said engineered meganuclease is expressed in said eukaryotic cell; or (b) said engineered meganuclease of any one of claims 23-51; wherein said engineered meganuclease produces a cleavage site at said recognition sequence comprising SEQ ID NO: 3 in mutant mitochondrial genomes of said eukaryotic cell. 89. The method of claim 88, wherein said mutant mitochondrial genomes comprising said recognition sequence are degraded in said genetically-modified eukaryotic cell. 90. The method of claim 89, wherein about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of mutant mitochondrial genomes comprising said recognition sequence are degraded in said genetically-modified eukaryotic cell. Attorney Docket No.: P893391190WO (01242) 91. The method of claim 89 or claim 90, wherein the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising said recognition sequence increases in said genetically-modified eukaryotic cell. 92. The method of any one of claims 89-91, wherein said ratio increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more. 93. The method of any one of claims 89-92, wherein the percentage of wild-type mitochondrial genomes in said genetically-modified eukaryotic cell is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, of the total mitochondrial genomes in said genetically-modified eukaryotic cell. 94. The method of any one of claims 89-93, wherein the percentage of mutant mitochondrial genomes comprising said recognition sequence in said genetically-modified eukaryotic cell decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. 95. The method of any one of claims 89-94, wherein cellular respiration in said genetically-modified eukaryotic cell increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. 96. The method of any one of claims 89-95, wherein cellular respiration in said genetically-modified eukaryotic cell increases by about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more. 97. A method for producing a population of eukaryotic cells comprising a plurality of genetically-modified eukaryotic cells, said method comprising introducing into a plurality of Attorney Docket No.: P893391190WO (01242) eukaryotic cells in said population: (a) a polynucleotide comprising a nucleic acid sequence encoding said engineered meganuclease of any one of claims 23-51, wherein said engineered meganuclease is expressed in said plurality of eukaryotic cells; or (b) said engineered meganuclease of any one of claims 23-51; wherein said engineered meganuclease produces a cleavage site at a recognition sequence comprising SEQ ID NO: 3 in mutant mitochondrial genomes of said plurality of eukaryotic cells. 98. The method of claim 97, wherein said mutant mitochondrial genomes comprising said recognition sequence are degraded in said plurality of genetically-modified eukaryotic cells. 99. The method of claim 98, wherein about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of mutant mitochondrial genomes comprising said recognition sequence are degraded in said plurality of genetically-modified eukaryotic cells. 100. The method of claim 98 or claim 99, wherein the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising said recognition sequence increases in said plurality of genetically-modified eukaryotic cells. 101. The method of any one of claims 98-100, wherein the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising said recognition sequence increases in said population of eukaryotic cells. 102. The method of claim 100 or claim 101, wherein said ratio increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more. 103. The method of any one of claims 98-102, wherein the percentage of wild-type mitochondrial genomes in said plurality of genetically-modified eukaryotic cells increases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about Attorney Docket No.: P893391190WO (01242) 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. 104. The method of any one of claims 98-103, wherein the percentage of wild-type mitochondrial genomes in said population of eukaryotic cells increases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. 105. The method of any one of claims 98-104, wherein the percentage of mutant mitochondrial genomes comprising said recognition sequence in said plurality of genetically- modified eukaryotic cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. 106. The method of any one of claims 98-105, wherein the percentage of mutant mitochondrial genomes comprising said recognition sequence in said population of eukaryotic cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. 107. The method of any one of claims 98-106, wherein cellular respiration in said plurality of genetically-modified eukaryotic cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. 