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WO2025137069A1 - Compositions et procédés pour l'édition améliorée de génome à l'aide de protéines de fusion cas9 - Google Patents

Compositions et procédés pour l'édition améliorée de génome à l'aide de protéines de fusion cas9 Download PDF

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WO2025137069A1
WO2025137069A1 PCT/US2024/060720 US2024060720W WO2025137069A1 WO 2025137069 A1 WO2025137069 A1 WO 2025137069A1 US 2024060720 W US2024060720 W US 2024060720W WO 2025137069 A1 WO2025137069 A1 WO 2025137069A1
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fragment
variant
sequence
cas9
nucleic acid
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Alexandros POULOPOULOS
Ryan Richardson
Colin Robertson
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University of Maryland Baltimore
University of Maryland College Park
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University of Maryland Baltimore
University of Maryland College Park
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    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • A61K48/0016—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the nucleic acid is delivered as a 'naked' nucleic acid, i.e. not combined with an entity such as a cationic lipid
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the field of the invention generally relates to biotechnology and medicine, in particular compositions and methods for genome editing.
  • Genome editing is a powerful technology that allows for the specific and often precise addition or removal of genetic material. Genome editing is initiated by making double stranded DNA breaks in the target cell. These double stranded DNA breaks can be created by several methods including meganucleases, Zine-Finger Nucleases, TALE-nucleases, and/or the CRISPR/Cas9 restriction modification system. Each of these systems creates a dsDNA break at a user designated genomic location. After the creation of the dsDNA break, the cellular machinery acts quickly to repair this dsDNA using either by the non-homologous end joining (NHEJ) pathway or by homologous recombination (HDR).
  • NHEJ non-homologous end joining
  • HDR homologous recombination
  • Cas9 targets genomic loci with high specificity. For knockin with doublestrand break repair, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible.
  • the present inventors disclose a high-throughput workflow to quantify editing outcomes for creating and identifying editing agents with increased performance and their optimal combinations for knockin applications. The inventors have established editing efficiency and precision as generalizable assessment metrics for comparisons of knockin performance across existing and novel agents. Using this platform, the inventors aimed to enhance DSB repair-based editing performance by combinatorial screens of Cas9 variants, DNA donors, and new compound fusions to DNA repair protein domains.
  • Cas9-RC a high-performance DSB repair-based editing agent with increased editing efficiency and precision.
  • Cas9- RC was tested for its editing performance in vivo in the embryonic mouse brain, where it enhanced fluorescent protein knockin in some cases by in utero electroporation. These improvements showcase the utility of this workflow for continued development and assessment of new precision editing tools for in vivo knockin applications, including expression vectors, host cells and methods of producing the recombinant Cas9 fusion protein.
  • the invention provides a nucleic acid comprising a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises Cas9 or a variant or fragment thereof, Radi 8 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the invention provides a nucleic acid comprising a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • Rad 18 or a variant or fragment thereof is fused to the amino terminus of Cas9 or a variant or fragment thereof and CtIP or a variant or fragment thereof is fused to the carboxy terminus of Cas9 or a variant or fragment thereof.
  • a Radi 8 fragment is fused to the amino terminus of Cas9 or a variant or fragment thereof and a HDR Enhancing N-terminal fragment of CtIP is fused to the carboxy terminus of Cas9 or a variant or fragment thereof.
  • the fusion protein comprises an amino acid sequence of SEQ ID NO:7.
  • the invention provides a nucleic acid sequence that encodes a polypeptide comprising SEQ ID NO:7.
  • the nucleic acid sequence of the fusion protein comprises SEQ ID NO: 8.
  • the Cas9 is a wild-type Cas9 from Streptococcus pyogenes.
  • the fusion protein comprises a fragment of Cas9 that lacks an N-terminal methionine.
  • the Cas9 or fragment or variant thereof comprises a nucleic acid sequence encoding a polypeptide comprising SEQ ID NO: 1.
  • the Cas9 or fragment or variant thereof nucleic acid sequence comprises SEQ ID NO:2 (DNA) or SEQ ID NO:36 (RNA).
  • Cas9 or fragment or variant thereof is a high fidelity variant (Cas9-HF).
  • the fusion protein comprises a fragment of CtIP.
  • the fusion protein comprises the HDR Enhancing N-terminal fragment of CtIP, comprising SEQ ID NO:3 (amino acids 1-296).
  • the CtIP fragment nucleic acid sequence comprises SEQ ID NO:4 (DNA) or SEQ ID NO:38 (RNA).
  • the CtIP, variant or fragment lacks an N-terminal methionine.
  • the fusion protein comprises a variant or fragment of Rad 18.
  • the fusion protein comprises a fragment of Radi 8, which contains a deletion of a putative DNA-binding domain.
  • this enhanced version of Radi 8 (also referred to herein as eRadl8) has a deletion in amino acids 242-282 in Radl8 and comprises SEQ ID NO:5.
  • the eRadl8 nucleic acid sequence comprises SEQ ID NO:6 (DNA) or SEQ ID NO:40 (RNA).
  • the Radi 8, variant or fragment lacks an N-terminal methionine.
  • the nucleic acid is selected from DNA or RNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is circular RNA (circRNA).
  • the nucleic acid encoding the fusion protein comprises a vector.
  • the vector is a viral vector.
  • the vector is a mammalian expression vector.
  • the invention provides a host cell transformed or transfected with a vector encoding a fusion protein, wherein the fusion protein comprises Cas9 or a variant or fragment thereof, Rad 18 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the invention provides a host cell transformed or transfected with a vector encoding a fusion protein, wherein the fusion protein comprises Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the invention provides a pharmaceutical composition comprising a nucleic acid encoding the fusion protein.
  • the composition comprises a delivery system, such as lipid nanoparticles or lipid-like nanoparticles.
  • the lipid or lipid-like nanoparticles are ionizable.
  • the invention provides a method of modifying a genomic sequence of a cell, comprising administering to the cell an effective amount of a nucleic acid encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad 18 or a variant or fragment thereof, and CUP or a variant or fragment thereof, and one or more additional agents to modify the genomic sequence in the cell.
  • the one or more additional agents include guide RNA(s) and/or template nucleic acid.
  • the invention provides a method of modifying a genomic sequence of a cell, comprising administering to the cell an effective amount of a nucleic acid encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof, and one or more additional agents to modify the genomic sequence in the cell.
  • the one or more additional agents include guide RNA(s) and/or template nucleic acid.
  • the invention provides a method of treating a disease of condition in a subject by administering to the subject an effective amount of the modified cells.
  • the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of a nucleic acid encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad 18 or a variant or fragment thereof, and CtIP or a variant or fragment thereof, and one or more additional agents to modify a genomic sequence in the subject.
  • the one or more additional agents include guide RNA(s) and/or template nucleic acid.
  • the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of a nucleic acid encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof, and one or more additional agents to modify genomic sequence in the subject.
  • the one or more additional agents include guide RNA(s) and/or template nucleic acid.
  • the present invention provides compositions and methods for expression and purification of Cas9 fusion proteins and variants thereof in prokaryotic host cells.
  • the invention provides a prokaryotic expression vector encoding a fusion protein, wherein the fusion protein comprises Cas9 or a variant or fragment thereof, Radi 8 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the invention provides a prokaryotic host cell transformed or transfected with a prokaryotic expression vector encoding a fusion protein, wherein the fusion protein comprises Cas9 or a variant or fragment thereof, Radi 8 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the invention provides a prokaryotic expression vector encoding a fusion protein, wherein the fusion protein comprises Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the invention provides a method for producing the fusion protein in a prokaryotic host cell, comprising culturing the prokaryotic host cell in a growth media under conditions suitable for the expression of the fusion protein and isolating the fusion protein.
  • FIG. 1 BFP-to-GFP editing as a platform to quantify knockin efficiency and precision.
  • A Schematic of BFP-to-GFP conversion assay screen. Genome editing agents are tested by transfecting HEK:BFP cells, a HEK293 knockin cell line that genomically expresses BFP driven by an EFla promoter. Successful editing knocks in the H62Y mutation into the BFP locus, thus producing GFP.
  • Genotyping of the three collected populations was performed by PCR of the BFP locus from genomic DNA, followed by Sanger sequencing of amplified fragment pools.
  • C Alignment of Sanger sequencing reads from BFP + , dark, and GFP + cell populations to the reference BFP locus sequence (top), showing wild-type, knockout (indel), and knockin genotypes, respectively.
  • D Editing outcomes (indels, knockin frequency) were quantified by decomposition of Sanger sequencing reads using the ICE algorithm and plotted on relative histograms binned by indel size. GFP + cells represent true knockins, and over 90% of dark cells contain indels predicted to cause knockout.
  • FIG. 2. Cas9-CtIP fusion and HMEJ donors additively improve knockin precision.
  • A Schema illustrating the combinatorial parameters of editing agents: dsDNA donor template HR or HMEJ variants in combination with Cas9 WT or HiFi variants, alone or fused to CtIP.
  • B Flow cytometry plots of HEK:BFP cells 7 days after transient transfection with gRNA, Cas9, and donor plasmids. GFP fluorescence is shown on the x-axis (log), and BFP fluorescence on the y-axis (log). Quantification gates are indicated on the plots for BFP + (WT), GFP + (KI), and dark (KO) cells.
  • Cas9 variants with HR (top row, yellow) or HMEJ (bottom row purple) donors were measured. Mean values from individual experiments (n >3) were normalized to those of the Cas9 WT with HR donor condition and presented as the mean ⁇ SEM.
  • C Heatmaps displaying statistical significance of pairwise comparisons of editing agent performance, calculated using one-way ANOVA with Tukey’s multiple comparison test and single pooled variance (* P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001).
  • Cas9-CtIP fusion with HMEJ donor outperforms Cas9 with HR donor in knockin precision by over 30-fold.
  • FIG. 3 Iterative screening of novel Cas9 fusions and compound fusions with DNA-repair domains for increased editing performance.
  • A Schema of the fusion screen. Candidate DNA-repair protein domains are fused N-terminally to either Cas9 alone or Cas9-CtIP and together with HMEJ donor assayed by BFP-to-GFP for knockin efficiency and precision.
  • B Bar graphs showing quantification of relative knockin efficiency, knockout frequency, and knockin precision for Cas9 fusion (light blue) or Cas9-CtIP compound fusion (dark blue) with the listed DNA-repair protein domains. Values from individual experiments (n > 3) were normalized to Cas9 only and presented as the mean + SEM.
  • FIG. 4 Expression of affinity-tagged Cas9-RC ribonucleoprotein.
