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WO2024227911A2 - Éditeurs de base crispr hautement actifs obtenus par évolution dirigée liée à un substrat assistée par cas (caslide) - Google Patents

Éditeurs de base crispr hautement actifs obtenus par évolution dirigée liée à un substrat assistée par cas (caslide) Download PDF

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WO2024227911A2
WO2024227911A2 PCT/EP2024/062227 EP2024062227W WO2024227911A2 WO 2024227911 A2 WO2024227911 A2 WO 2024227911A2 EP 2024062227 W EP2024062227 W EP 2024062227W WO 2024227911 A2 WO2024227911 A2 WO 2024227911A2
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seq
dna modifying
dna
modifying enzyme
enzyme
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WO2024227911A3 (fr
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Duran SÜRÜN
Frank Buchholz
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Technische Universitaet Dresden
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Technische Universitaet Dresden
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04004—Adenosine deaminase (3.5.4.4)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
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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 invention relates to a DNA modifying enzyme comprising or consisting of a DNA editing protein and a Cas protein and a peptide linker, wherein said DNA editing protein and said Cas protein are connected by said peptide linker; and wherein said DNA modifying enzyme has been obtained by directed molecular evolution of an original enzyme; and wherein said DNA modifying enzyme shows a total base editing activity on three defined target sites, which is at least 4-fold higher than the total base editing activity of said original enzyme.
  • the invention relates further to a method of directed molecular evolution of a DNA modifying enzyme and to the use evolved DNA modifying enzymes in research, medicine and agriculture.
  • DNA modifying enzymes such as CRISPR base editors hold tremendous potential to revolutionize therapies for genetic diseases.
  • CRISPR base editors e.g. spCas9- CBE or ABE
  • base editors e.g. spCas9- CBE or ABE
  • viral vectors e.g. AAV
  • miniature CRISPR systems e.g. Casl2f
  • Directed evolution applies the principles of Darwinian evolution to the laboratory to improve protein features (Li, H., 2021; Danecek, P. et al., 2021).
  • large gene variant libraries ⁇ 10 5 — ⁇ 10 8
  • Screening libraries to identify efficient variants is conventionally a manual process that is labor, resource, and time intensive.
  • the number of variants that can be tested is limited, reducing the probability of identifying optimal variants.
  • a high-throughput method for comparing large sets of enzymes would be desirable.
  • DNA modifying enzymes such as miniature CRISPR systems with improved DNA editing efficiency and in reliable methods to produce large gene variant libraries of such DNA modifying enzymes, and to screen these large libraries for variants with optimal activity.
  • the invention provides in first aspect a DNA modifying enzyme comprising or consisting of a DNA editing protein and a Cas protein and a peptide linker, wherein said base editing protein and said Cas protein are connected by said peptide linker; and wherein said DNA modifying enzyme has been obtained by directed molecular evolution of an original enzyme; and wherein said DNA modifying enzyme shows a editing rate which is at least 10%, 20 % or 30 %, preferably at least 40 %, more preferably at least 50 %, most preferably at least 60 % higher than the base editing rate of said original enzyme.
  • the invention provides a method of directed molecular evolution of a DNA modifying enzyme as claimed in any one of claims 1 to 15 or of any of the accompanying factors (or components) such as sgRNA scaffold, or pegRNA scaffold in the case of prime editing, said method comprising the steps of a. Generation of a library of said DNA modifying enzyme, b. Cloning said library in an expression vector containing the nucleic acid of at least one target site, preferably the nucleic acids of 2 or 3 target sites of said DNA modifying enzyme, c. Transformation of the expression vector of step b. into a host cell and expression of the library of said DNA modifying enzyme in said host cell; d.
  • the invention provides compositions comprising the DNA modifying enzymes of the invention and the use said DNA modifying enzymes or the compositions comprising the same in research, medicine and agriculture.
  • Fig. 1 schematically shows the CRISPR associated substrate linked directed evolution (CaSLiDE) method used in Examples 4 and 9.
  • the gene of a DNA modifying enzyme, which performs base editing is amplified using error-prone PCR or DNA shuffling. This results in multiple copies of the gene with mutations.
  • This gene library is then cloned into a bacterial expression vector, that contains target sites for the base editor, with restriction enzyme (RE) sites located at the position where base change is supposed to happen.
  • the expression vector is transformed into bacteria, which express the DNA modifying enzyme.
  • a sgRNA translated from the same vector guides the base editing enzyme to the target site and causes editing, dependent on the activity of the enzyme.
  • An example for editing can be seen in the bubble in the top right of the figure.
  • A is modified to a G which causes the loss of a RE-site.
  • a digest with the respective RE results in two possible outcomes, the plasmid is digested twice or once. Only single digested plasmids are valid templates for the error-prone PCR (performed with the primers that are indicated as arrows) to start a new cycle of evolution.
  • Fig. 2A is a schematic illustration of the base editing assay explained in detail in Example 8.
  • Fig. 2B shows agarose gel analysis of the base editing assays performed for Casl2f- ABE (original enzyme, SEQ ID NO: 1) and for the evolved library derived from the original enzyme (WT) on target sites 1 to 3 (SEQ ID NOs: 3521, 3522 and 3523).
  • Fig. 2C shows the base editing assay results for the evolved and enrich Casl2f-ABE library on different protein expression levels (induced with L-arabinose).
  • Fig. 3 shows an overview of the workflow of a further particularly preferred method according to the present invention.
  • Evolved variants of Casl2f-ABE are cloned together with a unique molecular identifier (UMI) into a vector containing three target sites.
  • UMI unique molecular identifier
  • FIG. 4A shows the results of a screen of evolved variants of Casl2f-ABE on three target sites, namely target site 1 (SEQ ID NO: 3521), target site 2 (SEQ ID NO: 3522), target site 3 (SEQ ID NO: 3523).
  • UMI-clusters containing evolved variants of Casl2f-ABE are indicated as grey spots, and original enzyme (WT, SEQ ID NO: 1) control clusters are indicated as black spots.
  • Three selected clusters are highlighted (2 (SEQ ID NO: 471), 3030 (SEQ ID NO: 20), 3301 (SEQ ID NO: 3))
  • Fig. 4B shows average editing rates of the original enzyme clusters.
  • Fig. 4A shows the results of a screen of evolved variants of Casl2f-ABE on three target sites, namely target site 1 (SEQ ID NO: 3521), target site 2 (SEQ ID NO: 3522), target site 3 (SEQ ID NO: 3523).
  • FIG. 4C shows that 58 non-original clusters identified with the method of the present invention had editing rates of more than 90% on all three target sites.
  • the clusters shown in Fig. 4C have the following SEQ ID NOs:
  • Fig. 4D shows the percentages of the reads of the Casl2f-ABE screen with correct editing, no editing or other editing outcomes of the original enzyme clusters (WT) and the three variants from clusters 2, 3030 and 3301 (SEQ ID NO: 471, SEQ ID NO: 20 and SEQ ID NO: 3, respectively) on the three target sites used in the screen (Figs. 3 and 4A, SEQ ID NOs: 3521, 3522 and 3523).
  • Fig. 5A shows respective agarose gel analysis of the base editing plasmid assays performed for four different DNA modifying enzymes (Clusters 2 (SEQ ID NO: 471), 3030 (SEQ ID NO: 20), and 3301 (SEQ ID NO: 3)and original enzyme (WT) control, SEQ ID NO: 1) on three different target sites simultaneously (target site 1 (SEQ ID NO: 3521), target site 2 (SEQ ID NO: 3522), target site 3 (SEQ ID NO: 3523)) in triplicate replicates (rep. 1-3).
  • Fig. 5B shows the quantified results of the base editing plasmid assay (Fig. 5A).
  • band intensities of the edited and non-edited products of the selected Casl2f-ABE clusters (2 (SEQ ID NO: 471), 3030 (SEQ ID NO: 20), and 3301 (SEQ ID NO: 3)) and the original enzyme (WT) control were determined using the image analysis software Fiji.
  • Band intensity values of the edited products were then divided by the combined values of the edited and non-edited bands to calculate a fraction, which was converted to a percentage value. Each dot represents one replicate of the assay of the respective variant.
  • Fig. 6A shows editing results of the DNA modifying enzymes from clusters 2 (SEQ ID NO: 471), 3030 (SEQ ID NO: 20), and 3301 (SEQ ID NO: 3) and the original (WT) Casl2f-ABE (SEQ ID NO: 1) on three E. coli genome target sites (SEQ ID NOs: 3533, 3534 and 3535) analyzed by Sanger sequencing. Included is also a negative control where no DNA modifying enzymes were expressed in the bacterium. The arrow indicates where base editing is supposed to occur. The sequence of the non-edited genomic DNA is displayed above the curves. Bases were also placed directly above each line graph at the position where editing is supposed to happen. In case of two bases at this position, the upper base corresponds to the higher curve.
  • Fig. 6B shows quantified base editing rates of the Sanger sequencing results from Fig. 6A using the EditR program (Kluesner et al., 2018).