108. The method of any one of claims 98-107, wherein cellular respiration in said plurality of genetically-modified eukaryotic cells increases by about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more. 109. The method of any one of claims 98-108, wherein cellular respiration in said population of eukaryotic cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. Attorney Docket No.: P893391190WO (01242) 110. The method of any one of claims 98-109, wherein cellular respiration in said population of eukaryotic cells increases by about 30-40%, about 40-50%, about 50-60%, about 60- 70%, about 70-80%, about 80-90%, about 90-100%, or more. 111. The method of any one of claims 98-110, wherein said recognition sequence is within a region of said mitochondrial genome associated with a mitochondrial disorder. 112. The method of claim 111, wherein said mitochondrial disorder is a mitochondrial DNA (mtDNA) common deletion disorder. 113. The method of claim 112, wherein said mutant mitochondrial genomes lack the nucleotides positioned between nucleotides 8,470 and 13,447 in a wild-type mitochondrial genome. 114. The method of any one of claims 88-113, wherein said method is performed in vivo. 115. The method of any one of claims 88-113, wherein said method is performed in vitro. 116. The method of any one of claims 88-115, wherein said polynucleotide is an mRNA. 117. The method of claim 116, wherein said polynucleotide is said mRNA of claim 51. 118. The method of any one of claims 88-115, wherein said polynucleotide is a recombinant DNA construct. 119. The method of any one of claims 88-115, wherein said polynucleotide is introduced into said eukaryotic cell by a lipid nanoparticle. 120. The method of any one of claims 88-115, wherein said polynucleotide is introduced into said eukaryotic cell by a recombinant virus. 121. The method of claim 120, wherein said recombinant virus is said recombinant virus of any one of claims 66-76. 122. The method of claim 120 or claim 121, wherein said recombinant virus is a recombinant AAV. Attorney Docket No.: P893391190WO (01242) 123. The method of claim 122, wherein said recombinant AAV is a muscle-tropic recombinant AAV or a CNS-tropic recombinant AAV. 124. The method of claim 122 or claim 123, wherein said recombinant AAV has an AAV9 capsid, a MyoAAV capsid or an AAVMYO capsid. 125. The method of any one of claims 88-124, wherein said polynucleotide comprises a promoter operably linked to said nucleic acid sequence encoding said engineered meganuclease. 126. The method of claim 125, wherein said promoter is a constitutive promoter. 127. The method of claim 126, wherein said constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. 128. The recombinant virus of claim 125, wherein said promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter. 129. The recombinant virus of claim 128, wherein said promoter is an MHCK7 promoter, a tMCK promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk-CRM4/DES promoter, or a desmin promoter. 130. The recombinant virus of claim 125, wherein said promoter is a CNS cell-specific promoter. 131. The method of any one of claims 88-130, wherein said eukaryotic cell is a mammalian cell. 132. The method of any one of claims 88-131, wherein said eukaryotic cell is a human cell. 133. The method of any one of claims 88-132, wherein said eukaryotic cell is a human muscle cell, a human muscle stem cell, or a human CNS cell. Attorney Docket No.: P893391190WO (01242) 134. A genetically-modified eukaryotic cell, or a population of genetically-modified eukaryotic cells, produced by the method of any one of claims 88-133. 135. A method for degrading mutant mitochondrial genomes in a target cell in a subject, or in a population of target cells in a subject, said method comprising delivering to said target cell or said population of target cells: (a) a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease of any one of claims 23-51, wherein said engineered meganuclease is expressed in said target cell or said population of target cells; or (b) said engineered meganuclease of any one of claims 23-51; wherein said engineered meganuclease produces a cleavage site in said mutant mitochondrial genomes at a recognition sequence comprising SEQ ID NO: 3, and wherein said mutant mitochondrial genomes are degraded. 136. The method of claim 135, wherein said mutant mitochondrial genomes comprise a mtDNA common deletion. 137. The method of claim 135 or claim 136, wherein said mutant mitochondrial genomes lack the nucleotides positioned between nucleotides 8,470 and 13,447 in a wild-type mitochondrial genome. 138. The method of any one of claims 135-137, wherein said subject is a mammal. 139. The method of any one of claims 135-138, wherein said subject is a human. 140. The method of claim 139, wherein said target cell or said population of target cells are human muscle cells, human muscle stem cells, or human CNS cells. 