  • A Schema of the Cas9-RC ribonucleoprotein N-terminally fused with cleavable tandem GST and MBP affinity tags. The molecular weights of each domain (kDa) and an HRV 3C protease cleavage site (red triangle) are indicated.
  • B Protein electrophoresis gel stained with Coomassie blue showing lysates from bacteria transformed with GST-MBP-Cas9-RC expression construct prior to (left lane) and after inducing expression with IPTG for 3h (right lane).
  • FIG. 5 Cas9-RC enhances knockin efficiency in vivo.
  • A Schema of the Cas9 and Cas9-RC agents compared for in vivo editing using in utero electroporation in the fetal mouse brain at embryonic day (E) 14.5, and analysis in the cerebral cortex at postnatal day (P) 7.
  • B Gene editing donor template DNA targets the endogenous ActB locus to insert mCherry downstream of the P-actin coding sequence separated by a 2A. Knockin efficiency was quantified as the number of mCherry-i- (knockin) neurons over the number of GFP+ (electroporated) neurons.
  • C Representative fluorescence images of the cerebral cortex at P7 with electroporated neurons receiving Cas9 or Cas9-RC. Images show plasmid GFP (green) and genomic ActB-2A-mCherry (red) expression. Scale bar 100 pm.
  • D Swarm plots showing quantification of in vivo knockin efficiency for Cas9 vs. Cas9-RC and effect size estimation. Points show means from each brain and are plotted on the left axis for both groups indicating knockin efficiency. The effect size on knockin efficiency of Cas9-RC versus Cas9 is plotted as a distribution on a floating axis on the right indicating standard deviations (S).
  • the effect size estimated by unpaired Cohen's d between Cas9 and Cas9-RC is 2.0 S (black dot), indicating a large effect size.
  • the 95% confidence interval is 0.739 to 3.97 (vertical error bar).
  • the P value is 0.0022.
  • FIG. 6 Knockin applications with Cas9-RC.
  • A Primary fibroblasts from wildtype newborn mice were cuvette electroporated (nucleofected, schema on the right) with Cas9-RC plasmid and ActB-2A-mCherry donor DNA. Fibroblasts show two intensities of mCherry fluorescence (red) from the mouse -Actin locus, indicating mCherry knockin on one (arrowheads) or both (arrows) ActB alleles.
  • Cortical projection neurons (arrows) and astrocytes (arrowheads) are knocked in with Cas9-RC plasmid and ActB-2A-mCherry donor DNA in utero as in Fig.
  • C mScarlet- fusion knockin onto Negri, which represents a locus with developmentally regulated levels of expression that vary between neurons.
  • Cas9-RC and mScarlet-Negrl donor DNA were in utero electroporated in E14.5 mouse brain to extracellularly tag the endogenous Negri GPI-linked membrane protein with mScarlet.
  • Knockin cells displayed variable levels of mScarlet fluorescence (red) with a wide range of high (arrows) and low (arrowheads) expressing cells.
  • Electroporated knockin positive (arrow) and knockin negative (arrowhead) cells can be seen in the Purkinje cell layer in P14 cerebellum.
  • Cas9-RC has versatile applications and may offer advantages over the disadvantage of its large size depending on the target gene and cell type. Color labels and scale bars as indicated.
  • FIG. 7 Map of bacterial expression vector encoding Cas9-RC.
  • FIG. 8 Gating strategy for flow cytometry analysis of editing in HEK:BFP cells. Live cells were first gated by size and granularity using FSC-A vs SSC-A and then singlets were gated using SSC-A vs SSC-H.
  • FIG. 9. Biological replicate data and statistical analysis related to Fig. 3.
  • (a- c) Quantification of flow cytometry data for biological replicates from HEK:BFP cells 7 days after transient transfection indicating (a) KI efficiency (% GFP+) (b) KO frequency (% dark) and (c) KI precision (KI/KO ratio) for Cas9 variants with HMEJ donor,
  • (d-f) Heatmap matrices showing statistical significance calculated using a oneway ANOVA with Tukey’s multiple comparison test and pooled variance (also shown in Fig. 3). Differences between conditions were judged to be significant at P ⁇ 0.05 (*), P ⁇ 0.01 (**), and P ⁇ 0.001 (***).
  • FIG. 10 Expression of affinity-tagged Cas9-RC ribonucleoprotein: A) Protein electrophoresis gel stained with Coomassie blue showing lysates from bacteria transformed with GST-MBP-Cas9-RC expression construct prior to (left lane) and after inducing expression with IPTG for 3h (right lane). Arrows indicate putative GST-MBP-Cas9RC (316 kDA) and Cas9-RC (250 kDa) bands. (B) Anti-Cas9 immunoblot of lysate lanes shows Cas9 immunoreactive bands after IPTG induction, including bands with electrophoretic mobilities corresponding to full-length GST- MBP-Cas9RC and Cas9-RC, as indicated.
  • the present invention generally relates to compositions and methods for modifying a genomic sequence of a cell, including nucleic acids and polypeptides encoding fusion proteins comprising Cas9 or a variant or fragment thereof, Rad52 or Rad 18 or a variant or fragment thereof, and CtIP or a variant or fragment thereof, and one or more additional agents to modify genomic sequence in the subject.
  • the term "about” means plus or minus 10% of the numerical value of the number with which it is being used.
  • nucleic acid and “polynucleotide,” are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
  • these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • loci locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched poly
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by nonnucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • chimeric RNA refers to the polynucleotide sequence comprising the guide sequence, the tracr sequence and the tracr mate sequence.
  • guide sequence refers to the about 20 bp sequence within the guide RNA that specifies the target site and may be used interchangeably with the terms “guide” or “spacer”.
  • tracr mate sequence may also be used interchangeably with the term “direct repeat(s)”.
  • Exemplary CRISPR-Cas system are provided in U.S. Pat. No. 8,697,359 and US 20140234972, both of which are incorporated herein by reference in their entirety.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally-occurring amino acids.
  • sequence relates to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded.
  • identity relates to an exact nucleotide- to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • Two or more sequences can be compared by determining their percent identity. Calculations of homology or sequence identity between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percentage identity between the two sequences is a function of the number of identical positions shared by the sequences.
  • Sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between homologous regions, followed by digestion with single- stranded- specific nuclease(s), and size determination of the digested fragments.
  • nucleic acids are written left to right in 5' to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.
  • recombinant includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention.
  • wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • variable should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature.
  • nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
  • “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non- traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • stringent conditions for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequencedependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part 1, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N.Y.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme.
  • a sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence.
  • expression refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • subject refers to a vertebrate, preferably a mammal, more preferably a human.
  • Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • therapeutic agent refers to a molecule or compound that confers some beneficial effect upon administration to a subject.
  • the beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
  • treatment or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
  • an effective amount refers to the amount of an agent that is sufficient to effect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein.
  • the specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
  • the present invention relates to compositions and methods for using nucleic acids and/or polypeptides encoding a Cas9 fusion protein to enhance genomic editing.
  • the compositions are useful in genomic or nucleic acid modification in vitro, ex vivo, and in vivo for a variety of research, screening, and therapeutic applications.
  • the invention provides a nucleic acid encoding a fusion protein comprising a Cas9 polypeptide or a variant or fragment thereof, Rad 18 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the invention provides a nucleic acid encoding a fusion protein comprising a Cas9 polypeptide or a variant or fragment thereof, a nucleic acid encoding Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is a circular RNA molecule.
  • the invention provides a polypeptide encoding a fusion protein comprising a Cas9 polypeptide or a variant or fragment thereof, Radi 8 or a variant or fragment thereof, and CtIP or a variant or fragment thereof. In some embodiments, the invention provides a polypeptide encoding a fusion protein comprising a Cas9 polypeptide or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the nucleic acid or polypeptide encodes a fusion protein that comprises Cas9 or a variant or fragment thereof.
  • the Cas9 or variant or fragment thereof is not particularly limiting.
  • Cas molecules of a variety of species can be used in nucleic acids, polypeptides, vectors and methods described herein.
  • the Cas9 is from Staphylococcus aureus. In some embodiments, the Cas9 is from S', pyogenes, S. thermophiles, or Neisseria meningitides. Additional Cas9 species include: Acidovorax avenae, Actinobacillus pleuropneumoniae.
  • Actinobacillus succinogenes Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus Puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Coryn ebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae
  • Cas9 molecule refers to a molecule that can interact with a gRNA molecule and, in concert with the gRNA molecule, localize (e.g., target or home) to a site which comprises a target domain and PAM sequence.
  • the Cas9 molecule is capable of cleaving a target nucleic acid molecule.
  • the ability of a Cas9 molecule to interact with and cleave a target nucleic acid is PAM sequence dependent.
  • a PAM sequence is a sequence in the target nucleic acid.
  • cleavage of the target nucleic acid occurs upstream from the PAM sequence.
  • Cas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences).
  • a Cas9 molecule of S. pyogenes recognizes the sequence motif NGG and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence.
  • meningitidis recognizes the sequence motif NNNNGATT and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Hou et al., PNAS Early Edition 2013, 1-6.
  • the ability of a Cas9 molecule to recognize a PAM sequence can be determined, e.g., using a transformation assay described in Jinek et al., Science 2012, 337:816.
  • Cas9 molecules include Cas9 molecules of a cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, a cluster 7 bacterial family, a cluster 8 bacterial family, a cluster 9 bacterial family, a cluster 10 bacterial family, a cluster 11 bacterial family, a cluster 12 bacterial family, a cluster 13 bacterial family, a cluster 14 bacterial family, a cluster 15 bacterial family, a cluster 16 bacterial family, a cluster 17 bacterial family, a cluster 18 bacterial family, a cluster 19 bacterial family, a cluster 20 bacterial family, a cluster 21 bacterial family, a cluster 22 bacterial family, a cluster 23 bacterial family, a cluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26 bacterial family, a
  • Exemplary naturally occurring Cas9 molecules include a Cas9 molecule of a cluster 1 bacterial family.
  • Examples include a Cas9 molecule of: S. pyogenes (e.g., strain SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5OO5, MGAS6180, MGAS9429, NZ131 and SSI-1), S. thermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. mutans (e.g., strain UA159, NN2025), S.
  • S. pyogenes e.g., strain SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5OO5, MGAS6180, MGAS9429, NZ131 and SSI-1
  • S. thermophilus e.g., strain LMD-9
  • macacae e.g., strain NCTC11558
  • S', gallolyticus e.g., strain UCN34, ATCC BAA-2069
  • S. equines e.g., strain ATCC 9812, MGCS 124
  • S. dysdalactiae e.g., strain GGS 124
  • 5. bovis e.g., strain ATCC 7003378
  • S. anginosus e.g.; strain F0211
  • S. agalactiae e.g., strain NEM316, A909
  • Listeria monocytogenes e.g., strain F6854
  • Listeria innocua L.