  • Fig. 7 shows an overview of the workflow of a further particularly preferred method according to the present invention. Sequences encoding different variants of a DNA modifying enzyme are cloned into plasmids comprising respective enzyme target sites. E. coli cells are transformed with the vectors. Transformed bacteria are cultured to express the encoded DNA modifying enzymes. Plasmid DNA is isolated. From all plasmids isolated from the cell cultures, the region of interest (comprising regions 1 and 2) is excised and sequenced by nanopore sequencing.
  • Fig. 8A shows comparative base editing efficiencies between ABE8e and new evolved TadA with spCas9. Schematic illustration of the GFP-to-BFP conversion.
  • Fig. 8B shows FACS profiles of HEK293-eGFP cells 7 days post co-transfection with mock transfected (Neg. Control) or ABE8e and new ABEs mRNA transfected cells in combination with GFP-sgRNA-Y66H.
  • Fig. 8C shows the Quantification of GFP-to-BFP and GFP to no fluorescent protein (NoFP) edited cells. In dark grey the GFP-to-BFP conversion is shown and in light grey the bystander editing GFP-to-NoFP is shown.
  • the clusters shown in Fig. 8B and Fig 8C have the following SEQ ID NOs:
  • Fig. 8D shows the comparison of the TadA domains (clusters 2 (SEQ ID NO: 471), 3030 (SEQ ID NO: 20), and 3301 (SEQ ID NO: 3)) to Tad8r (SEQ ID NO: 10115)and with TadA8e as control (SEQ ID NO: 1) in a plasmid-based assay on three different target sites (target site 1 (SEQ ID NO: 3521), target site 2 (SEQ ID NO: 3522), target site 3 (SEQ ID NO: 3523)) in triplicate replicates.
  • UnlCasl2fl-WT of SEQ ID NO. 1 was used as Cas protein in the DNA modifying enzymes tested.
  • Fig. 9 shows a schematic illustration of the 3 different CaSLiDE approaches of the invention.
  • Fig. 10 shows an amino acid sequence alignment of the top 30 clusters according to Approach II.
  • the top-performing clusters from the evolved library are aligned against the original TadA-ABE8e sequence.
  • the modified amino acids are marked in grey.
  • the clusters shown in Fig. 10 have the following SEQ ID NOs:
  • Fig. 11 shows a scatter plot of the evolved clusters obtained with Approach II.
  • A shows the results of a screen of evolved variants of TadA as UnlCasl2fl-ABE on three target sites, namely target site 1 (SEQ ID NO: 3521), target site 2 (SEQ ID NO: 3522), target site 3 (SEQ ID NO: 3523) at two L-Arabinose concentration (Ara2 and
  • UMI-clusters containing evolved variants of TadA are indicated as grey spots, and original enzyme (ABE8e) control clusters are indicated as black spots.
  • Fig. 12 shows an amino acid sequence alignment of the top 30 clusters obtained by Approach III.
  • the top-performing clusters from the evolved library are aligned against the original WT UnlCasl2fl sequence.
  • the mortified amino acids are marked in grey.
  • the clusters shown in Fig. 10 have the following SEQ ID NOs:
  • Fig. 13 shows a scatter plot of the evolved clusters obtained with Approach III.
  • the figure shows the results of a screen of evolved variants of UnlCasl2fl as ABE on three target sites, namely target site 1 (SEQ ID NO: 3521), target site 2 (SEQ ID NO: 3522), target site 3 (SEQ ID NO: 3523).
  • UMI-clusters containing evolved variants of UnlCasl2fl are indicated as grey spots, and original enzyme (WT) control clusters are indicated as black spots.
  • Table II shows base editing activities of variants of the DNA modifying enzyme of the invention, which were obtained by Approach I.
  • Table III shows base editing activities of variants of the DNA modifying enzyme of the invention, which were obtained by Approach II.
  • Table IV shows base editing activities of variants of the DNA modifying enzyme of the invention, which were obtained by Approach III.
  • nucleic acid and “nucleic acid molecule” are used synonymously herein and are understood as well-accepted in the art, i.e. as single or double- stranded oligo- or polymers of deoxyribonucleotide or ribonucleotide bases or both.
  • nucleic acids as used herein includes not only deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), but also all other linear polymers in which the bases adenine (A), cytosine (C), guanine (G) and thymine (T) or uracil (U) are arranged in a corresponding sequence (nucleic acid sequence).
  • the invention also comprises the corresponding RNA sequences (in which thymine is replaced by uracil), complementary sequences and sequences with modified nucleic acid backbone or 3 'or 5'- terminus. Nucleic acids in the form of DNA are however preferred.
  • target site refers to a specific nucleotide sequence which a DNA modifying enzyme recognizes, and at which or in the vicinity of which a DNA modification such as breakage and strand exchanges occur.
  • a target site is a target site for Cas like gene editors.
  • the target site for the UnlCasl2fl enzyme for example has a 4 bp PAM site (TTTR) followed by a 20 bp recognition site for the sgRNA.
  • TTTR 4 bp PAM site
  • Further target sites of DNA modifying enzymes are e.g. UnlCasl2fl target sites (disclosed e.g.
  • UMI unique molecular identifier
  • a UMI is an oligonucleotide comprising at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 random nucleotides.
  • a UMI is an oligonucleotide comprising at least 50 random nucleotides.
  • a UMI may preferably form part of a UMI-tag, which may further comprise one or more sequences down-stream and/or upstream of the random nucleotides of the UMI (e.g. flanking the random nucleotides), which sequences may serve as one or more primer binding sites.
  • a UMI-tag may further comprise one or more and preferably at least two restriction sites preferably down-stream and/or upstream of the random nucleotides of the UMI.
  • a UMI-tag comprises a first primer binding site, a first restriction site, the random nucleotides of the UMI, a second restriction site, and a second primer binding site.
  • cell or "host cell” as used herein relates to an intact cell, i.e. a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material.
  • An intact cell preferably is a viable cell, i.e. a living cell capable of carrying out its normal metabolic functions.
  • said term relates to any cell which can be transfected or transformed with an exogenous nucleic acid.
  • regulatory nucleic acid sequence refers to gene regulatory regions of DNA. In addition to promoter regions, this term encompasses operator regions more distant from the gene as well as nucleic acid sequences that influence the expression of a gene, such as cis-elements, enhancers or silencers.
  • promoter region refers to a nucleotide sequence on the DNA allowing a regulated expression of a gene. The promoter region allows regulated expression of the nucleic acid encoding for the respective protein. The promoter region is located at the 5'-end of the gene and thus before the coding region. Both, bacterial and eukaryotic promoters are applicable for the present invention.
  • essentially identical used in the context of two nucleic acids or polypeptides means that the two sequences compared share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity over the specified sequence, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the percentage identity for "essentially identical” likewise applies for sequences that are "essentially reverse complementary" to each other.
  • the invention provides a DNA modifying enzyme comprising or consisting of a DNA editing protein and a Cas protein and a peptide linker, wherein said base editing protein and said Cas protein are connected by said peptide linker; and wherein said DNA modifying enzyme has been obtained by directed molecular evolution of an original enzyme; and wherein said DNA modifying enzyme shows an editing rate which is at least 30 %, preferably at least 40 %, more preferably at least 50 %, most preferably at least 60 % higher than the base editing rate of said original enzyme.
  • base editing rate is defined as the number of base editing events in percent (%) caused by a DNA modifying enzyme of the invention on a target site.
  • An “average base editing rate” represents the arithmetic mean (in %) of the base editing rates estimated for more than 1 target sites.
  • the “original enzyme” can be a wildtype enzyme or an enzyme that has already been subject to mutation or molecular evolution, and which has been used as starting point to produce the DNA modifying enzyme of the invention.
  • a wildtype enzyme comprising or consisting of a DNA editing protein and a Cas protein and a peptide linker has been subjected to directed molecular evolution.
  • mutations in the amino acid sequence of the resulting DNA modifying enzyme occurred after directed molecular evolution randomly over the entire amino acid sequence, including in the DNA editing protein and/or the Cas protein and/or peptide linker sequences. Mutations were not restricted to only the DNA editing protein sequence, the linker sequence or the Cas protein sequence.
  • a wildtype enzyme comprising or consisting of a DNA editing protein and a Cas protein and a peptide linker has been used as starting point for directed molecular evolution, but only the Cas protein domain was subjected to directed molecular evolution.
  • mutations in the amino acid sequence of the resulting DNA modifying enzyme occurred after directed molecular evolution only in the Cas protein sequence, but not in the DNA editing protein and not in the peptide linker sequences.
  • Said DNA modifying enzyme preferably shows an editing rate which is at least 30 %, preferably at least 40 %, more preferably at least 50 %, most preferably at least 60 % higher, or even 70%, 80% or 90 % higher than the editing rate of the original enzyme on at least one target site, preferably on two target sites, more preferably on three target sites of said DNA modifying enzyme.
  • “Original enzyme” in accordance with the invention means the DNA modifying enzyme variant that is used as starting material for the directed molecular evolution.
  • the DNA modifying enzyme can be a naturally occurring DNA modifying enzyme or it can be a variant of a naturally occurring DNA modifying enzyme.