141. The method of any one of claims 135-140, wherein said polynucleotide is an mRNA. 142. The method of claim 141, wherein said polynucleotide is said mRNA of claim 51. 143. The method of any one of claims 135-140, wherein said polynucleotide is a recombinant DNA construct. Attorney Docket No.: P893391190WO (01242) 144. The method of claim 143, wherein said polynucleotide is said recombinant DNA construct of any one of claims 54-65. 145. The method of any one of claims 135-140, wherein said polynucleotide is introduced into said target cell or said population of target cells by a lipid nanoparticle. 146. The method of any one of claims 135-140, wherein said polynucleotide is introduced into said target cell or said population of target cells by a recombinant virus. 147. The method of claim 146, wherein said recombinant virus is said recombinant virus of any one of claims 66-76. 148. The method of claim 146 or claim 147, wherein said recombinant virus is a recombinant AAV. 149. The method of claim 148, wherein said recombinant AAV is a muscle-tropic recombinant AAV or a CNS-tropic recombinant AAV. 150. The method of claim 148 or claim 149, wherein said recombinant AAV has an AAV9 capsid, a MyoAAV capsid or an AAVMYO capsid. 151. The method of any one of claims 135-150, wherein said polynucleotide comprises a promoter operably linked to said nucleic acid sequence encoding said engineered meganuclease. 152. The method of claim 151, wherein said promoter is a constitutive promoter. 153. The method of claim 152, wherein said constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. 154. The method of claim 151, wherein said promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter. 155. The method of claim 154, wherein said promoter is an MHCK7 promoter, a tMCK promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk- CRM4/DES promoter, or a desmin promoter. Attorney Docket No.: P893391190WO (01242) 156. The method of claim 151, wherein said promoter is a CNS cell-specific promoter. 157. The method of any one of claims 135-156, wherein about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of mutant mitochondrial genomes comprising said recognition sequence are degraded in said target cell or said population of said target cells. 158. The method of any one of claims 135-157, wherein the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising said recognition sequence increases in said target cell or said population of target cells. 159. The method of any one of claims 135-158, wherein said ratio increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more. 160. The method of any one of claims 135-159, wherein the percentage of wild-type mitochondrial genomes in said target cell or said population of target cells is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, of the total mitochondrial genomes in said target cell or said population of target cells. 161. The method of any one of claims 135-160, wherein the percentage of mutant mitochondrial genomes comprising said recognition sequence in said target cell or said population of target cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. Attorney Docket No.: P893391190WO (01242) 162. The method of any one of claims 135-161, wherein cellular respiration in said target cell or said population of target cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. 163. The method of any one of claims 135-162, wherein cellular respiration in said target cell or said population of target cells increases by about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more. 164. A method for treating a condition associated with a mitochondrial mtDNA common deletion in a subject, said method comprising administering to said subject: (a) a therapeutically- effective amount of a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease of any one of claims 23-51, wherein said polynucleotide is delivered to a target cell, or a population of target cells, in said subject; wherein said engineered meganuclease is expressed in said target cell or said population of target cells; or (b) a therapeutically-effective amount of said engineered meganuclease of any one of claims 23-51, wherein said engineered meganuclease is delivered to a target cell, or a population of target cells, in said subject, wherein said engineered meganuclease produces a cleavage site in mutant mitochondrial genomes at a recognition sequence comprising SEQ ID NO: 3, and wherein said mutant mitochondrial genomes are degraded. 165. The method of claim 164, wherein said mutant mitochondrial genomes comprise said mtDNA common deletion. 166. The method of claim 164 or claim 165, wherein said mutant mitochondrial genomes lack the nucleotides positioned between nucleotides 8470 and 13,447 in a wild-type mitochondrial genome. 