  • Additional exemplary Cas9 molecules are a Cas9 molecule of Neisseria meningitidis (Hou et al. PNAS Early Edition 2013, 1-6) and a 5. aureus Cas9 molecule.
  • the polynucleotide may include the coding sequence for the full-length polypeptide or a fragment thereof, by itself; the coding sequence for the full-length polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro or prepro-protein sequence, nuclear localization signal or other fusion peptide portions.
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, signals, ribosome binding sites and sequences that stabilize mRNA.
  • the nucleic acid sequence of Cas9, or variant or fragment thereof contains a nucleotide sequence that is highly identical, at least 90% identical, with a nucleotide sequence encoding Cas9 polypeptide.
  • the nucleic acid sequence of Cas9 comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100% identical with the encoding nucleotide sequence set forth in SEQ ID NOS:2, 36, 14 or 37.
  • the molecule is a wild-type Cas9.
  • the Cas9 is a wild-type Cas9 from Streptococcus pyogenes and comprises SEQ ID NO: 1.
  • the expression vector encodes a variant of the Cas9 protein referred to herein as a high fidelity Cas9.
  • the high fidelity Cas9 comprises an amino acid sequence comprising SEQ ID NO: 13.
  • the Cas9 comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOS: 1 or 13.
  • the nucleotide sequence encoding Cas9 or a biologically active fragment or derivative thereof includes nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100% identical to a nucleotide sequence encoding Cas9 having the amino acid sequence in SEQ ID NO: 1 or 13.
  • the Cas9 portion of the fusion protein is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOS:2, 36, 14 or 37, which lacks the codon for the N-terminal methionine.
  • SEQ ID NO:13 is encoded by SEQ ID NO: 14 or SEQ ID NO:37.
  • the nucleic acid or polypeptide encodes a biologically active fragment of Cas9 protein.
  • a Cas9 molecule comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NOS:1 or 13 (either full length or lacking the N-terminal methionine) or a naturally occurring Cas9 molecule sequence, e.g., a Cas9 molecule from a species listed herein or described in Chylinski et al., RNA Biology 2013, 10:5, 727-737; Hou et al. PNAS Early Edition 2013, 1-6.
  • the Cas9 polypeptide comprises an amino acid sequence that differs from a sequence of SEQ ID NOS:1 or 13 by as many as 1, but no more than 2, 3, 4, or 5 residues.
  • the nucleic acid or fusion protein encodes a Cas9 fragment.
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of one of a Cas9 polypeptide or variant. Fragments may be continuous or discontinuous. In some embodiments, the fragment may constitute from about 1000 contiguous amino acids identified in SEQ ID NOS: 1 or 13 In some embodiments, the fragment is about 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1360, 1361, 1362, 1363, 1364, 1365, or 1367 contiguous amino acids identified in SEQ ID NOSH or 13. In some embodiments, the fragment comprises SEQ ID NO:1 but lacks the N-terminal methionine residue (i.e., comprises amino acids 2-1368 of SEQ ID NO: 1).
  • the fragments include, for example, truncation polypeptides having the amino acid sequence of Cas9 polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus.
  • Naturally occurring Cas9 molecules possess a number of properties, including: nickase activity, nuclease activity (e.g., endonuclease and/or exonuclease activity); helicase activity; the ability to associate functionally with a gRNA molecule; and the ability to target (or localize to) a site on a nucleic acid (e.g., PAM recognition and specificity).
  • a Cas9 molecule can include all or a subset of these properties.
  • Cas9 molecules have the ability to interact with a gRNA molecule and, in concert with the gRNA molecule, localize to a site in a nucleic acid.
  • Other activities e.g., PAM specificity, cleavage activity, or helicase activity can vary more widely in Cas9 molecules.
  • Cas9 molecules with desired properties can be made in a number of ways, e.g., by alteration of a parental, naturally occurring Cas9 molecule to provide an altered Cas9 molecule having a desired property.
  • One or more mutations or differences relative to a parental Cas9 molecule can be introduced. Such mutations and differences can comprise substitutions e.g., conservative substitutions or substitutions of non-essential amino acids); insertions; or deletions.
  • a Cas9 molecule can comprise one or more mutations or differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations but less than 200, 100, or 80 mutations relative to a reference Cas9 molecule.
  • Candidate Cas9 molecules can be evaluated by art-known methods or as described herein. For example, exemplary methods for evaluating the endonuclease activity of Cas9 molecule are described, e.g., in Jinek et al., Science 2012; 337(6096):816-821.
  • the nucleic acids or fusion proteins comprise a nucleic acid sequence encoding CtIP or a variant or fragment thereof.
  • CtIP is a DNA repair protein. See e.g., Huertas et al. J. Biol. Chem. 284, 9558-9565 (2009) and Charpentier are incorporated by reference herein.
  • human CtIP is an 897 amino acid protein identified in NCBI Accession No. Q99708.
  • CtIP has an amino acid sequence of SEQ ID NO: 10.
  • the CtIP, variant or fragment thereof is from a mammal, such as human, mouse, rat, or the like.
  • the nucleic acid or fusion protein encodes a fragment of CtIP.
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of one of a CtIP polypeptide or variant. Fragments may be continuous or discontinuous.
  • the nucleic acid or fusion protein encodes a CtIP fragment comprising an amino acid sequence of SEQ ID NO:3, corresponding to amino acids 1-296 of CtIP.
  • SEQ ID NO:3 is encoded by a nucleotide sequence comprising SEQ ID NOS:4 or 38.
  • the fragment may constitute about 150 contiguous amino acids identified in SEQ ID NOS:3 or 10. In some embodiments, the fragment is about 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, or 290 contiguous amino acids or more identified in SEQ ID NOS:3 or 10.
  • the fragments include, for example, truncation polypeptides having the amino acid sequence of CtIP polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus.
  • the fragment is a homology-directed repair enhancing (HDR Enhancing) N-terminal fragment of CtIP.
  • HDR Enhancing homology-directed repair enhancing
  • the HDR Enhancing N-terminal fragment of CtIP comprises an amino acid sequence at least 90% identical to SEQ ID NOS:3 or 10 (amino acids 1-296).
  • the HDR Enhancing N-terminal fragment of CtIP comprises an amino acid sequence of at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOS:3 or amino acids 1-296 of SEQ ID NO: 10.
  • the homology-directed repair enhancing (HDR Enhancing) N-terminal fragment of CtIP comprises one or more mutations or differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations but less than 200, 100, or 80 mutations relative to SEQ ID NOS:3 or amino acids 1-296 of SEQ ID NO: 10.
  • the homology-directed repair enhancing (HDR Enhancing) N-terminal fragment of CtIP comprises an amino acid sequence that differs from a sequence of SEQ ID NOS:3 or amino acids 1-296 of SEQ ID NO: 10 by as many as 1, but no more than 2, 3, 4, or 5 residues.
  • the nucleic acid sequence of CtIP, a variant or fragment thereof contains a nucleotide sequence that is highly identical, at least 90% identical, with a nucleotide sequence encoding CtIP, a variant or fragment thereof polypeptide.
  • the nucleic acid sequence of CtIP, a variant or fragment thereof comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100% identical with the encoding nucleotide sequence set forth in SEQ ID NOS :4 or 38.
  • the nucleic acid or fusion protein comprises Rad 18 or a variant or fragment thereof or Rad52 or a variant or fragment thereof.
  • Rad 18 is a E3 ubiquitin-protein ligase involved in postreplication repair of UV-damaged DNA. which are incorporated by reference herein. See Nambiar et al., Nat. Commim. 10, 3395 (2019), which is incorporated by reference herein
  • human Rad 18 is a 495 amino acid protein identified in NCBI Accession No. AAF86618.
  • Radi 8 has an amino acid sequence of SEQ ID NO:11.
  • Rad52 or a variant or fragment thereof can be used in place of the Rad 18 component in the fusion protein.
  • the Rad52 or a variant or fragment thereof has an amino acid sequence of SEQ ID NO: 15.
  • SEQ ID NO: 15 is encoded by a nucleotide sequence comprising SEQ ID NOS: 16 or 39.
  • the Radl8 or Rad52, variant or fragment thereof is from a mammal, such as human, mouse, rat, or the like.
  • the nucleic acid or fusion protein encodes a Radi 8 fragment.
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of one of a Rad 18 polypeptide or variant. Fragments may be continuous or discontinuous.
  • the fragment of Radi 8 comprises a deletion of a putative DNA-binding domain, and optionally lacks the N-terminal methionine.
  • the fragment of Rad 18 (also referred to herein as eRad 18) has a deletion in amino acids 242-282 in Radi 8 and is encoded by an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5 or SEQ ID NO:43.
  • the fragment may constitute about 150 contiguous amino acids identified in SEQ ID NOS:5 or 1 1. In some embodiments, the fragment is about 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, or 450 contiguous amino acids or more identified in SEQ ID NO:5 or SEQ ID NO:43.
  • the fragments include, for example, truncation polypeptides having the amino acid sequence of Rad 18 polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus.
  • the Rad 18 variant or fragment comprises one or more mutations or differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations but less than 200, 100, or 80 mutations relative to SEQ ID NO:5 or SEQ ID NO:43.
  • the Rad 18 variant or fragment comprises an amino acid sequence that differs from a sequence of SEQ ID NO:5 or SEQ ID NO:43 by as many as 1, but no more than 2, 3, 4, or 5 residues.
  • the nucleic acid sequence of Radi 8, a variant or fragment thereof contains a nucleotide sequence that is highly identical, at least 90% identical, with a nucleotide sequence encoding Radi 8, a variant or fragment thereof polypeptide.
  • the nucleic acid sequence of Radi 8, a variant or fragment thereof comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100% identical with the encoding nucleotide sequence set forth in SEQ ID NOS:6 or 40.
  • the Rad 18 or a variant or fragment thereof is fused to the amino terminus of the Cas9 or a variant or fragment thereof and the CtIP or a variant or fragment thereof is fused to the carboxy terminus of Cas9.
  • the Rad52 or a variant or fragment thereof is fused to the amino terminus of the Cas9 or a variant or fragment thereof and the CtIP or a variant or fragment thereof is fused to the carboxy terminus of Cas9.
  • the fusion protein comprises one or more linker sequences between Radi 8 or a variant or fragment thereof, Cas9 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the linker sequence comprises a nuclear localization sequence.
  • the linker sequence comprises GAAPKKKRKVGIHGVPAA (SEQ ID NO:21) and/or KRPAATKKAGQAKKKKEFGSGGAAS (SEQ ID NO:22).