  • the DNA modifying enzyme is an evolved enzyme.
  • the DNA modifying enzyme has been evolved applying Cas-assisted substrate linked directed evolution (CaSLiDE).
  • CaSLiDE Cas-assisted substrate linked directed evolution
  • CaSLiDE Cas-assisted SLiDE
  • directed molecular evolution in accordance with the invention is preferably Substrate Linked Directed Evolution (SLiDE) as known in the art or which has been adapted as detailed herein in the second aspect of the invention and in examples 4 and 9 hereinbelow.
  • a “target site” of a DNA modifying enzyme is a nucleotide sequence.
  • the target site can be the target site known for the respective DNA modifying enzyme.
  • the target site is not limited to the specific target site that might be known in the art for the DNA modifying enzyme.
  • the target site can be a modified version of a known target site.
  • the target site can be a naturally occurring target site of a DNA modifying enzyme, or it can be an artificially created target site such as a modified version of a known target site, preferably of a known naturally occurring target site.
  • Such modified versions of a known target site preferably differ in one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve nucleotides from the known target site.
  • a modified target site may alternatively differ from a known target site in about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or in about 50% of the nucleotides.
  • Said DNA editing protein as part of the DNA modifying enzyme of the invention is preferably selected from the group consisting of deoxycytidine deamination-derived editors (CBEs) which facilitate C ⁇ G to T ⁇ A mutations, deoxycytidine deamination and DNA glycosylase derived editors (CGBEs) which facilitate C ⁇ G to G ⁇ C mutations, deoxycytidine deamination and DNA glycosylase derived editors (GBEs) which facilitate C ⁇ G to A ⁇ T, deoxyadenosine deamination- derived base editors (ABEs) which facilitate A ⁇ T to G ⁇ C mutations, deoxyadenosine deamination and DNA glycosylase derived base editors (AYBE) which facilitate A ⁇ T to C ⁇ G and A ⁇ T to T ⁇ A mutations and prime editing enzymes such as reverse transcriptase in combination with a prime editing guide (peg)RNA, which facilitate all types of DNA substitution, insertions and deletions on a target site.
  • CBEs deoxycyt
  • the DNA modifying enzyme of the invention is a deoxycytidine deamination-derived editor (CBE).
  • the DNA modifying enzyme of the invention is a deoxyadenosine deamination-derived base editor (ABE).
  • DNA editing generally means any type of DNA substitution, insertions and deletions on a target site.
  • DNA editing relates to “base editing”, i.e. base exchanges of DNA and “DNA editing rate” relates to “base editing rate” of DNA.
  • Said Cas protein as part of the DNA modifying enzyme of the invention is preferably selected from the group consisting of Cas9; IscB; HEARO; Cas 12, such as Cas 12a, Cas 12b, Cas 12c, Casl2d, Casl2f, Casl2i, Casl2j, Casl2g, Casl2h, TnpB, Casp, Casl2e, Casl2k, Casl21, C2c4, C2c8, C2c9; and Casl3, such as Casl3a, Casl3b, Casl3c, Casl3d.
  • said Cas protein is Casl2f.
  • said DNA modifying enzyme comprises or consists of a base editing protein, preferably an ABE base editor protein, a Casl2f protein and a peptide linker.
  • Said peptide linker typically comprises or consists of 5 to 35 amino acids, preferably of 32 amino acids.
  • a DNA modifying enzyme which comprises an ABE base editor protein, a Casl2f protein and a peptide linker.
  • the invention therefore provides in a preferred embodiment a DNA modifying enzyme comprising or consisting of a DNA editing protein and a Cas protein and a peptide linker, wherein said DNA editing protein and said Cas protein are connected by said peptide linker; and wherein said DNA modifying enzyme has been obtained by directed molecular evolution of an original enzyme, wherein said original enzyme is selected from the group consisting of the enzymes of SEQ ID NO: 1 and SEQ ID NO: 7547; characterized in that said Cas protein in said DNA modifying enzyme is UnlCasl2f; said DNA editing protein is TadA; said DNA modifying enzyme comprises single amino acid substitutions at least at one or at more positions when compared to said original enzyme of SEQ ID NO: 1 or SEQ ID NO: 7547.
  • DNA modifying enzymes comprising or consisting of an ABE base editor protein such as TadA, a Casl2f protein such as UnlCasl2f, and a peptide linker, namely the DNA modifying enzymes of SEQ ID Nos. 2 to 3520.
  • an ABE base editor protein such as TadA
  • Casl2f protein such as UnlCasl2f
  • a peptide linker namely the DNA modifying enzymes of SEQ ID Nos. 2 to 3520.
  • the efficacy of the DNA modifying enzymes could be improved in all three parts of the DNA modifying enzymes, i.e. in the ABE base editor protein, the peptide linker and in the Casl2f protein.
  • the protein with SEQ ID NO: 1 was used as wildtype protein.
  • said ABE base editor protein such as TadA is selected from the group consisting of proteins consisting of amino acids 1 to 166 of SEQ ID Nos. 2 to 3520.
  • Said ABE base editor protein such as TadA preferably comprises at least one single amino acid substitution, preferably wherein the single amino acid substitution is comprised in the catalytic region, preferably wherein the single amino acid substitution is at a position of a conserved amino acid in the catalytic region of said ABE base editor such as TadA, when compared to the original ABE base editor protein such as TadA, which has the amino acid sequence of amino acids 1 to 166 of SEQ ID NO. 1.
  • said Casl2f protein such as UnlCasl2f is selected from the group consisting of amino acids 199 to 726 of SEQ ID NOs. 2 to 3520.
  • Said Casl2f protein such as UnlCasl2f preferably comprises at least one single amino acid substitution, preferably wherein the single amino acid substitution is comprised in the catalytic region, preferably wherein the single amino acid substitution is at a position of a conserved amino acid in the catalytic region of said Casl2f protein such as UnlCasl2f, when compared to the amino acid sequence of the original Casl2f protein such as UnlCasl2f, which has the amino acid sequence of amino acids 199 to 726 of SEQ ID NO. 1.
  • said peptide linker is selected from the group consisting of amino acids 167 to 198 of SEQ ID NOs. 2 to 3520 and comprises at least one single amino acid substitution, when compared to the amino acid sequence of the original linker, which has the amino acid sequence of amino acids 167 to 198 of SEQ ID NO. 1.
  • Amino acid substitutions in the amino acid sequence of SEQ ID NO. 1, which lead to superior base editing efficacy of the DNA modifying enzyme of the invention are selected from those listed in Table I. These amino acid substitutions occurred in 30 % of the 30 DNA modifying enzymes with the highest base edition rates provided by the invention.
  • Table I-I Amino acid substitutions in SEQ ID NO: 1 which lead to superior base editing efficacy of the DNA modifying enzyme of the invention according to Approach I of the directed molecular evolution.
  • the invention provides in a preferred embodiment DNA modifying enzymes comprising single amino acid substitutions at least at one or at more positions selected from the group consisting of those listed in table I.
  • DNA modifying enzymes comprising or consisting of an ABE base editor protein such as TadA, a Casl2f protein such as UnlCasl2f and a peptide linker, where mutations are found in the ABE base editor proteins such as TadA only, namely the DNA modifying enzymes, which comprise a ABE base editor protein such as TadA of SEQ ID Nos. 3551 to 7546.
  • the efficacy of the DNA modifying enzymes could be improved especially in one part of the DNA modifying enzymes, namely in the ABE base editor protein such as TadA.
  • the protein with SEQ ID NO: 1 was used as wildtype protein.
  • said ABE base editor protein such as TadA is selected from the group consisting of proteins consisting of SEQ ID Nos. 3551 to 7546, wherein said proteins of SEQ ID Nos. 3551 to 7546 represent amino acids 1 to 166 of the entire DNA modifying protein.
  • the remaining amino acids 167 to 726 of the DNA modifying enzymes obtained by Approach II are identical with amino acids 167 to 726 of the protein of SEQ ID NO. 1.
  • Said ABE base editor protein such as TadA preferably comprises at least one single amino acid substitution, preferably wherein the single amino acid substitution is comprised in the catalytic region, preferably wherein the single amino acid substitution is at a position of a conserved amino acid in the catalytic region of said ABE base editor such as TadA, when compared to the original ABE base editor protein such as TadA, which has the amino acid sequence of amino acids 1 to 166 of SEQ ID NO. 1.
  • Amino acid substitutions in the amino acid sequence of SEQ ID NO. 1, which lead to superior base editing efficacy of the DNA modifying enzyme of the invention are selected from those listed in Table I- II. These amino acid substitutions occurred in 30 % of the 30 DNA modifying enzymes of Approach II with the highest base edition rates provided by the invention.
  • Table I-II Amino acid substitutions in SEQ ID NO: 1 which lead to superior base editing efficacy of the DNA modifying enzyme of the invention according to Approach II of the directed molecular evolution.
  • the invention provides in a preferred embodiment DNA modifying enzymes comprising single amino acid substitutions at least at one or at more positions selected from the group consisting of those listed in table I-II.