167. The method of any one of claims 164-166, wherein said method reduces or ameliorates one or more symptoms associated with said mtDNA common deletion. 168. The method of any one of claims 164-167, wherein said subject is a mammal. 169. The method of any one of claims 164-168, wherein said subject is a human. Attorney Docket No.: P893391190WO (01242) 170. The method of claim 169, wherein said target cell or said population of target cells are human muscle cells, human muscle stem cells, or human CNS cells. 171. The method of any one of claims 164-170, wherein said condition is a condition of the bone marrow, the pancreas, muscle, skeletal muscle, central nervous system, the eye, or the ears. 172. The method of any one of claims 164-171, wherein said condition is Pearson Syndrome, Kearns-Sayre Syndrome (KSS), Progressive External Ophthalmoplegia (PEO), diabetes, or kidney dysfunction. 173. The method of any one of claims 164-172, wherein said polynucleotide is an mRNA. 174. The method of claim 173, wherein said polynucleotide is said mRNA of claim 51. 175. The method of any one of claims 164-172, wherein said polynucleotide is a recombinant DNA construct. 176. The method of claim 175, wherein said polynucleotide is said recombinant DNA construct of any one of claims 54-65. 177. The method of any one of claims 164-172, wherein said polynucleotide is introduced into said target cell or said population of target cells by a lipid nanoparticle. 178. The method of any one of claims 164-172, wherein said polynucleotide is introduced into said target cell or said population of target cells by a recombinant virus. 179. The method of claim 178, wherein said recombinant virus is said recombinant virus of any one of claims 66-76. 180. The method of claim 178 or claim 179, wherein said recombinant virus is a recombinant AAV. Attorney Docket No.: P893391190WO (01242) 181. The method of claim 148, wherein said recombinant AAV is a muscle-tropic recombinant AAV or a CNS-tropic recombinant AAV. 182. The method of claim 180 or claim 181, wherein said recombinant AAV has an AAV9 capsid, a MyoAAV capsid or an AAVMYO capsid. 183. The method of any one of claims 164-182, wherein said polynucleotide comprises a promoter operably linked to said nucleic acid sequence encoding said engineered meganuclease. 184. The method of claim 183, wherein said promoter is a constitutive promoter. 185. The method of claim 184, wherein said constitutive promoter is a CMV promoter, a CAG promoter, an EF1 alpha promoter, or a UbC promoter. 186. The method of claim 183, wherein said promoter is a muscle cell-specific promoter or a muscle stem cell-specific promoter. 187. The method of claim 186, wherein said promoter is an MHCK7 promoter, a tMCK promoter, a CK8 promoter, an SPc-512 promoter, an SP-301 promoter, an MH promoter, an Sk- CRM4/DES promoter, or a desmin promoter. 188. The method of claim 183, wherein said promoter is a CNS cell-specific promoter. 189. The method of any one of claims 164-188, wherein about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of mutant mitochondrial genomes comprising said recognition sequence are degraded in said target cell or said population of said target cells. 190. The method of any one of claims 164-189, wherein the ratio of wild-type mitochondrial genomes to mutant mitochondrial genomes comprising said recognition sequence increases in said target cell or said population of target cells. 191. The method of any one of claims 164-190, wherein said ratio increases to about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, Attorney Docket No.: P893391190WO (01242) about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 20:1, about 50:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, or more. 192. The method of any one of claims 164-191, wherein the percentage of wild-type mitochondrial genomes in said target cell or said population of target cells is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more, of the total mitochondrial genomes in said target cell or said population of target cells. 193. The method of any one of claims 164-192, wherein the percentage of mutant mitochondrial genomes comprising said recognition sequence in said target cell or said population of target cells decreases by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more. 194. The method of any one of claims 164-193, wherein cellular respiration in said target cell or said population of target cells increases by about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more. 195. The method of any one of claims 164-194, wherein cellular respiration in said target cell or said population of target cells increases by about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, about 90-100%, or more.
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