  • GAAPKKKRKVGIHGVPAA is a linker between the Rad52 or eRadl8 portion and the Cas9 portion of the fusion protein.
  • KRPAATKKAGQAKKKKEFGSGGAAS (SEQ ID NO:22) is a linker between the Cas9 portion and the CtIP portion of the fusion protein.
  • the fusion protein (eRadl8-Cas9-CtIP) has an amino acid sequence that is at least 80% , 85 % , 90% , 91 % , 92% , 93 % , 94% , 95 % , 96% , 97 % , 98%, 99% or 100% identical to an amino acid sequence comprising SEQ ID NO: 17.
  • an N-terminal methionine is added to the amino acid sequence of SEQ ID NO:17 at the 1 position.
  • the nucleic acid encoding a fusion protein has a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence comprising SEQ ID NOS: 18 or 41.
  • an N-terminal start methionine codon (AUG/ATG)) is added to sequence of SEQ ID NOS: 18 or 41.
  • the fusion protein (RAD52-Cas9-QIP) has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence comprising SEQ ID NO:19.
  • an N-terminal methionine is added to the amino acid sequence of SEQ ID NO: 19 at the 1 position.
  • the nucleic acid encoding a fusion protein has a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence comprising SEQ ID NOS:20 or 42.
  • an N-terminal start methionine codon (AUG/ATG)) is added to sequence of SEQ ID NQS:20 or 42.
  • the fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains.
  • protein domains that may be included in the fusion protein include, without limitation, epitope tags, reporter gene sequences, and nucleic acid repair proteins described herein.
  • the fusion protein can include any sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • Lex A DNA binding domain (DBD) fusions Lex A DNA binding domain
  • GAL4A DNA binding domain fusions GAL4A DNA binding domain fusions
  • HSV herpes simplex virus
  • the fusion protein comprises one or more affinity or purification tags.
  • the affinity or purification tag comprises glutathione S-transferase.
  • the affinity or purification tag comprises a maltose binding protein (MBP) or a variant or fragment thereof.
  • the glutathione S-transferase comprises SEQ ID NO:23.
  • the glutathione S-transferase is encoded by SEQ ID NO:24.
  • the maltose binding protein or a variant or fragment thereof comprises SEQ ID NO:25.
  • the maltose binding protein or a variant or fragment thereof is encoded by SEQ ID NO:26.
  • the affinity or purification tag comprises glutathione S- transferase and a maltose binding protein or a variant or fragment thereof.
  • the fusion protein comprises an N-terminal GST and MBP tandem affinity tag.
  • the expression vector comprises a linker sequence following the affinity/purification tag and prior to any cleavage site.
  • the linker is an N10 linker sequence (SEQ ID NO:27), and in some embodiments, the N10 linker is encoded by SEQ ID NO:28.
  • the affinity/purification tag and optional linker sequence is followed by a protease recognition site.
  • the recognition site is an HRV 3C protease recognition site.
  • the HRV 3C protease recognition site comprises the amino acid sequence LEVLFQGP (SEQ ID NO: 12), where cleavage occurs between the Q and G residues.
  • SEQ ID NO: 12 is encoded by SEQ ID NO:29.
  • the fusion protein comprises a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • the NLS is a from SV40 and comprises SEQ ID NO:30, which in some embodiments is encoded by SEQ ID NO:31.
  • the NLS is followed by a fragment of Rad 18 that has a deletion of a putative DNA-binding domain and lacking the N-terminal methionine encoded by SEQ ID NO:5, wild-type Cas9 from Streptococcus pyogenes comprising amino acids 2-1368 of SEQ ID NO: 1, and a homology-directed repair enhancing (HDR Enhancing) N-terminal fragment of CtIP encoded by SEQ ID NO:3.
  • HDR Enhancing homology-directed repair enhancing
  • the invention provides a vector encoding nucleic acids and fusion proteins herein.
  • the vector comprises a nucleic acid encoding a fusion protein comprising a Cas9 polypeptide or a variant or fragment thereof, Rad 18 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the vector comprises a nucleic acid encoding a fusion protein comprising a Cas9 polypeptide or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • expression of the fusion protein in a mammalian cell can be achieved by transient transfection of a vector encoding the fusion protein into the cell.
  • expression of the fusion protein in a mammalian cell can be achieved by integration of the polynucleotides containing the fusion protein into the nuclear genome of the mammalian cell.
  • a variety of vectors and systems for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of a mammalian cell have been developed. Examples of expression vectors are disclosed in, e.g., WO 1994/011026 and are incorporated herein by reference.
  • expression vectors for use in the compositions and methods described herein contain a polynucleotide sequence of the fusion protein, as well as, e.g., additional sequence elements used for the expression of these agents and/or the integration of these polynucleotide sequences into the genome of a mammalian cell.
  • Certain vectors that can be used for the expression of the fusion protein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
  • Other useful vectors for expression of the fusion protein contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription.
  • sequence elements include, e.g., 5’ and 3’ untranslated regions and a polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • the expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.
  • nucleic acids or vectors for therapeutic application in the treatment of conditions described herein or modifying the genome they can be directed to the interior of the cell, and, in particular, to specific cell types.
  • Nucleic acids and vectors can be introduced into a cell by a variety of methods, including transformation, transfection, transduction, direct uptake, projectile bombardment, and by encapsulation of the vector or nucleic acid in a liposome or nanoparticle, such as a lipid nanoparticle.
  • suitable methods of transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection and direct uptake. Such methods are described in more detail, for example, in Green, et al.
  • the fusion protein can also be introduced into a mammalian cell by targeting vectors.
  • vectors can be targeted to the phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to a VSV-G protein, a viral protein with affinity for all cell membrane phospholipids.
  • VSV-G protein a viral protein with affinity for all cell membrane phospholipids.
  • RNA polymerase Recognition and binding of the polynucleotide encoding the fusion protein by mammalian RNA polymerase is important for gene expression. As such, one may include sequence elements within the polynucleotide that exhibit a high affinity for transcription factors that recruit RNA polymerase and promote the assembly of the transcription complex at the transcription initiation site.
  • sequence elements include, e.g., a mammalian promoter, the sequence of which can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase.
  • Polynucleotides suitable for use in the compositions and methods described herein also include those that encode the fusion protein downstream of a mammalian. Promoters that are useful for the expression in mammalian cells include ubiquitous promoters such as the CAG promoter, or the cytomegalovirus (CMV) promoter. Cell type and tissue specific promoters can also be utilized.
  • CAG CAG promoter
  • CMV cytomegalovirus
  • promoters derived from viral genomes can also be used for the stable expression of these agents in mammalian cells.
  • functional viral promoters that can be used to promote mammalian expression of these agents include adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, Epstein barr virus (EBV) promoter, and the Rous sarcoma virus (RSV) promoter.
  • the fusion protein is delivered by a viral vector.
  • a “viral vector” is a virus that can be used to deliver genetic material into target cells. This can be done either in vivo or in vitro. In general, viral vectors are either inherently safe or are modified to present a low handling risk and have low toxicity with respect to the targeted cells.
  • a “retrovirus” is a virus of the family Retroviridae that inserts a copy of its RNA genome into the DNA of a host cell, then uses a reverse transcriptase enzyme to produce DNA from its RNA genome. Retroviruses are known in the art to be useful in gene delivery systems.
  • a “lentivirus” is a type of retrovirus; they are known as slow retroviruses.
  • An “adenovirus” is a virus of the family Adenoviridae that lacks an outer lipid bilayer and includes a double stranded DNA genome. Adenoviruses are well established in the art as viral vectors for gene therapy, and delivering genes coding proteins of interest to particular locations, as to selected cell types, is possible.
  • An “adeno-associated virus” is of the genus Dependoparvovirus, which is of the family Parvoviridae. These are nonenveloped viruses having a single-stranded DNA genome. Adeno-associated viruses are well known in the art as attractive candidates for use as viral vectors for gene therapy. Unlike adenoviruses, they have the advantage that they do not cause disease.
  • nucleic acids of the compositions and methods described herein are incorporated into recombinant AAV (rAAV) vectors and/or virions in order to facilitate their introduction into a cell.
  • rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs that include (1) a heterologous sequence to be expressed (e.g., a polynucleotide encoding the fusion protein) and (2) viral sequences that facilitate stability and expression of the heterologous genes.
  • the viral sequences may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional ITRs) of the DNA into a virion.
  • Such rAAV vectors may also contain marker or reporter genes.
  • useful rAAV vectors have one or more of the AAV wild-type genes deleted in whole or in part but retain functional flanking ITR sequences.
  • the AAV ITRs may be of any serotype suitable for a particular application.
  • the ITRs can be AAV2 ITRs. Methods for using rAAV vectors are described, for example, in Tai et al., J. Biomed. Sci. 1 :279 (2000), and Monahan and Samulski, Gene Delivery' . A (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.
  • the nucleic acids and vectors described herein can be incorporated into a rAAV virion in order to facilitate introduction of the nucleic acid or vector into a cell.
  • the capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene.
  • the cap gene encodes three viral coat proteins, VP1, VP2 and VP3, which are required for virion assembly.
  • the construction of rAAV virions has been described, for instance, in U.S. Pat. Nos. 5,173,414; 5,139,941 ; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitz et al., J. Virol.
  • rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including, without limitation, AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, rhlO, rh39, rh43, rh74, Anc80, Anc80L65, DJ/8, DJ/9, 7m8, PHP.B, PHP.eb, and PHP.S.
  • pseudotyped rAAV vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.).
  • AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for instance, in Duan et al., J. Virol. 75:7662 (2001); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).
  • AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions.
  • suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types.
  • the construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635 (2000).
  • Other rAAV virions that can be used in methods described herein include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biolechnol. 19:423 (2001).
  • the vector is a yeast expression vector.
  • yeast Saccharomyces cerivisae examples include pYepSecl (Baldari, et al., 1987. EMBO J 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933- 943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif).
  • the vector drives protein expression in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • the vector is a prokaryotic expression vector comprising a promoter sequence operably linked to a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises i) Cas9 or a variant or fragment thereof; ii) Rad 18 or a variant or fragment thereof; and iii) CtIP or a variant or fragment thereof.
  • the vector is a prokaryotic expression vector comprising a promoter sequence operably linked to a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises i) Cas9 or a variant or fragment thereof; ii) Rad52 or a variant or fragment thereof; and iii) CtIP or a variant or fragment thereof.
  • Appropriate prokaryotic vectors are typically equipped with a selectable marker-encoding nucleic acid sequence, insertion sites, and suitable control elements, such as termination sequences.