  • DNA modifying enzymes comprising or consisting of an ABE base editor protein such as TadA, a Casl2f protein such as UnlCasl2f and a peptide linker, where mutations are found in the Casl2f protein such as UnlCasl2f only, namely the DNA modifying enzymes, which comprise a Casl2f protein such as UnlCasl2f of SEQ ID Nos. 7547 to 10114, wherein said proteins of SEQ ID Nos. 7547 to 10114 represent amino acids 199 to at most 726 of the entire DNA modifying protein.
  • the remaining amino acids 1 to 198 of the DNA modifying enzymes obtained by Approach III are identical with amino acids 1 to 198 of the protein of SEQ ID NO. 7547.
  • the efficacy of the DNA modifying enzymes could be improved especially in one part of the DNA modifying enzymes, namely in the Casl2f protein such as UnlCasl2f.
  • the protein with SEQ ID NO: 7547 was used as wildtype protein.
  • said Casl2f protein such as UnlCasl2f is selected from the group consisting of amino acids consisting of SEQ ID NOs. 7547 to 10114.
  • Said Casl2f protein such as UnlCasl2f preferably comprises at least one single amino acid substitution, preferably wherein the single amino acid substitution is comprised in the catalytic region, preferably wherein the single amino acid substitution is at a position of a conserved amino acid in the catalytic region of said Casl2f protein such as UnlCasl2f, when compared to the amino acid sequence of the original Casl2f protein such as UnlCasl2f, which has the amino acid sequence of amino acids 199 to 726 of SEQ ID NO. 7547.
  • Amino acid substitutions in the amino acid sequence of SEQ ID NO. 7547, which lead to superior base editing efficacy of the DNA modifying enzyme of the invention are selected from those listed in Table I-III. These amino acid substitutions occurred in 30 % of the 30 DNA modifying enzymes of Approach III with the highest base edition rates provided by the invention.
  • Table I-III Amino acid substitutions in SEQ ID NO. 7547 which lead to superior base editing efficacy of the DNA modifying enzyme of the invention according to Approach III of the directed molecular evolution.
  • the invention provides in a preferred embodiment DNA modifying enzymes comprising single amino acid substitutions at least at one or at more positions selected from the group consisting of those listed in table I-III.
  • TadA in the DNA modifying enzymes of Approaches I, II and III is Tad8e.
  • the DNA modifying enzymes of the invention comprising or consisting of an ABE base editor protein such as TadA, a Casl2f protein such as UnlCasl2f and a peptide linker, namely the DNA modifying enzymes of SEQ ID Nos.
  • target site 1 SEQ ID NO: 3521
  • target site 2 SEQ ID NO: 3522
  • target site 3 SEQ ID NO: 3523
  • an editing activity on each of target site 1 SEQ ID NO: 3521
  • target site 2 SEQ ID NO: 3522
  • target site 3 SEQ ID NO: 3523
  • base editing events in % are shown in Tables II, III and IV for the best performing DNA modifying enzymes of the invention in comparison to the original enzymes of SEQ ID NO: 1 or SEQ ID NO: 7547 for target site 1 (SEQ ID NO: 3521), target site 2 (SEQ ID NO: 3522), and target site 3 (SEQ ID NO: 3523).
  • the present invention provides in a further embodiment a DNA modifying enzyme which shows an editing activity on each of target site 1 (SEQ ID NO: 3521), target site 2 (SEQ ID NO: 3522), target site 3 (SEQ ID NO: 3523), wherein said DNA modifying enzyme is selected from the group consisting of SEQ ID NO. 2 to 3520, 3551 to7546 and 7548 to 10114.
  • the invention provides a DNA modifying enzyme comprising or consisting of a DNA editing protein and a Cas protein and a peptide linker, wherein said DNA editing protein and said Cas protein are connected by said peptide linker; and wherein said DNA modifying enzyme has been obtained by directed molecular evolution of an original enzyme, wherein said original enzyme is selected from the group consisting of the enzymes of SEQ ID NO: 1 and SEQ ID NO: 7547; characterized in that said Cas protein in said DNA modifying enzyme is UnlCasl2f; said DNA editing protein is TadA; said DNA modifying enzyme comprises single amino acid substitutions at least at one or at more positions when compared to said original enzyme of SEQ ID NO: 1 or SEQ ID NO: 7547; and wherein said DNA modifying enzyme shows a total base editing activity which is at least 4- fold higher than the total base editing activity of said original enzyme on the three target sites having the SEQ ID NO: 3521, SEQ ID NO: 3522 and SEQ ID NO: 3523.
  • total base editing activity is a value expressed in “fold” and represents the improvement of the “average base editing rate” (definition see above) of the DNA modifying enzyme of the invention over the average base editing rate of the original enzyme.
  • Said total base editing activity is measured with a method described in Figures 3 and 5 herein and which comprises the steps of: i) Providing a plasmid comprising a nucleic acid encoding for said DNA modifying enzyme, a unique molecular identifier (UMI) and the three target sites of SEQ ID NO: 3521, SEQ ID NO: 3522 and SEQ ID NO: 3523; ii) Expressing the DNA modifying enzyme of step i) and performing base editing on the three target sites of SEQ ID NO: 3521, SEQ ID NO: 3522 and SEQ ID NO: 3523; iii) Isolating the plasmid DNA and excising DNA fragments comprising the UMI, the DNA modifying enzyme and the three target sites of SEQ ID NO: 3521, SEQ ID NO: 3522 and SEQ ID NO: 3523; iv) Sequencing the fragments of step iii); v) Clustering the UMIs and determining the base editing events for the DNA modifying enzyme on each of the
  • DNA modifying enzyme of the invention shows a total base editing activity which is at least 4- fold higher than the total base editing activity of said original enzyme on the three target sites having the SEQ ID NO: 3521, SEQ ID NO: 3522 and SEQ ID NO: 3523.
  • the DNA modifying enzyme obtained by approach I shows a total base editing activity which is at least 8-fold higher, more preferably at least 10-fold or higher, most preferably at least 12-fold or higher than the total base editing activity compared to original enzyme of SEQ ID NO: 1 on all three target sites having the SEQ ID NO: 3521, SEQ ID NO: 3522 and SEQ ID NO: 3523.
  • the DNA modifying enzyme obtained by approach I is selected from a protein having an amino acid sequence of one of the SEQ ID NOs shown in Table II.
  • the DNA modifying enzyme obtained by approach II shows a total base editing activity (Ara5) which is at least 4-fold higher, more preferably at least 5-fold or higher or at least 6-fold or higher, most preferably at least 7-fold or higher than the total base editing activity compared to original enzyme of SEQ ID NO: 1, or a total base editing activity (Ara2) which is at least 4-fold higher, more preferably at least 6-fold or higher or at least 8-fold or higher, most preferably at least 7-fold or higher than the total base editing activity compared to original enzyme of SEQ ID NO: 1 on all three target sites having the SEQ ID NO: 3521, SEQ ID NO: 3522 and SEQ ID NO: 3523.
  • the DNA modifying enzyme obtained by approach II is selected from a protein having an amino acid sequence of one of the SEQ ID NOs shown in Table III.
  • the DNA modifying enzyme obtained by approach III shows a total base editing activity which is at least 8-fold higher, more preferably at least 10-fold or higher or at least 15-fold or higher, most preferably at least 20-fold or higher than the total base editing activity compared to original enzyme of SEQ ID NO: 7547 on all three target sites having the SEQ ID NO: 3521, SEQ ID NO: 3522 and SEQ ID NO: 3523.
  • the DNA modifying enzyme obtained by approach III is selected from a protein having an amino acid sequence of one of the SEQ ID NOs shown in Table IV.
  • the DNA modifying enzyme obtained by approach I shows an editing activity on all three of target site 1 (SEQ ID NO: 3521), target site 2 (SEQ ID NO: 3522), target site 3 (SEQ ID NO: 3523) of equally to or more than 90 %, and wherein said DNA modifying enzyme is selected from the group consisting of SEQ ID NOs.
  • the DNA modifying enzyme of the invention belongs to the 30 DNA modifying enzymes with the highest editing activity obtained by Approach I herein and is selected from SEQ ID NOs. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 33, 101, 471.
  • the DNA modifying enzyme of the invention obtained by Approach II belongs to the 30 DNA modifying enzymes with the highest editing activity obtained by Approach II herein and is selected from SEQ ID NOs. 3551, 3566, 3567, 3563, 3568, 3569, 3580, 3572, 3565, 3575, 3560, 3558, 3564, 3554, 3556, 3552, 3573, 3571, 3574, 3557, 3559, 3553, 3578, 3570, 3579, 3576, 3555, 3562, 3561, and 3577.
  • the DNA modifying enzyme of the invention obtained by Approach III belongs to the 30 DNA modifying enzymes with the highest editing activity obtained by Approach III herein and is selected from SEQ ID NOs. 7548, 7549, 7550, 7551, 7552, 7553, 7554, 7555, 7556, 7557, 7558, 7559, 7560, 7561, 7562, 7563, 7564, 7565, 7566, 7567, 7568, 7569, 7570, 7571, 7572, 7573, 7574, 7575, 7576, and 7577.