  • the vectors comprise regulatory sequences, including, for example, control elements (i.e., promoter and terminator elements or 5' and/or 3' untranslated regions), effective for expression of the coding sequence in host cells (and/or in a vector or host cell environment in which a modified protein coding sequence is not normally expressed), operably linked to the coding sequence.
  • the expression vector is derived from the pGEX-6P-l commercial vector from Addgene.
  • the prokaryotic expression vector comprises a plasmid.
  • plasmid refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal selfreplicating genetic element in some eukaryotes or prokaryotes, or integrates into the host chromosome.
  • the expression vector is a bacterial expression vector plasmid. In some embodiments, the expression vector is capable of expressing the fusion protein in Escherichia coli.
  • expression refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene.
  • the process includes both transcription and translation.
  • expression of the fusion protein refers to transcription and translation of the fusion protein to be expressed, the products of which can include precursor RNA, mRNA, polypeptide, post-translation processed polypeptide, and derivatives thereof.
  • the terms “vector” and “cloning vector” refer to nucleic acid constructs designed to transfer nucleic acid sequences into cells.
  • expression vector refers to nucleic acid constructs generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell.
  • the vector comprises a recombinant expression cassette, and can be incorporated into a plasmid, chromosome, virus, or nucleic acid fragment.
  • the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.
  • the expression cassette comprises a promoter sequence operably linked to a nucleotide sequence encoding a fusion protein.
  • a "promoter sequence” refers to a DNA sequence which is recognized by a cell for expression purposes.
  • Exemplary promoters include both constitutive promoters and inducible promoters. Such promoters are well known to those of skill in the art. Those skilled in the art are also aware that a natural promoter can be modified by replacement, substitution, addition or elimination of one or more nucleotides without changing its function. The practice of the present invention encompasses and is not constrained by such alterations to the promoter.
  • it is operably linked to a DNA sequence encoding the fusion polypeptide. Such linkage comprises positioning of the promoter with respect to the translation initiation codon of the DNA sequence encoding the fusion DNA sequence.
  • the promoter is an inducible promoter.
  • the promoter is induced by a change in temperature, e.g., an increase of in temperature from 37 degrees Celsius to 42 degrees Celsius.
  • the promoter is induced by an agent, such as a small molecule such as IPTG.
  • the inducible promoter is a lad or lacZ promoter.
  • the lacl gene may also be present in the system.
  • the lacl gene (usually a constitutively expressed gene) encodes the Lac repressor protein Lacl protein that binds to the rack operator of the lac family promoter. Therefore, in some embodiments, when the lac family promoter is utilized, the lac gene can also be included and expressed in the expression system.
  • the expression vector comprises a lac operator.
  • the lac operator comprises SEQ ID NO:32.
  • the lac operator is derived from the pGEX-6P-l commercial vector (Addgene). An operator sequence located at the 5' end serves as a binding site for a repressor protein that blocks RNA polymerase.
  • the expression vector comprises a tac promoter.
  • the tac promoter comprises SEQ ID NO:33.
  • the tac promoter is derived from the pGEX-6P-l commercial vector (Addgene).
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA encoding a secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • Operably linked DNA sequences are usually contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is typically accomplished by ligation at convenient restriction sites. If such sites do not exist, in some embodiments, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the expression constructs of the invention encode a recombinant fusion protein fused to a secretory leader capable of transporting the recombinant fusion protein to the cytoplasm of cells.
  • the expression construct encodes a recombinant fusion protein fused to a secretory leader capable of transporting the recombinant fusion protein to the periplasm.
  • the secretory leader is cleaved from the recombinant fusion protein.
  • transcription enhancer sequences include, but are not limited to, transcription enhancer sequences, translation enhancer sequences, other promoters, activators, translation start and stop signals, transcription terminators, cistron regulators, polycistronic regulators, expression as described above.
  • tag sequences such as the nucleotide sequence "tags” and "tag” polypeptide coding sequences that facilitate identification, separation, purification, and/or isolation of the polypeptide.
  • the expression construct in addition to the protein coding sequence, operably binds to one of the following regulatory elements: promoter, ribosome binding site (RBS), transcription terminator, and translation initiation and termination signals.
  • Useful RBSs can also be obtained from any of the species useful as host cells in, for example, the expression systems of US Patent Application Publication Nos. 2008/0269070 and 2010/0137162. Many specific and various consensus RBSs are known. See Frishman et al., Gene 234 (2): 257-65 (8 Jul. 1999); and B. E. Suzek et al., Bioinformatics 17 (12): 1123-30 (December 2001).
  • a secretory signal or leader coding sequence is fused to the N-terminus of the sequence encoding the recombinant fusion protein.
  • the use of a secretory signal sequence can increase the production of recombinant proteins in bacteria.
  • By utilizing a secretory leader it is possible to increase the yield of properly folded proteins by secreting proteins from the intracellular environment.
  • proteins secreted from the cytoplasm may end up in the peri-cell membrane cavity, attached to the outer membrane, or to the extracellular culture medium. These methods also avoid the formation of inclusion bodies.
  • the recombinant fusion protein targets the peripheral or extracellular space of a host cell.
  • the expression vector further comprises a nucleotide sequence encoding a secretory signal polypeptide operably linked to a nucleotide sequence encoding the recombinant fusion protein.
  • the expression vector further comprises a transcription termination signal downstream of a nucleotide sequence encoding the fusion protein.
  • terminatator sequence refers to a DNA sequence which is recognized by the io expression host to terminate transcription. It is operably linked to the 3' end of the fusion DNA encoding the fusion polypeptide to be expressed.
  • the termination region is obtained from the same gene as the promoter sequence, while in other embodiments it is obtained from another gene. The selection of suitable transcription termination signals is well-known to those of skill in the art.
  • the expression vector comprises a rrnB T1 terminator comprising SEQ ID NO:34.
  • the vector comprises a T7Te terminator comprising SEQ ID NO: 35.
  • the vector comprises a rmB T1 terminator sequence followed by a T7Te terminator sequence.
  • the expression vector comprises a selectable marker encoding nucleic acid sequence.
  • selectable markerencoding nucleotide sequence refers to a nucleotide sequence which is capable of expression in prokaryotic cells and where expression of the selectable marker confers to cells containing the expressed gene the ability to grow in the presence of is a corresponding selective condition.
  • selectable marker will depend on the host cell. Appropriate markers for different bacterial hosts are well known in the art. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, methotrexate, tetracycline, neomycin mycophenolic acid, puromycin, zeomycin, or hygromycin); or (b) complement an auxotrophic mutation or a naturally occurring nutritional deficiency in the host strain. In some embodiments, the selectable marker gene encodes a gene capable of conferring antibiotic resistance. In some embodiments, the selectable marker gene encodes a gene capable of conferring resistance to ampicillin.
  • antibiotics or other toxins e.g., ampicillin, methotrexate, tetracycline, neomycin mycophenolic acid, puromycin, zeomycin, or hygromycin
  • the selectable marker gene encodes a gene capable of conferring antibiotic resistance.
  • the selectable marker gene encodes
  • the expression vector encodes a fusion protein (GST- MBP-eRAD18-Cas9-CtIP) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7.
  • a fusion protein GST- MBP-eRAD18-Cas9-CtIP
  • the fusion protein (GST-MBP-eRAD18-Cas9-CtIP) is encoded by a nucleic acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8.
  • the prokaryotic expression vector comprises a nucleic acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9.
  • the vector map is shown in FIG. 7.
  • the present invention provides host cells which have been transduced, transformed or transfected with a vector as described herein.
  • the host cells are prokaryotic.
  • the culture conditions such as temperature, pH and the like, are those previously used for the parental host cell prior to transduction, transformation or transfection and are apparent to those skilled in the art.
  • the nucleotide sequence encoding a fusion protein is operably linked to a promoter sequence functional in the host cell.
  • a bacterial culture is transformed with an expression vector having a promoter or biologically active promoter fragment or one or more (e.g., a series) enhancers which functions in the host cell, operably linked to a nucleic acid sequence encoding the fusion protein, such that the fusion protein is expressed in the cell.
  • hosts include bacterial cells, such as streptococci, staphylococci, Escherichia coli, Streptomyces and Bacillus subtilis cells.
  • the host cell is Escherichia coli.
  • the invention provides a CRISPR/Cas9 system for modifying a nucleic acid sequence in cells or in cells of a subject comprising: a) a sgRNA molecule comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome b) a nucleic acid or vector encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Radi 8 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the system further comprises a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • the invention provides a CRISPR/Cas9 system for modifying a nucleic acid sequence in cells or in cells of a subject comprising: a) a sgRNA comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome b) a nucleic acid or vector encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof.
  • the system further comprises a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • the invention provides a CRISPR/Cas9 system for modifying a nucleic acid sequence in cells or in cells of a subject comprising, wherein the system comprises a vector comprising a) a sequence encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Radi 8 or a variant or fragment thereof, and CtIP or a variant or fragment thereof and b) a sequence encoding a sgRNA comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome.
  • the system further comprises a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • the invention provides a CRISPR/Cas9 system for modifying a nucleic acid sequence in cells or in cells of a subject comprising, wherein the system comprises a vector comprising a) a sequence encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof and b) a sequence encoding a sgRNA comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome.
  • the system further comprises a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • a CRISPR/CAS9 system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system) to effect the modification of a nucleic acid sequence.
  • the CRISPR/CAS9 system includes one or more nucleic acids or polypeptides encoding fusion proteins and one or more sgRNAs as described herein.
  • the CRISPR/CAS9 system includes a nucleic acid template encoding a sequence of interest for purposes of editing a nucleic acid sequence.
  • target sequence refers to a sequence to which a guide RNA sequence is designed to have complementarity, where hybridization between a target sequence and a guide RNA sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast.
  • a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template,” “editing polynucleotide,” “editing sequence,” or as a “nucleic acid template encoding a sequence of interest” herein.
  • an exogenous template polynucleotide may be referred to as a “nucleic acid template encoding a sequence of interest.”
  • the recombination is homologous recombination.
  • a coding sequence encoding the fusion protein is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • Codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database”, and these tables can be adapted in a number of ways.
  • codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available.
  • one or more codons e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.
  • the sgRNA sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence- specific binding of a CRISPR complex to the target sequence.
  • the degree of complementarity between a sgRNA sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • Burrows-Wheeler Transform e.g. the Burrows Wheeler Aligner
  • ClustalW Clustal X
  • BLAT Novoalign
  • SOAP available at soap.genomics.org.cn
  • Maq available at maq.sourceforge.net
  • a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • Other assays are possible, and will occur to those skilled in the art.