  • the invention provides a method of directed molecular evolution of a DNA modifying enzyme or of any of the accompanying factors (or components) such as sgRNA scaffold, or pegRNA scaffold in the case of prime editing, said method as described hereinbefore, said method comprising the steps of a. Generation of a library of said DNA modifying enzyme, b. Cloning said library in expression vectors containing the nuclei acid of at least one target site, preferably of 2 or 3 target sites of said DNA modifying enzyme, c. Transformation of the expression vector of step b. into a host cell and expression of the library of said DNA modifying enzyme in said host cell; d.
  • said library of a DNA modifying enzyme that is generated in step a) is a library of DNA modifying enzymes comprising or consisting of an ABE base editor protein, a Casl2f protein and a peptide linker (a Casl2f-ABE library).
  • said library of DNA modifying enzymes may be generated using error-prone-PCR, e.g. with a low- fidelity DNA polymerase.
  • an expression vector in accordance with the present invention is not limited to any specific expression vector.
  • an expression vector comprises an origin of replication, a promoter, as well as specific gene sequences that allow phenotypic selection of host cells comprising the vector.
  • the vector comprises a first region encoding a variant of a DNA modifying enzyme, and a second region comprising at least one, at least two, at least three or more target sites of a DNA modifying enzyme and as defined herein above.
  • a particularly preferred expression vector to be used in accordance with the present invention is the pEVO vector as described in Buchholz and Stewart 2001.
  • the gene encoding the DNA modifying enzyme is preferably excised from a respective evolution library as shown in Fig. 3.
  • Respective evolution libraries are described e.g. in Buchholz and Stewart 2001, and in Lansing et al. 2022.
  • the library of expression vectors is introduced into the host cells.
  • a host cell within the meaning of the invention is a naturally occurring cell or a cell line (optionally transformed or genetically modified) that comprises at least one vector as described above.
  • the invention thus includes host cells that include at least one expression vector according to the invention as a plasmid.
  • a host cell includes one expression vector according to the invention as a plasmid. This embodiment is particularly useful if bacteria are used as host cells.
  • the host cell is selected from the group consisting of bacterial cells, yeast cells, fungal cells, and mammalian cells.
  • Suitable bacterial cells include cells from gram-negative bacterial strains such as strains of Escherichia coli, Proteus, and Pseudomonas, and gram-positive bacterial strains such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus.
  • Suitable fungal cells include cells from species of Trichoderma, Neurospora, and Aspergillus .
  • Suitable yeast cells include cells from species of Saccharomyces (for example Saccharomyces cerevisiae), Schizosaccharomyces (for example Schizosaccharomyces pombe), Pichia (for example Pichia pastoris and Pichia methanolica), and Hansenula.
  • Suitable mammalian cells include for example CHO cells, BHK cells, HeLa cells, COS cells, HEK293T and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for the expression can be used as well.
  • the host cell is a bacterial cell.
  • the host cell is an E. coli cell.
  • the introduction of the nucleic acids into the host cells is performed using techniques of genetic manipulation known by a person skilled in the art. Among suitable methods are cell transformation, transfection or viral infection, whereby a nucleic acid sequence encoding the DNA modifying enzyme is introduced into the cell as a component of the vector.
  • the cell culturing is carried out by methods known to a person skilled in the art for the culture of the respective cells. Therefore, cells are preferably transferred into a conventional culture medium, and cultured at temperatures and in a gas atmosphere that is conducive to the survival of the cells and allows expression of the encoded protein.
  • a preferred method for introducing the library of expression vectors into host cells is transformation, preferably by electroporation.
  • E. coli cells can be transformed with the vectors using electroporation.
  • the host cells comprising the expression vectors are plated on a suitable medium such as an agar plate to allow growing and selection of individual cell colonies. This step may also be used for selecting a specific number of individual colonies instead of culturing all cells simultaneously, thereby decreasing the overall number of the members of the library and reducing the number of enzyme variants to be screened.
  • the host cells comprising the expression vectors can be briefly cultured in a respective culture medium (e.g. for a limited time, for example 0.5 to 1 hour) and an amount of the culture medium equal to the desired number of variants can be taken from this culture medium for further culturing as described in the following.
  • the number of transformed bacteria present per pl of culture medium can be estimated based on the number of colonies on the plates. If for example 10 pl of the medium were spread on a plate and the plate after incubation shows 100 distinct colonies, one pl of medium contains ten transformed bacteria.
  • the host cells comprising the library of expression vectors or the cells selected as explained above are cultured to allow the expression of the plasmid. Culturing conditions depend on the cell line used and are not particularly limited. It is, however, not necessary to culture each selected colony individually. The selected colonies can be cultured together in a single cell culture. Alternatively, the host cells comprising the library of expression vectors or the cells selected as explained above are cultured in more than one separate cell culture.
  • the nucleic acid encoding for the DNA modifying enzyme further comprises a regulatory nucleic acid sequence, preferably a promoter region.
  • expression of the nucleic acid encoding for DNA modifying enzyme is initiated or regulated by activating the regulatory nucleic acid sequence.
  • the cells are preferably cultured under conditions allowing cell growth and protein expression.
  • the cells are grown for more than two hours up to 12 hours or longer, allowing more than two, preferably more than three, more than four, more than five, more than six, more than seven, more than eight, more than nine or more than ten cell doublings.
  • the DNA modifying enzymes Upon expression of the DNA modifying enzymes in the culture of host cells, the DNA modifying enzymes start modifying the DNA at the one or more target sites in the expression vector, if said DNA modifying enzymes recognizes the respective target site(s).
  • plasmids are isolated and digested with restriction enzymes, which target the target sites. Applying a restriction digest to the plasmids results in one linear fragment for the edited plasmids and in two fragments for non-edited plasmids. An error-prone PCR using the indicated primers (arrows) will exclusively generate a product of the edited plasmids.
  • These amplified and mutated active Casl2fl-ABE variants are then subjected to the next evolution cycle.
  • the expression of said DNA modifying enzymes in said host cell is usually performed in presence of L-arabinose.
  • L-arabinose levels and thus induction of enzyme expression are lowered from 200 pg/ml (in the initial evolution cycle) to 1 pg/ml in further evolution cycles.
  • the method of the invention may be performed such that it includes 2 to 100, such as 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cycles of directed molecular evolution.
  • said library of said DNA modifying enzyme may be further enriched by high fidelity PCR.
  • said further enriched library of said DNA modifying enzyme may be screened for the DNA editing rate, preferably the base and/or prime editing rate of the DNA modifying enzymes on at least one, preferably 2 or 3 target sites, wherein said screening comprising sequencing of the DNA modifying enzymes contained in said library and activity testing in a base editing assay in comparison to the original enzyme of said DNA modifying enzyme.
  • plasmid DNA is isolated from the culture(s) of host cells after culturing of the host cells and expressing the encoded library of DNA modifying enzymes. Isolating of plasmid DNA from a culture of cells can be done by any conventional methods well known in the art. After isolation, the plasmid DNA is sequenced to determine its nucleotide sequence at least in the region coding for the DNA modifying enzyme (first region) and in the region coding for the target site(s) (second region). It will be appreciated that more parts or all of the plasmid can be sequenced than regions containing the sequences encoding the one or more DNA modifying enzyme and the one or more target sites.
  • the first and the second region are sequenced since the information obtained by sequencing the first and the second region is sufficient for the method of the present invention.
  • This sequencing step allows determining the sequence of the specific DNA modifying enzyme, the sequence of the target region and whether not the DNA sequence in the second region on the expression vector has been altered by the DNA modifying enzyme.
  • the first and the second region of the vector are sequenced in a single method step, i.e. they are sequenced together and not subsequently.
  • the sequencing method is not particularly limited. However, for particularly high-throughput methods, long-read sequencing methods are preferred.
  • a particularly preferred sequencing method in accordance with the present invention is nanopore sequencing as described in Wang et al., 2021 (incorporated herein by reference), or as provided by e.g. Oxford Nanopore Technologies, UK.
  • nanopore sequencing works by monitoring changes to an ionic current as nucleic acids are passed through a protein nanopore. The resulting signal is decoded to provide the specific DNA sequence. Based on the sequencing results, an activity rate for each DNA modifying enzyme on the respective target site(s) can be determined.
  • sequence comparisons of the screen sequence data to the known sequences of the DNA modifying enzymes are performed to identify which sequence reads belong to which DNA modifying enzyme.
  • DNA modifying enzyme sequences can be known because they have been sequenced previously, or because they were synthesized according to a defined sequence. The sequence reads are separated according to their sequence matches and from here on processed separately for consensus sequence generation and determination of the activity rate. It is preferred that in such cases no UMI and no UMI-tag are used in the method of the invention.
  • the method according to the present invention may further comprise the steps of clustering the unique molecular identifiers, generating and polishing consensus sequences, determining the number of DNA modification events for each DNA modifying enzyme, and determining an activity rate for each DNA modifying enzyme.
  • the UMI sequence of the sequencing reads can be identified by comparing the sequences upstream and/or downstream of the UMI.