  • a sgRNA sequence may be selected to target any target sequence.
  • the target sequence is a sequence within a genome of a cell.
  • Exemplary target sequences include those that are unique in the target genome.
  • a sgRNA sequence is selected to reduce the degree of secondary structure within the guide sequence.
  • Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
  • the invention provides a method of modifying a genomic sequence of a cell, comprising administering to the cell an effective amount of a nucleic acid or vector encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad 18 or a variant or fragment thereof, and CtIP or a variant or fragment thereof, and one or more additional agents to modify the genomic sequence in the cell.
  • the one or more additional agents include guide RNA(s) and/or template nucleic acid.
  • the invention provides a method of modifying a genomic sequence of a cell, comprising administering to the cell an effective amount of a nucleic acid or vector encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof, and one or more additional agents to modify the genomic sequence in the cell.
  • the one or more additional agents include guide RNA(s) and/or template nucleic acid.
  • the fusion protein comprising Cas9 or a variant or fragment thereof, Radi 8 (or Rad52) or a variant or fragment thereof, and CtIP or a variant or fragment thereof, with optimized (HMEJ) donor DNA can improve knockin performance (both KI precision and efficiency) up to 40-fold (e.g., in cultured human cells) compared to conventional Cas9 knockin (with homologous recombination donor DNA).
  • the invention provides a method of modifying a genomic sequence of a cell, comprising administering to the cell an effective amount of a CRISPR/Cas9 system, wherein the system comprises a) a nucleic acid or vector encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad 18 or a variant or fragment thereof, and CtIP or a variant or fragment thereof; and b) a sgRNA comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome.
  • the cell is further administered an effective amount of a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • the invention provides a method of modifying a genomic sequence of a cell, comprising administering to the cell an effective amount of a CRISPR/Cas9 system, wherein the system comprises a) a nucleic acid or vector encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof; and b) a sgRNA comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome.
  • the cell is further administered an effective amount of a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • the invention provides a method of modifying a genomic sequence of a cell, comprising administering to the cell an effective amount of a CRISPR/Cas9 system, wherein the system comprises a vector comprising a) a sequence encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad 18 or a variant or fragment thereof, and CtIP or a variant or fragment thereof and b) a sequence encoding a sgRNA comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome.
  • the system further comprises a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • the invention provides a method of modifying a genomic sequence of a cell, comprising administering to the cell an effective amount of a CRISPR/Cas9 system, wherein the system comprises a vector comprising a) a sequence encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof and b) a sequence encoding a sgRNA comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome.
  • the system further comprises a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • the invention provides a method of treating a disease of condition in a subject by administering to the subject an effective amount of the cells having a modified genomic sequence.
  • the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of a nucleic acid or vector encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad 18 or a variant or fragment thereof, and CtIP or a variant or fragment thereof, and one or more additional agents to modify a genomic sequence in cells of the subject.
  • the one or more additional agents include guide RNA(s) and/or template nucleic acid.
  • the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of a nucleic acid or vector encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof, and one or more additional agents to modify genomic sequence in the subject.
  • the one or more additional agents include guide RNA(s) and/or template nucleic acid.
  • the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of a CRISPR/Cas9 system, wherein the system comprises a) a nucleic acid or vector encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad 18 or a variant or fragment thereof, and CtIP or a variant or fragment thereof; and b) a sgRNA comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome.
  • the subject is further administered an effective amount of a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of a CRISPR/Cas9 system, wherein the system comprises a) a nucleic acid or vector encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof; and b) a sgRNA comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome.
  • the subject is further administered an effective amount of a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of a CRISPR/Cas9 system, wherein the system comprises a vector comprising a) a sequence encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad 18 or a variant or fragment thereof, and CtIP or a variant or fragment thereof and b) a sequence encoding a sgRNA comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome.
  • the system further comprises a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • the invention provides a method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of a CRISPR/Cas9 system, wherein the system comprises a vector comprising a) a sequence encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Rad52 or a variant or fragment thereof, and CtIP or a variant or fragment thereof and b) a sequence encoding a sgRNA comprising a targeting domain which is complementary with a target domain sequence of a gene or region interest in a genome.
  • the system further comprises a nucleic acid template encoding a sequence of interest (e.g., on a vector).
  • the invention provides a method of modifying a genomic sequence of a cell comprising: introducing a system described herein into a cell.
  • the introducing results in disruption, deletion, or insertion of a target nucleic acid (e.g., gene) in the cell.
  • gene editing results in an increase or decrease in expression of an endogenous or exogenous gene in the cell.
  • the cell is a eukaryotic cell (e.g., a mammalian such as a human) cell).
  • the cell is in vitro, ex vivo, or in vivo.
  • the method treats a disease or condition in a subject.
  • the genomic editing comprises HDR or NHEJ.
  • the invention provides methods comprising delivering one or more polynucleotides, such as or one or more vectors, nucleic acids, or systems, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a cell or to a subject.
  • the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.
  • a CRISPR/Cas9 system is delivered to a cell.
  • Viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CR1SPR/Cas9 system to cells in culture, or in a host organism.
  • Non-viral vector delivery systems include DNA plasmids, RNA (including circular RNA), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome, or lipid nanoparticle.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipidmucleic acid conjugates, naked DNA, lipid nanoparticles, lipid- like nanoparticles, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
  • lipid nucleic acid complexes, including targeted liposomes such as immunolipid complexes
  • the preparation of lipid: nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995): Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
  • RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo).
  • Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
  • Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol.
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIV Simian Immuno deficiency virus
  • HAV human immuno deficiency virus
  • adenoviral based systems may be used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
  • Adeno- associated virus (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641 ; Kotin, Human Gene Therapy 5:793- 801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No.
  • Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and W 2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line.
  • AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line may also be infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.
  • a host cell is transiently or non-transiently transfected with one or more vectors described herein.
  • a cell is transfected as it naturally occurs in a subject.
  • a cell that is transfected is taken from a subject.
  • the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art.
  • cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, C1R, Ratb, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01 , LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, C0S-M6A, BS-C-1 monkey kidney epithelial
  • a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences.
  • a cell transiently transfected with the components of a CRISPR system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a CRISPR complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.
  • cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.
  • one or more vectors described herein are used to produce a non-human transgenic animal or transgenic plant.
  • the transgenic animal is a mammal, such as a mouse, rat, or rabbit.
  • the organism or subject is a plant.
  • the organism or subject or plant is algae.
  • Methods for producing transgenic plants and animals are known in the art, and generally begin with a method of cell transfection, such as described herein.
  • Transgenic animals are also provided, as are transgenic plants, especially crops and algae. The transgenic animal or plant may be useful in applications outside of providing a disease model.
  • transgenic plants especially pulses and tubers, and animals, especially mammals such as livestock (cows, sheep, goats and pigs), but also poultry and edible insects, are preferred.
  • Transgenic algae or other plants such as rape may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol), for instance. These may be engineered to express or overexpress high levels of oil or alcohols for use in the oil or biofuel industries.
  • Certain embodiments provide a method (e.g., gene editing method), comprising: introducing a system described herein into a cell.
  • the introducing results in disruption, deletion, or insertion of a target nucleic acid (e.g., gene) in the cell.
  • the gene editing results in an increase or decrease in expression of an endogenous or exogenous gene in the cell.
  • the cell is a eukaryotic cell (e.g., a mammalian (e.g., human) cell). In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the method treats a disease or condition in a subject.
  • a eukaryotic cell e.g., a mammalian (e.g., human) cell.
  • the cell is in vitro, ex vivo, or in vivo.
  • the method treats a disease or condition in a subject.
  • the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro.
  • the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including micro-algae), and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant (including micro-algae).
  • the target polynucleotide in the cells or cells of a subject in the methods described herein can be any polynucleotide endogenous or exogenous to the cell.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of a eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA).
  • a PAM protospacer adjacent motif
  • PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). Persons skilled in the art will be able to identify PAM sequences for use with a given CRISPR enzyme.
  • target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide.
  • target polynucleotides include a disease associated gene or polynucleotide.
  • a “disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease- affected tissues compared with tissues or cells of a non-disease control.
  • a disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.
  • the transcribed or translated products may be known or unknown, and may be at a normal or abnormal level.
  • nucleic acid or expression vector may be employed for delivering a nucleic acid or expression vector into cells in vitro.
  • Methods of introducing nucleic acids into cells for expression of heterologous nucleic acid sequences are also known to the ordinarily skilled artisan, including, but not limited to electroporation; protoplast fusion with intact cells; transduction; high velocity bombardment with DNA-coated microprojectiles; infection with modified viral (e.g., phage) nucleic acids; chemically- mediated transformation, competence, etc.
  • the host cells can be cultured in suitable nutrient media.
  • the media can be modified as appropriate for any activating promoters, selecting transformants, and/or amplifying expression of the fusion protein by modifying culture conditions, such as temperature, pH and the like.
  • the invention provides a method for producing the fusion protein in a prokaryotic host cell, comprising culturing the prokaryotic host cell comprising the expression vector in a growth media under conditions suitable for the expression of the fusion protein and isolating the fusion protein.
  • the fusion protein is produced and isolated as described in Example 2.
  • isolated and purified refer to a nucleic acid or polypeptide that is removed from at least one component with which it is associated.
  • the isolated protein is substantially free of other cellular components.
  • the term “substantially free” encompasses preparations of the desired fusion polypeptide having less than about 20% (by dry weight) other proteins (i.e., contaminating protein), less than about 10% other proteins, less than about 5% other proteins, or less than about 1% other proteins.
  • the term "substantially pure" when applied to the fusion proteins or fragments thereof of the present invention means that the proteins are essentially free of other substances to an extent practical and appropriate for their intended use.
  • the proteins are sufficiently pure and are sufficiently free from other biological constituents of the host cells so as to be useful in, for example, protein sequencing, and/or producing pharmaceutical preparations.
  • a culture of the prokaryotic host cells that harbors the expression vector is used to inoculate a growth media.
  • the growth media is not limiting, provided it is suitable for promoting growth of the host cells.
  • the growth media comprises Luria broth.
  • the culture of prokaryotic host cells used to inoculate the growth media is an overnight culture. In some embodiments, the host cells are cultured at 37°C.
  • the prokaryotic host cells are incubated in the growth media for a period of time to achieve a certain density prior to inducing expression of the fusion protein.
  • the prokaryotic host cells are incubated in the growth media until the optical density at 600 nanometers reaches a value between about 0.6 to about 0.8, at which point, expression of the fusion protein is induced by addition of an agent or a change in culture conditions.
  • the inducing agent is IPTG.
  • the change in conditions is a change in temperature. In some embodiments, the change in temperature is an increase in temperature, e.g., to about 42 degrees Celsius.
  • the growth media is cooled following incubation of the cells in the growth media and prior to or subsequent to inducing expression of the fusion protein by the addition of an agent, such as IPTG.
  • IPTG is added to a final concentration in the growth media of about 0.25 mM to about 1 .0 mM. In some embodiments, the concentration of IPTG in the growth media is about 0.5 mM.
  • the growth media is incubated at a temperature of between about 14-24 degrees Celsius, wherein the fusion protein is expressed in the prokaryotic host cells in the presence of the agent.
  • the incubation time for cells in the media following induction of expression of the fusion protein is not necessarily limiting provide a sufficient quantity of the fusion protein is produced.
  • the cells are incubated for about 12 to about 24 hours to allow expression of the fusion protein.
  • the cells are incubated in the growth media for about 18 hours at about 16 degrees Celsius to allow for expression of the fusion protein.
  • the prokaryotic cells are lysed following incubation to make a lysate.
  • the cells are pelleted prior to lysis.
  • the cells can be pelleted by centrifugation.
  • the cells are lysed by sonication.
  • the cells are lysed by addition of a solution that promotes lysis.
  • cellular debris is removed from the lysate, e.g., by centrifugation and/or filtration.
  • the fusion protein can be isolated/purified from the lysate.
  • the proteins are precipitated from the lysate prior to purification.
  • the lysate is subjected to chromatography to isolate and purify the fusion protein, e.g., by passing it through a column or other apparatus or composition that is able to capture the fusion protein.
  • the lysate is passed through a chromatography column that comprises an agent that binds to the fusion protein, e.g., glutathione in the case of GST-tagged proteins or Ni 2+ or Co 2+ in the case of 6x-His tagged proteins.
  • the agent can be immobilized on beads or a resin to aid in the purification.
  • a protease is added to cleave the fusion protein.
  • the protease recognizes a HRV 3C protease recognition site (e.g., SEQ ID NO: 12).
  • the protease is a human rhinovirus (HRV) type 14 3C protease.
  • the human rhinovirus (HRV) type 14 3C protease is fused to GST.
  • the protease is added while the fusion protein is bound to an agent on the column, and the fusion protein is subsequently eluted from the column following cleavage by the protease.
  • the fusion protein eluate is filtered.
  • the fusion protein is further concentrated.
  • glycerol is added (e.g., 20%) to the fusion protein and the fusion protein can be stored for later use under appropriate storage conditions.
  • the invention provides pharmaceutical compositions comprising effective amounts of the nucleic acids, polypeptides, vectors encoding the polypeptides, or the CRISPR/Cas9 system herein in combination with a pharmaceutically acceptable excipient.
  • the composition comprises a delivery system, such as liposomes, lipid nanoparticles or lipid-like nanoparticles for delivery of nucleic acids.
  • the lipid or lipid- like nanoparticles are ionizable.
  • a “pharmaceutically acceptable excipient” is a material that acts in concert with an active ingredient of a medication to impart desirable qualities to a drug intended to be introduced into the body of a subject.
  • the desirable qualities could include enhancing long term stability, acting as a diluent for an active ingredient that must be administered in small amounts, enhancement of therapeutic qualities of an active ingredient, facilitating absorption of an active ingredient into the body, adjusting viscosity, enhancing solubility of an active ingredient, or modifying macroscopic properties of a drug such as flowability or adhesion.
  • compositions can comprise but are not limited to diluents, binders, pH stabilizing agents, disintegrants, surfactants, glidants, dyes, flavoring agents, preservatives, sorbents, sweeteners and lubricants. These materials can take many different forms. See, e.g., Nema, et al., Excipients and their use in injectable products, PDA J. Pharm. Sci. & Tech. 1997, 51(4): 166-171.
  • nucleic acids, vectors (e.g., AAV vectors) and CRISPR/Cas9 system described herein may be incorporated into a vehicle for administration into a patient, such as a human patient suffering from any of the conditions described herein.
  • Pharmaceutical compositions containing nucleic acids such as RNA or vectors, such as viral vectors, that contain a polynucleotide encoding the fusion protein can be prepared using methods known in the art.
  • such compositions can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.
  • nucleic acids or vectors may be prepared in water suitably mixed with one or more excipients, carriers, or diluents.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference).
  • the formulation may be sterile and may be fluid to the extent that easy syringability exists. Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • a solution containing a pharmaceutical composition described herein may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • compositions described herein may be administered to a subject by a variety of routes, such as local administration, ocular, retinal, intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration.
  • routes such as local administration, ocular, retinal, intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration.
  • routes such as local administration, ocular, retinal, intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration.
  • Treatment may include administration of a composition containing the nucleic acids or vectors (e.g., AAV vectors) described herein in various unit doses.
  • Each unit dose will ordinarily contain a predetermined quantity of the therapeutic composition.
  • the quantity to be administered, and the particular route of administration and formulation, are within the skill of those in the clinical arts.
  • a unit dose need not be administered as a single injection but may include continuous infusion over a set period of time. Dosing may be performed using a syringe pump to control infusion rate in order to minimize damage to the tissue administered.
  • the nucleic acids, fusion proteins, or vectors described herein are provided in the form of a kit or system that optionally comprises one or more guide RNAs (e.g., sgRNAs), and/or a template nucleic acid encoding a sequence of interest.
  • guide RNAs e.g., sgRNAs
  • a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein.
  • Reagents may be provided in any suitable container.
  • a kit may provide one or more reaction or storage buffers.
  • Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form).
  • a buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof.
  • the buffer is alkaline.
  • the buffer has a pH from about 7 to about 10.
  • the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element.
  • the kit comprises a homologous recombination template polynucleotide.
  • a prokaryotic expression vector comprising a promoter sequence operably linked to a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises i) Cas9 or a variant or fragment thereof; ii) Rad 18 or a variant or fragment thereof; and iii) CtIP or a variant or fragment thereof.
  • HDR Enhancing N-terminal fragment of CtIP comprises an amino acid sequence at least 90% identical to SEQ ID NO:3 (amino acids 1-296).
  • a nucleic acid comprising a sequence encoding a fusion protein comprising Cas9 or a variant or fragment thereof, Radi 8 or a variant or fragment thereof, and CtlP or a variant or fragment thereof.
  • nucleic acid of any of paragraphs 56-62, wherein the fusion protein comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 17.
  • nucleic acid of any of paragraphs 79-87, wherein the fusion protein is encoded by a nucleotide sequence comprising SEQ ID NO:20 or SEQ ID NO:42.
  • nucleic acid of any of paragraphs 79-87, wherein the fusion protein is encoded by a nucleotide sequence at least about 60% identical to SEQ ID NO:20 or SEQ ID NO:42.
  • Cas9 targets genomic loci with high specificity. For knockin with doublestrand break repair, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible.
  • This example demonstrates a high-throughput workflow to comparatively assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations yielding orders-of- magnitude increases in knockin precision, increased knockin performance in vitro and in vivo in the developing mouse brain. Continued comparative assessment of editing efficiency and precision with this framework will further the development of high- performance editing agents for in vivo knockin and future genome therapeutics. Results
  • the genotype of the BFP + population matched that of the WT BFP sequence.
  • the dark population exhibited a complex mixture of sequence results in the vicinity of the BFP gRNA cleavage site, representing unintended on-target edits due to imprecise repair.
  • Sequencing decomposition using the ICE algorithm 15 on amplicons from the dark sorted population revealed a predominance of deleterious indels (79% frameshift vs. 11% in-frame), in line with a loss of fluorescence due to knockout (Brinkman et al., Nucleic Acids Res., (2014), 42:el68).
  • the GFP + population exhibited a single genotype containing the desired H26Y point mutation knockin from the donor template (Fig. 1C-D).
  • the sequencing results matched the fluorescence surrogates, thus validating this platform for high-throughput ratiometric screening of knockin agents.
  • knockin donors were provided on plasmids with the knockin sequence flanked by ⁇ 800bp homology arms, the length of which did not significantly affect results within a range of 500 to 1500 bp (data not shown).
  • the HMEJ donor differed from the HR donor by the insertion of gRNA Binding Sites (GRBS) flanking the homology arms, which are cleaved by Cas9 to create linear dsDNA donors in cells. The orientation of the GRBSs did not have significant effects on editing performance.
  • GRBS gRNA Binding Sites
  • Cas9 HF variants were generally not significantly different from Cas9 WT , although Cas9 HF -CtIP showed a 1.6-fold improvement in knockin efficiency specifically with the HR donor (Fig. 2D). Knockout rates did not significantly differ among the Cas9 variants (Fig. 2C), and thus, the KI/KO ratios (knockin precision) mirror the differences seen in knockin efficiency (Fig. 2D).
  • Double fusions on Cas9 improve editing performance
  • dn53BPl-, TIP60-, and RNF169- fused Cas9 did not significantly differ from the Cas9-only control. Rad52 and eRad 18 fusion, however, showed 18% and 28% reductions in knockout frequency, respectively (Fig. 3B-D).
  • compound fusion of each of the five DNA repair proteins with CtIP led to significant reductions in the knockout rate, with eRadl8, Rad52, and TIP60 showing the most pronounced decreases (45%, 38% and 38%, respectively).
  • eRadl8 led to significant improvements in overall knockin precision.
  • Fig. 5A To test the knockin efficiency of Cas9-RC in vivo, we used in utero plasmid electroporation in the embryonic mouse brain (Fig. 5A) targeting integration of a 2A mCherry cassette at the 3’ end of the endogenous [3- Actin (ActB) locus (Fig. 5B) (Saito etal., Dev. Biol., (240), 237-246, (2001 ); Mikuni et al., Cell, (2016), 165:1803-1817).
  • a combination of four plasmids containing Cas9 or Cas9-RC, HMEJ donor with the 2A mCherry knockin, ActB gRNA, and a GFP transfection marker were electroporated into embryonic day 14.5 (E14.5) wild-type mice targeting progenitors of projection neurons of sensorimotor cortex.
  • E7 postnatal day 7
  • electroporation of Cas9-RC led to an increase in mCherry + knockin neurons compared to Cas9 (Fig. 5C).
  • HMEJ donor and gRNA constructs to fuse the fluorescent protein mScarlet onto the N-terminus of the GPI-linked membrane protein Neuronal growth regulator 1 (Negri), a protein with variable expression in the mouse brain (Miyata et al., Neuroscience, (2003), 117:645-658). Knockin efficiency was significantly lower than actin knockin. Lower efficiencies may result from the fact that, unlike actin, not all knockin cells will express Negri, or because many cells express it at levels below our detection threshold. When comparing Cas9-RC efficiency to Cas9, there was no significant difference in knockin efficiency (Fig. 6C). This may indicate that increases of Cas9-RC performance in vivo may be locusspecific.