  • the DNA sequence with the length of the UMI after the match is collected with the sequence read ID.
  • the DNA sequence with the length of the UMI before the match is collected with the sequence read ID.
  • the DNA sequence between the matches is collected with the sequence read ID.
  • the collected UMI sequences are then clustered based on sequence identity, while maintaining their association to their read IDs, for example with vsearch (Rognes et al., 2016). Based on these clusters and their associated read IDs, the sequence reads are then separated into different files, where each file contains reads that corresponds to one UMI- cluster. From each of these clusters of separated reads, the part of the sequence that contains the gene for the DNA modifying enzyme is aligned to a reference gene that contains a high similarity to the screened variants. Typically, such a gene is determined through sequencing of the screened DNA modifying enzyme library or by selecting the DNA modifying enzyme gene that was modified to produce the screened variant library.
  • a software for consensus sequence generation and sequence polishing can be used to determine the most likely gene sequence of the DNA modifying enzyme associated to this cluster based on all sequence reads. Polishing in this respect refers to additional rounds of processing of the reads, leading to improved sequence accuracy.
  • the method for determining the activity rate is not particularly limited.
  • the sequencing reads which have been separated through sequence comparison or UMI clustering, are compared to expected sequences from the second region in the changed and non-changed sequences. Based on this sequence comparison, the number of reads matched to the changed DNA can be counted. The counted changes divided by the total number of reads matching the second region gives rise to an activity rate of the DNA modifying enzyme which is associated to the separated reads.
  • additional references for the second region can be provided to associate the activity rate for the identified DNA modifying enzyme to multiple target sites.
  • the additional references are sequences of the second region with the target sites replaced with the alternative target sites that were used in the screen. These references can also be provided in a changed and non-changed version to determine the activity rate as described above.
  • Genes encoding DNA modifying enzymes are cloned together with a unique molecular identifier (UMI) into an expression vector, which further contains one or more of the target sites of interest.
  • UMI unique molecular identifier
  • the barcoded plasmids are transformed into respective cells, and the cells are spread on one or more culture plates. Based on the number of colonies on the plates, a defined number of clones is cultured in medium that induces the expression of the genes encoding the DNA modifying enzymes. This results in multiple copies of the transformed plasmids, with a fraction of the plasmids being modified by the DNA modifying enzymes.
  • the plasmids are isolated and sequenced, preferably using nanopore sequencing. Using the UMIs, the sequences are then clustered.
  • clusters are used to construct accurate consensus sequences of the variant genes.
  • a cluster thus refers to an enzyme variant identified with the methods of the present invention. Counting of the recombined plasmids generates an editing rate for the particular variant of the DNA modifying enzyme on the respective target site(s).
  • the method of the invention as laid out above can be likewise used for screening and sequencing of a plurality of target sites for DNA modifying enzymes. Accordingly, all embodiments and descriptions provided herein above apply likewise to a further aspect of the present invention, which provides a method for screening and sequencing of a plurality of target sites for DNA modifying enzymes, the method comprising the steps of: providing a library of expression vectors comprising a first region encoding a DNA modifying enzyme, and a second region comprising variants of one or more two target sites of a DNA modifying enzyme; introducing the library of expression vectors into host cells; culturing the host cells and expressing the DNA modifying enzymes; isolating plasmid DNA from the culture of host cells; sequencing the first and the second region of the expression vectors; and determining, based on the sequencing results, whether the DNA sequence of the second region on the expression vector has been altered by the DNA modifying enzyme.
  • the target sites in the second region of the vector can be different naturally occurring target site of different DNA modifying enzymes.
  • the target sites can be an artificially created target sites such as modified versions of a known target site.
  • modified versions of a known target site preferably differ in one, two, three, four, five, six, seven, eight, nine or ten nucleotides from the known target site.
  • a modified target site may alternatively differ from a known target site in about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or in about 50% of the nucleotides.
  • the method preferably utilizes nanopore technology for sequencing full-length enzyme variants at fast turn-around times.
  • the DNA editing rate for each enzyme variant can be quantified.
  • the sequencing results can be clustered to a highly accurate consensus sequence.
  • the inventors were able to select DNA modifying enzymes from the enriched library which show a DNA editing rate, preferably a base and/or prime editing rate which is at least 10 %, 20% or 30 %, preferably at least 40 %, more preferably at least 50 %, most preferably at least 60 % higher, or 70 % or higher or 80 % higher or 90 % higher than the base editing rate of the original DNA modifying enzyme.
  • a DNA editing rate preferably a base and/or prime editing rate which is at least 10 %, 20% or 30 %, preferably at least 40 %, more preferably at least 50 %, most preferably at least 60 % higher, or 70 % or higher or 80 % higher or 90 % higher than the base editing rate of the original DNA modifying enzyme.
  • the invention further relates to a DNA modifying enzyme as described in the first aspect of the invention, which has been prepared and selected by the method of the second aspect of the invention.
  • the DNA modifying enzyme described in the first aspect of the invention may be comprised in in a pharmaceutical composition, which is suitable for use in certain medical therapies, such as gene therapy.
  • the invention further relates to use of the DNA modifying enzyme as described in the first aspect of the invention, or a pharmaceutical composition comprising the same in medicine.
  • the DNA modifying enzyme as described in the first aspect of the invention are also useful tools in research applications.
  • the DNA modifying enzyme as described in the first aspect of the invention are also useful tools in the generation of engineered plants in agriculture.
  • a single stranded DNA oligonucleotide was ordered containing primer binding sites, restriction sites, and 50 random bases. There were two variants of this oligonucleotide depending on which pEVO plasmid (Buchholz and Stewart, 2001) it was intended to be used for.
  • the "Casl2f UMI-tag” was used for screening of the evolved Casl2f-ABE variants.
  • a 50 pl PCR was performed with 20 pM of the primers UMIprimer F and UMIprimer R (SEQ ID NOs: 3525 and 3526), 10 pM of the oligonucleotide, 10 pl of 5x MyTaq buffer and 1 pl MyTaq polymerase (Bioline).
  • the PCR-cycler was set 94°C for 90 seconds, followed by 10 cycles of 15 seconds at 94°C, 15 seconds at 54°C and 15 seconds at 72°C.
  • the resulting PCR product was digested with Sbfl and Xbal for the Casl2f UMI-tag. The digest was then again cleaned up with the Isolate II PCR and Gel Kit (Bioline) and measured with a Qubit HS dsDNA Kit on a Qubit 2.0 (Thermo Fisher Scientific).
  • Example 2 Enzyme variant barcoding Evolved libraries of Casl2f-ABE were obtained by directed evolution of the fusion protein UnlCasl2fl-Tad8e (short name: Casl2f-ABE, SEQ ID NO: 1). The resulting enzyme variants contain randomly acquired mutations in comparison to their origin. The DNA editing enzyme gene variants were acquired from the pEVO plasmids used in the evolution by digesting with Xbal and BsrGI-HF for the evolved Casl2f-ABE library.
  • the Casl2f-ABE library and the Casl2f-ABE controls were ligated in a ratio of 60 ng of Casl2f-ABE fragment, 4.8 ng UMI- tag and 100 ng of Bsrgl-HF and Sbfl digested pEVO-BE plasmid.
  • Ligated plasmids were desalted with MF-Millipore membrane filters (Merck) on distilled water for 30 minutes and transformed into XL-1 Blue E. coli (Agilent) via electroporation.
  • the transformed bacteria were cultured in SOC medium for 30 minutes at 37°C. 2 pl of this culture was spread on agarose plates with 15 mg/ml chloramphenicol and incubated over night at 37°C. The number of colonies on the plates were counted to calculate the number of transformed bacteria present per pl of SOC culture.
  • Barcoded plasmid extracts from the Casl2f-ABE library were digested with Seal and BsrgI, The resulting fragments containing the evolved gene, the UMI-tag, and the target sites were isolated via agarose gel excision with the Isolate II PCR and Gel Kit (Bioline).
  • Nanopore sequencing library preparation for the Casl2f-ABE library was performed using the protocol "Amplicons by Ligation (SQK-LSK112)".
  • the Casl2f library was loaded onto a MinlON FLO-MINI 10 flow cell with rl0.4 pores (Oxford Nanopore Technologies). Sequencing was performed for 72 hours. Each screen was performed on one flow cell.
  • the sequences were then aligned with minimap2 (Ei, 2021) to a reference sequence containing UnlCasl2fl-ABE8e (Casl2f-ABE screen, SEQ ID NO: 3540) and the UMI consisting of 50 random bases.
  • the aligned reads were filtered with samtools (Danecek et al., 2021) based on coordinates at the beginning of the enzyme gene and the end of the UMI.
  • UMIs were then extracted from the filtered alignment using the stackStringsFromBam function from the R package GenomicAlignments (Lawrence et al., 2013). UMIs were subsequently clustered with VSEARCH (Rognes et al., 2016) with a cluster_identity value of 0.7. Sequence reads from clusters with a minimum size of 50 reads were then transferred to separate files and aligned to the gene-UMI reference sequence. These separate read files and alignments were used to construct consensus sequences with racon (Vaser et al. 2017) followed by further polishing with medaka (https://github.com/nanoporetech/medaka), both with standard settings. The polishing process was run in parallel with GNU parallel (Tange 2023). Finally, gene sequences were extracted using the R package GenomicAlignments and translated to amino acids.