  • Cas9-RC with tritrode electroporation and supercoiled constructs to target Purkinje cells in the mouse embryonic cerebellum, as a case to examine a difficult to transduce cell type that we were not able to knock in with Cas9.
  • HMEJ donor and gRNA constructs to knock in the fluorescent protein mGreenLantem downstream of the Pvalb locus, expressed by parvalbumin+ Purkinje cells. While efficiency was low, we consistently detected sparse knockin parvalbumin+ Purkinje cells (Fig. 6D).
  • Negri did not show increases in efficiency of Cas9-RC over Cas9, possibly due to the increased size of Cas9-RC resulting in the targeting of fewer cells.
  • Knockin on a highly expressed cell-type- specific locus allowed us to demonstrate the use of Cas9-RC on Purkinje cells, a highly differentiated cell type.
  • optimal agents can be selected based on the experimental need. For example, with ex vivo editing, one might prioritize efficiency if there are facile methods for post hoc selection of properly edited cells, whereas precision may be prioritized for contexts where edited cells cannot be selected, such as with in vivo editing.
  • Cas9-RC high- performance DSB repair editor
  • Cas9-RC When paired with HMEJ donor templates, Cas9-RC outperformed other knockin agents by over 30-fold in human cells and showed potential for 3 -fold increases in the mouse brain, albeit not at all loci tested.
  • Cas9-RC enables high performance for large genomic edits, such as fluorescent protein knockin.
  • This complements parallel developments in base editors and Prime editing, which offer high performance but are limited to smaller edits.
  • a diverse toolkit of precision editors will be useful to broaden the scope of in vivo editing applications. As presented in our study, having standardized platforms for quantitative comparison of new tools and novel combinations will further support efforts towards precision in vivo editing for both basic research and the development of future human therapeutics.
  • Cas9-RC is a new DSB repair genome editor demonstrating enhanced knockin performance in vitro and in vivo.
  • Mammalian expression plasmids and knockin donor template plasmids were constructed with a combination of standard cloning techniques.
  • oligos Integrated DNA Technologies
  • GGA Golden Gate Assembly
  • Cas9 expression constructs were assembled by a modified mMoClo system (Duportet et al., Nucleic Acids Res., (2014), 42:13440-13451).
  • CD1 mouse pups were euthanized via decapitation at postnatal day 0.
  • the skin was sterilized and removed from the pup’s back using sterile surgical tools. Skins were placed dermis-side down on cold 0.25% Trypsin with EDTA (Invitrogen) and incubated at 4°C overnight.
  • the epidermis was separated from the dermis in a sterile hood. Dermis was minced with a razor blade and triturated in warm 10% FBS lx GlutaMAX DMEM using a glass pipette 10-20 times to separate individual cells. The suspension was then transferred to a 50 mL conical tube and centrifuged at 150 g.
  • the cell pellet was resupsended in 10% FBS lx Glutamax DMEM and filtered through a 100 pm cell strainer (BD Biosciences). Cells were counted using a hemocytometer and cell viability was estimated using Trypan Blue (Sigma). Approximately 4-5xl0 6 cells were used for each electroporation. Cells were centrifuged at 150 g and resuspended in 100 pl AM AXA Nucleofection solution (Lonza) at the proper concentration and combined with 1-3 pg of desired DNA mixture in a cuvette. The cuvette was electroporated with the AMAXA biosystems Nucelofector II (Lonza) using the manufacturers settings for Mouse Embryonic Fibroblasts.
  • the solution was immediately transferred to 12- well glass bottom plates (#1.5H; Cellvis) that were pretreated with poly-L-lysine (Sigma Aldrich) diluted 1 :12 in sterile PBS the night before, containing prewarmed sterile-filtered Dulbecco’s Modified Eagle Medium (DMEM; ThermoFisher) supplemented with 10% Fetal Bovine Serum (FBS; Gibco) and lx GlutaMAX (Gibco) at the desired density and incubated at 37°C/5% CO2. Half volume fresh medium was exchanged the next day.
  • DMEM Modified Eagle Medium
  • FBS Fetal Bovine Serum
  • Gibco Fetal Bovine Serum
  • Gibco lx GlutaMAX
  • Electroporations of plasmid DNA were performed in utero on embryonic day 14.5 (E14.5) to target cortical layer II/III, as previously described (Saito et al., Dev. Biol., (240), 237-246, (2001); Poulopoulos et al., Nature, (2019), 565:356-360.).
  • the triple electrode in utero electroporation approach was utilized (dal Maschio et al., Nat. Commun., (2012), 3:960; Szczurkowska, J. et al., Nat. Protoc., (2016), 11:399-412).
  • DNA solutions were prepared to 4 pg/pL total DNA, with 1 pg/pL of each of the relevant plasmids (donor, guide, Cas9, and fluorescent protein). Symptoms were deeply anesthetized with isoflurane under a vaporizer with thermal support (Patterson Scientific Link7 & Heat Therapy Pump HTP-1500). The abdominal area was prepared for surgery with hair removal, surgical scrub, and 70% ethanol and 10% Betadine solution. A midline incision was made to expose the uterine horns.
  • DNA solution was injected into one lateral brain ventricle for cerebral cortex electroporation at E14.5, or in the 4th ventricle for cerebellar Pukinje cell electroporation at El 1.5.
  • 4 x 50 ms square pulses of 35 V were applied to target the nascent sensorimotor areas of the cortical plate.
  • 6 x 50 ms square pulses at 35V was performed at E14.5 and 25 V at El 1.5.
  • 4-6 pups were electroporated per dame.
  • Uterine horns were placed back inside the abdominal cavity, and monofilament nylon sutures (AngioTech) were used to close muscle and skin incisions.
  • electroporated mouse pups were non-invasively screened for unilateral cortical or cerebellar fluorescence using a fluorescence stereoscope (Leica MZIOf with X-Cite FIRE LED light source) and returned to their dame until postnatal day 7 (P7) or P14.
  • fluorescence stereoscope Leica MZIOf with X-Cite FIRE LED light source
  • Tissue was prepared by intracardial perfusion with PBS and 4% paraformaldehyde. Brains were cut to 80 pm coronal sections on a vibrating microtome (Leica VT1000). Sections were immunolabeled in blocking solution consisting of 5% bovine serum albumin and 0.2% Triton X-100 in PBS for 30 minutes, then incubated overnight at 4°C with primary antibodies diluted in blocking solution. Sections were washed in PBS, incubated for 3-4h at room temperature with secondary antibodies diluted 1 :400-l : 1000 in blocking solution. Following PBS washes, sections were mounted on slides with Fluoromount- G Mounting Medium with DAPI (ThermoFisher Scientific).
  • Fluorescence images were acquired using a Nikon Ti2-E inverted microscope fitted with an automated registered linear motor stage (HLD117, Pior Scientific), a Spectra-X 7 channel LED light engine (Lumencor), and standard filter sets for DAPI, FITC, TRITC, and Cy5. Images were stitched and analyzed with NIS-Elements (Nikon) using an automated script to identify and count electroporated cells in brain sections. Knockin-positive neurons were counted manually using ImageJ (NIH) and independently by at least two blinded investigators. Five 80 pm sections, centered at the middle of the anteroposterior axis of the electroporation field and taken every other section, were analyzed per brain, with counts aggregated across sections from the same brain.
  • NIS-Elements NIS-Elements
  • Example 2 Identifying protein domains that add their enhancements to editing performance even when fused to Cas9 enables the production of a single fusion protein for use in ribonucleoprotein (RNP) editing applications, without the need for expression or delivery of these as additional co-factors.
  • RNP ribonucleoprotein
  • IPTG-specific Cas9 immunoreactive bands corresponding to the predicted electrophoretic mobilities of full-length GST-MBP-Cas9-RC (316 kDa) and Cas9-RC (250 kDa) were abundant in lysates, indicating that Cas9-RC can be bacterially expressed for RNP applications.

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Abstract

La présente invention concerne des acides nucléiques contenant une séquence codant pour une protéine de fusion comprenant Cas9 ou un variant ou un fragment de celle-ci, Radi 8 ou Rad52 ou un variant ou un fragment de celle-ci, et CtIP ou un variant ou un fragment de celle-ci, des vecteurs codant pour celles-ci, des compositions pharmaceutiques associées et des procédés de modification d'une séquence génomique dans des cellules par administration des compositions.
PCT/US2024/060720 2023-12-18 2024-12-18 Compositions et procédés pour l'édition améliorée de génome à l'aide de protéines de fusion cas9 Pending WO2025137069A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170073695A1 (en) * 2014-12-31 2017-03-16 Synthetic Genomics, Inc. Compositions and methods for high efficiency in vivo genome editing
US20210403922A1 (en) * 2019-01-07 2021-12-30 Crisp-Hr Therapeutics, Inc. Non-toxic cas9 enzyme and application thereof
US20230203178A1 (en) * 2020-07-06 2023-06-29 Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. Chimeric antigen receptor car or car construct targeting bcma and cd19 and application thereof
US20230357798A1 (en) * 2020-10-12 2023-11-09 The Board Of Trustees Of The Leland Stanford Junior University Gene correction for x-cgd in hematopoietic stem and progenitor cells

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
US20170073695A1 (en) * 2014-12-31 2017-03-16 Synthetic Genomics, Inc. Compositions and methods for high efficiency in vivo genome editing
US20210403922A1 (en) * 2019-01-07 2021-12-30 Crisp-Hr Therapeutics, Inc. Non-toxic cas9 enzyme and application thereof
US20230203178A1 (en) * 2020-07-06 2023-06-29 Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. Chimeric antigen receptor car or car construct targeting bcma and cd19 and application thereof
US20230357798A1 (en) * 2020-10-12 2023-11-09 The Board Of Trustees Of The Leland Stanford Junior University Gene correction for x-cgd in hematopoietic stem and progenitor cells

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Title
RICHARDSON RYAN R., STEYERT MARILYN, KHIM SAOVLEAK N., CRUTCHER GARRETT W., BRANDENBURG CHERYL, ROBERTSON COLIN D., ROMANOWSKI AND: "Enhancing Precision and Efficiency of Cas9-Mediated Knockin Through Combinatorial Fusions of DNA Repair Proteins", THE CRISPR JOURNAL, vol. 6, no. 5, 1 October 2023 (2023-10-01), US, pages 447 - 461, XP093331874, ISSN: 2573-1599, DOI: 10.1089/crispr.2023.0036 *

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