  • the base editing rate of the enzyme variants was determined by aligning the clustered reads to a reference sequence that contains the unedited target site (SEQ ID NO: 3541).
  • a reference sequence that contains the unedited target site SEQ ID NO: 3541.
  • the aligned stacks of four bases from position two to five counting from the TTTG PAM sequence on all three target sites were extracted.
  • the base editing of Casl2f-ABE is expected on position three or four after the PAM sequence. Comparing this region can therefore provide information about potential editing of adjacent positions. Editing rates can then be determined by counting the correctly edited reads, non-edited reads and other editing outcome reads.
  • Results from each screen were combined and filtered for clusters with 50 reads or more. All further data processing and visualization was performed in R with the tidy verse (Wickham et al. 2019) and stringdist packages (van der Loo, 2014).
  • enzyme variants were extracted from the screened libraries using PCR.
  • Reverse primers specific for the UMI of the cluster of interest were designed ("Casl2f UMI-2 R (SEQ ID NO: 3542), "Casl2f UMI-3030 R (SEQ ID NO: 3543), “Casl2f UMI-3301 R (SEQ ID NO: 3544)) and used together with a universal forward primer (binding on the plasmid before the sequence encoding the enzyme in region 1 ("Evolution F (SEQ ID NO: 3545) for the Casl2f-ABE screen) to amplify enzyme genes.
  • PCRs were performed with a high-fidelity polymerase (Herculase II Fusion DNA Polymerase, Agilent) and PCR products were digested with Xbal and BsrgI (Casl2f-ABE enzymes).
  • oligonucleotides containing the target sites were annealed to form a DNA fragment that was cloned into a Bglll digested pEVO backbone via Cold Fusion (System Biosciences).
  • SgRNA scaffold fragments were synthesized (Twist Bioscience) and cloned into pEVO vector containing the target sites utilizing Nsil and Notl restriction enzymes.
  • the pEVO plasmid containing the sgRNA scaffold and the target sites was then used as a template in a PCR reaction where three different sgRNA spacers were included in the reverse primers.
  • sgRNA fragments were introduced to the pEVO vector in a stepwise manner: by cloning sgRNA 1 using Notl and Nsil, by cloning sgRNA2 using Nsil, and by cloning sgRNA3 using Xhol and Sall.
  • UnlCasl2fl fragments were produced by Twist Bioscience and amplified via PCR, and the TadA gene was prepared via PCR by using the pABE8e-protein plasmid as template (Addgene plasmid # 161788). To create Casl2f-ABE, both PCR products were used as template for an overlap-PCR.
  • the Casl2f-ABE was then cloned into the pEVO-TS-sgl-sg2-sg3 vector using BsrGI and Xbal restriction enzymes.
  • the expression of the Casl2f-ABEs was controlled by the araBAD L- arabinose inducible promoter system.
  • Fig. 1 A schematic illustration of the procedure is shown in Fig. 1.
  • the Casl2f-ABE library was generated using error-prone-PCR with a low-fidelity DNA polymerase (MyTaq, Bioline and Primers Evolution F and Evolution R (SEQ ID NOs: 3545 and 3546) and cloned into the vector using BsrGI and Xbal restriction enzymes.
  • MyTaq Bioline and Primers Evolution F and Evolution R (SEQ ID NOs: 3545 and 3546)
  • BsrGI and Xbal restriction enzymes After transformation into XL-1 blue E. coli, expression of the enzymes was induced with 200 pg/ml L-arabinose.
  • the plasmids were isolated and digested with restriction enzymes (RE), which target the sgRNAs and their target sites.
  • RE restriction enzymes
  • FIG. 2A A schematic illustration of the base editing assay is shown in Fig. 2A.
  • Edited and non-edited pEVO plasmids (circles in top part) for base editing are digested with restriction enzymes (RE) that are specific for a sequence in the target site (quadratic box) and in the sgRNA array (grey). If the target site is edited (quadratic box with line), the RE-site is lost. Due to the different number of cuts, the number and sizes of the fragment changes. Digestion of non-edited plasmids results in two DNA fragments (smallest two fragments), digestion of edited plasmids results in one DNA fragment (biggest fragment). These fragments are visualized using agarose gel electrophoresis (schematic shown at the bottom).
  • a mixture of edited and non-edited plasmids is shown on the right lane of the gel schematic. To the right of the gel, pictograms are used to indicate to which editing status the fragments belong to. A line with a box filled with a horizontal line indicates the edited single DNA fragment, the shorter lines with half a box indicate the two non-edited fragments that have been cut. For the assay, the same pEVO plasmid that was used in the examples before has been used.
  • the Casl2f-ABE variants were cloned utilizing BsrGI and Xbal restriction enzymes. Expression of the variants was controlled by an L-arabinose inducible promoter system (araBAD).
  • sgRNA Three different sgRNA were cloned into the pEVO plasmid, which target genomic DNA of E. Coli. Casl2f-ABE WT and the three variants were cloned into these vectors utilizing BsrGI and Xbal restriction enzymes. Modified vectors were subsequently transformed into E. Coli cells. After an overnight culture, the cells were spun down and resuspended in 200 pl ddH2O. This suspension was then heated to 95°C for 10 minutes and spun down again.
  • the PCR products were sequenced via Sanger sequencing and the editing rates were analyzed with EditR (Kluesner et al., 2018).
  • the method of the present invention was further assessed on a CRISPR-Cas based system, a schematic representation of CRISPR associated substrate linked directed evolution (CaSLiDE) method is shown in Fig. 3.
  • CaSLiDE CRISPR associated substrate linked directed evolution
  • 46 directed evolution cycles were performed to generate a library of UnlCasl2fl-ABE8e (Casl2f-ABE) with improved editing efficiency compared to the original Casl2f-ABE (Fig. 2B).
  • the evolved library was then further enriched for four cycles, by performing CaSLiDE with a high-fidelity PCR (Fig. 2C).
  • This library was then screened with the method of the present invention, in which the original UnlCasl2fl- ABE8e (wild type, WT) was used as control.
  • the screen yielded more than 3.600 UMI- clusters with 50 or more reads, of which about 3.4 % clusters were identified as WT control (Fig. 4A).
  • the determined average editing rates of the WT were 0.5% (target site 1), 16% (target site 2), and 6.2% (target site 3) (Fig. 4B).
  • 58 had editing rates of more than 90% on all 3 target sites (Fig. 4C).
  • Table II shows the base editing rates of the 3.519 non-WT clusters (SEQ ID Nos. 2-3520) obtained by Approach I compared to the original (“WT”) enzyme of SEQ ID NO. 1.
  • TS means target site.
  • Table III shows the base editing rates of the non-WT clusters (SEQ ID Nos. 3551-7546) obtained by Approach II compared to the original (“WT”) enzyme of SEQ ID NO. 1.
  • TS means target site
  • Ara2 means induction with 2 pg/ml L-arabinose
  • Ara5 means induction with 5 pg/ml L- arabinose.
  • Table IV shows the base editing rates of the non-WT clusters (SEQ ID Nos. 7548- 10114) obtained by Approach III compared to the original (“WT”) enzyme of SEQ ID NO. 7547.
  • TS means target site.
  • Fig. 4A three variants with high activity on all three target sites (clusters 2 (SEQ ID NO: 471), 3030 (SEQ ID NO: 20), 3301 (SEQ ID NO: 3)); Fig. 4A) from the screened Casl2f-ABE library were extracted and evaluated using a base editing assay, schematically shown in Fig. 2A.
  • the assay is performed by digesting the CaSLiDE evolution plasmid with restriction enzymes that recognize the target sites (quadratic box, Fig 2A).
  • Successfully edited plasmids (quadratic box with dash, Fig 2A) do not contain the restriction sites.
  • Non- edited target sites are cut by the restriction enzyme (RE), while edited target sites are not cut due to the change in the restriction site.
  • RE restriction enzyme
  • TadA domains of the invention c2 (SEQ ID NO: 10107), c3030 (SEQ ID NO: 4498), c3301 (SEQ ID NO: 4511) to published Tad8r (SEQ ID NO: 10115, published in WO 2023034959 A2 as SEQ ID NO. 308) and TadA8e (SEQ ID NO: 10118, published in Richter M.F. et al, 2020)
  • a plasmid-based assay on three different target sites target site Ndel (SEQ ID NO: 3521), target site Hpal (SEQ ID NO: 3522), target site Psil (SEQ ID NO: 3523) in triplicate replicates was used (Fig. 8D).
  • UnlCasl2fl-WT (SEQ ID NO: 1) was used as Cas protein in the DNA modifying protein constructs.
  • the editing rate was measured by Sanger sequencing and analyzed with EditR program (Kluesner et al., 2018). Quantified results show, that Tad8r has ⁇ 1% editing on all sites, wherein the TadAs according to the invention show significant higher editing rates.

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Abstract

L'invention concerne une enzyme de modification d'ADN comprenant une protéine d'édition d'ADN et une protéine Cas et un lieur peptidique, ou en étant constitués, ladite protéine d'édition d'ADN et ladite protéine Cas étant reliées par ledit lieur peptidique ; et ladite enzyme de modification d'ADN ayant été obtenue par évolution moléculaire dirigée d'une enzyme d'origine ; et ladite enzyme de modification d'ADN présentant un taux d'édition de base qui est au moins 4 fois supérieur au taux d'édition de base de ladite enzyme de type sauvage. L'invention concerne en outre un procédé d'évolution moléculaire dirigée d'une enzyme de modification d'ADN et l'utilisation d'enzymes de modification d'ADN évoluées dans la recherche, la médecine et l'agriculture.
PCT/EP2024/062227 2023-05-04 2024-05-03 Éditeurs de base crispr hautement actifs obtenus par évolution dirigée liée à un substrat assistée par cas (caslide) Pending WO2024227911A2 (fr)

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Family Cites Families (6)

* Cited by examiner, † Cited by third party
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CN111801345A (zh) * 2017-07-28 2020-10-20 哈佛大学的校长及成员们 使用噬菌体辅助连续进化(pace)的进化碱基编辑器的方法和组合物
WO2020181195A1 (fr) * 2019-03-06 2020-09-10 The Broad Institute, Inc. Édition de base t : a à a : t par excision d'adénine
US20220290121A1 (en) * 2019-08-30 2022-09-15 The General Hospital Corporation Combinatorial Adenine and Cytosine DNA Base Editors
CA3198422A1 (fr) * 2020-10-08 2022-04-14 Genkore Inc. Arn guide modifie comprenant une queue riche en u pour augmenter l'efficacite d'un systeme crispr/cas12f1 et utilisation correspondante
AU2022246176A1 (en) * 2021-03-26 2023-10-05 Beam Therapeutics Inc. Adenosine deaminase variants and uses thereof
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023034959A2 (fr) 2021-09-03 2023-03-09 The University Of Chicago Polypeptides et procédés de modification d'acides nucléiques

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
BUCHHOLZ F., HAUBER J.: " In vitro evolution and analysis of HIV-1 LTR-specific", METHODS, vol. 53, 2011, pages 102 - 109, XP002643857, DOI: 10.1016/j.ymeth.2010.06.014
BUCHHOLZ, F., STEWART, A.F.: "Alteration of Cre recombinase site specificity by substrate-linked protein evolution.", NAT BIOTECHNOL, vol. 19, 2001, pages 1047 - 1052, XP002289807, DOI: 10.1038/nbt1101-1047
CONG LANN RAN FCOX DLIN S ET AL.: "Multiplex Genome Engineering Using CRISPR/Cas Systems", SCIENCE, vol. 339, no. 6121, 2013, pages 819 - 823, XP055871219, DOI: 10.1126/science.1231143
DANECEK P., BONFIELD J.K., LIDDLE J., MARSHALL J., OHAN V., POLLARD M.O.: "Twelve years of SAMtools and BCFtools", GIGASCIENCE, vol. 10, no. 2, 2021, pages 1 - 4
GAJ TGERSBACH C.ABARBAS, C.F: "ZFN, TALEN and CRISPR/Cas-based methods for genome engineering", TRENDS BIOTECHNOL, vol. 31, no. 7, 2013, pages 397 - 405
HOERSTEN J., RUIZ-GOMEZ G., LANSING F., ROJO-ROMANOS T., SCHMITT L.T., SONNTAG J.,PISABARRO M.T., BUCHHOLZ F.: "Pairing of single mutations yields obligate Cre-type site-specific recombinases", NUCLEIC ACIDS RESEARCH, 2021, Retrieved from the Internet <URL:https://doi.org/10.1093/nar/gkab1240.>
KARST S.M., ZIELS R.M., KIRKEGAARD R.H., SORENSEN E.A., MCDONALD D., ZHU Q., KNIGHT R.,ALBERTSEN M.: "High-accuracy long-read amplicon sequences using unique molecular identifiers with nanopore or PacBio sequencing", NAT METHODS, vol. 18, 2021, pages 165 - 169, XP037359604, Retrieved from the Internet <URL:https://doi.org/10.1038%2Fs41592-020-01041-y.> DOI: 10.1038/s41592-020-01041-y
KLUESNER M.G., NEDVECK D.A., LAHR W.S., GARBE J.R., ABRAHANTE J.E., WEBBER B.R.: "EditR: A Method to Quantify Base Editing from Sanger Sequencing", CRISPR J., vol. 1, no. 3, 2018, pages 239 - 50, XP055715954, DOI: 10.1089/crispr.2018.0014
LANSING F., PASZKOWSKI-ROGACZ M., SCHMITT L.T., SCHNEIDER P.M., ROMANOS T.R., SONNTAG J., BUCHHOLZ F.: " A heterodimer of evolved designer-recombinases precisely excises a human genomic DNA locus", NUCLEIC ACIDS RESEARCH, vol. 48, 2019, pages 472 - 485
LANSING FMUKHAMETZYANOVA LROJO-ROMANOS TIWASAWA KKIMURA MPASZKOWSKI-ROGACZ MKARPINSKI JGRASS TSONNTAG JSCHNEIDER P.M: "Correction of a Factor VIII genomic inversion with designer-recombinases", NATURE COMMUNICATIONS, 2022, pages 13, Retrieved from the Internet <URL:https://doi.org/10.1038%2Fs41467-022-28080-7>
LAWRENCE M., HUBER W., PAGES H., ABOYOUN P., CARLSON M., GENTLEMAN R., MORGAN M.T.,CAREY V.J.: "Software for computing and annotating genominc ranges.", COMPUT. BIOL, vol. 9, pages 1 - 10
LI H.: "New strategies to improve minimap2 alignment accuracy", BIOINFORMATICS, vol. 37, no. 23, 2021, pages 4572 - 4
RICHTER M.F., ZHAO K.T., ETON, E., LAPINAITE A., NEWBY G.A., THURONYI B.W., WILSON C.,KOBLAN L.W., ZENG J., BAUER D.E., DOUDNA J.A: "Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity", NAT BIOTECHNOL., vol. 38, no. 7, June 2020 (2020-06-01), pages 883 - 891, XP037523981, DOI: 10.1038/s41587-020-0453-z
RICHTER, M. F ET AL.: "Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity", NAT BIOTECHNOL., vol. 38, no. 7, July 2020 (2020-07-01), pages 883 - 891, XP037523981, DOI: 10.1038/s41587-020-0453-z
ROGNES T., FLOURI T., NICHOLS B., QUINCE C., MAHE, F.: "VSEARCH: a versatile opeN source tool for metagenomics", PEERJ, 22 January 2016 (2016-01-22)
SCHINDELIN J., ARGANDA-CARRERAS I., FRISE, E., KAYNIG V.: "Fiji: an open-source platform for biological-image analysis", NATURE METHODS, vol. 9, pages 676 - 682, XP055343835, DOI: 10.1038/nmeth.2019
SHERIFF AGURI IZEBROWSKA P ET AL.: "ABE8e adenine base editor precisely and efficiently corrects a recurrent COL7A1 nonsense mutation", NATURE: SCIENTIFIC REPORTS, vol. 12, 2022, pages 19643
STODDARD B.L: "Homing endonuclease structure and function. Quaterly Reviews of Biophysics", vol. 38, 2006, CAMBRIDGE UNIVERSITY PRESS, pages: 1
TANGE O, GNU PARALLEL 2018, 2018, Retrieved from the Internet <URL:https://zenodo.org/record/1146014>
VAN DER LOO M.P.J.: "The stingdist package for approximate string matching.", JOURNAL, vol. 6, 2014, pages 111 - 122
VASER R., SOVIC I., NAGARAJAN N., SIKIC, M: " Fast and de novo genome assembly from long uncorrected reads.", GENOME RES, vol. 27, 2017, pages 737 - 746, XP055608901, DOI: 10.1101/gr.214270.116
WANG Y., ZHAO Y., BELLAS A., WANG Y, AU K.F.: " Nanopore sequencing technology,bioinformatics and applications.", NATURE BIOTECHNOLOGY, vol. 39, 2012, pages 1348 - 1365, XP037616214, DOI: 10.1038/s41587-021-01108-x
WICKHAM H.AVERICK MBRYAN J ET AL.: "Welcome to the Tidyverse", J. OPEN SOURCE, vol. 4, 2019, pages 1686
XIN CYIN JYUAN SOU LLIU MZHANG WHU J: "Comprehensive assessment of miniature CRISPR-Cas 12f nucleases for gene disruption", NATURE COMMUNICATIONS, 2022, pages 13
ZUREK P.JKNYPHAUSEN PNEUFELD KPUSHPANATH AHOLLFELDER F: "UMI-linked consensus sequencing enables phylogenetic analysis of directed evolution", NATURE, 2020, pages 11, Retrieved from the Internet <URL:https://doi.org/10.1038/s41467-020-19687-9.>

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