Site-specific genome modification technology

Abstract

The present disclosure provides compositions, methods and systems for template-mediated genome editing and modification.In particular, the present disclosure provides novel genome modification techniques involving site-specific chemical modification of nucleotides to introduce replication-blocking lesions.The compositions, methods and systems described herein facilitate efficient site-specific genome modification of DNA targets while minimizing unintended editing and cytotoxicity associated with current genome editing approaches.

Description

[Technical field]

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 149,419, filed February 15, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing The text of the computer readable sequence listing filed herewith, entitled "39212-601_SEQUENCE_LISTING_ST25", created on February 14, 2022, having a file size of 144,908 bytes, is hereby incorporated by reference in its entirety into this specification.

[0003] Government funding This invention was made with Government support under Grant No. GM119561 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0004] Field The present disclosure provides compositions, methods and systems for template-mediated genome modification.In particular, the present disclosure provides a novel genome modification technique that involves site-specific chemical modification of nucleotides to introduce replication-blocking lesions.The compositions, methods and systems described herein facilitate efficient site-specific genome modification of DNA targets while minimizing unintended editing and cytotoxicity associated with current genome editing approaches. [Background technology]

[0005] CRISPR-based genome editing tools have found widespread applications, relying on their easily programmable targeting and robust activity. The initial use of such CRISPR-based tools focused on the ability of Cas nucleases to cleave DNA. In the process of repairing the cleaved DNA, genome edits are introduced via homologous recombination with a supplied DNA repair template. However, DNA breaks are one of the most toxic cellular events; they activate the cellular alarm system, which can lead to mutations, DNA rearrangements, or loss of cell viability. Subsequent CRISPR-Cas genome editing tools have explored alternative approaches by targeted modification of individual bases or by incorporating short templates encoded within guide RNAs. Summary of the Invention [Problem to be solved by the invention]

[0006] Nevertheless, these methods are limited in the range of edits that can be produced and can result in unwanted edits. Thus, there is a need for efficient genome editing and modification platforms that overcome the limitations of current systems. [Means for solving the problem]

[0007] Summary of the Invention The present disclosure includes a composition for targeted genome modification. According to such an embodiment, the composition includes a gap editor complex comprising a DNA recognition domain and a DNA modifying domain, wherein the DNA recognition domain binds to a DNA target sequence in a genome, and the DNA modifying domain induces the formation of a replication blocking portion at at least one nucleotide in the genome.

[0008] In some embodiments, the composition further comprises a donor nucleic acid template. In some embodiments, the donor nucleic acid template comprises a polynucleotide derived from an endogenous homologous sequence corresponding to the DNA target sequence. In some embodiments, the donor nucleic acid template comprises an exogenous single-stranded DNA (ssDNA) molecule or a double-stranded DNA (dsDNA) molecule. In some embodiments, the donor nucleic acid template is an RNA molecule. In some embodiments, the presence of the donor nucleic acid template facilitates homology-directed gap repair and / or recombination, and the donor nucleic acid template or a fragment thereof is recombined into the genome of the DNA target sequence.

[0009] In some embodiments, the DNA recognition domain comprises at least one Cas protein or fragment thereof that lacks deoxyribonuclease activity. In some embodiments, the DNA recognition domain comprises a complex of Cas proteins that lack deoxyribonuclease activity. In some embodiments, the DNA recognition domain comprises a Cas protein or fragment thereof that has nickase activity. In some embodiments, the Cas protein or Cas protein complex comprises a type I Cascade, a type II Cas9, a type IV effector module, a type V Cas12, a Cas9-associated IscB, a Cas9-associated TnpB, and combinations thereof.

[0010] In some embodiments, the DNA recognition domain and the DNA modification domain are operably coupled, which in some embodiments includes a polypeptide fusion, a peptide tag, a peptide linker, an RNA tag, and any combination thereof.

[0011] In some embodiments, the DNA-modifying domain blocks DNA replication by adding a replication-blocking moiety to: (i) at least one nucleotide in the DNA strand that is complementary to the DNA target sequence; (ii) at least one nucleotide in the DNA strand that contains the DNA target sequence; or (iii) both at least one nucleotide in the DNA strand that is complementary to the DNA target sequence and at least one nucleotide in the DNA strand that contains the DNA target sequence.

[0012] In some embodiments, the DNA recognition domain induces a single-stranded break in a DNA target strand and the DNA modifying domain adds a replication-blocking moiety to at least one nucleotide in the DNA strand complementary to the DNA target sequence.

[0013] In some embodiments, the DNA-modifying domain is engineered to have reduced DNA binding, increased specificity for single-stranded DNA, and / or decreased enzymatic activity.

[0014] In some embodiments, the DNA-modifying domain catalyzes the addition of ADP-ribose to thymine or guanine nucleotides. In some embodiments, the DNA-modifying domain comprises a DarT enzyme or a functional fragment, derivative, or variant thereof. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 70% amino acid sequence identity to any of SEQ ID NOs: 18-21. In some embodiments, the DarT enzyme comprises one or more of the following amino acid substitutions: G49D, K56A, M86L, R92A, and / or R193A.

[0015] In some embodiments, the DNA-modifying domain comprises a Scabin enzyme or a functional fragment, derivative, or variant thereof. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 70% amino acid sequence identity to any of SEQ ID NOs: 22-24. In some embodiments, the Scabin enzyme comprises an amino acid substitution that is K130A.

[0016] In some embodiments, the DNA-modifying domain catalyzes the methylcarbamoylation of adenine nucleotides. In some embodiments, the DNA-modifying domain comprises a Mom enzyme, or a functional fragment, derivative, or variant thereof. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 70% amino acid sequence identity to SEQ ID NOs: 25-27. In some embodiments, the Mom enzyme comprises an amino acid substitution that is D149A.

[0017] In some embodiments, the DNA-modifying domain catalyzes the addition of a replication-blocking moiety selected from the group consisting of glucose, threonylcarbamoyladenosine, acetate, glyceryl, L-ascorbic acid, uridine, adenosine monophosphate, a lipid, an amino acid, agmatine, L-threonylcarbamoyladenylate, L-threonylcarbamoyl, methylthiolate, sulfur, a methyl group, S-adenosyl-L-methione or a subgroup of S-adenosyl-L-methionine, and dimethylallyl diphosphate or a subgroup thereof.

[0018] In some embodiments, the DNA modifying enzyme domain is selected from the group consisting of Pierisin, Scabin, cell cycle and apoptosis regulator 1 (CARP-1), SCO5461 protein (ScARP), adenine modifying enzymes, acetyltransferases, amino acid transferases, nucleotidyltransferases, uridyltransferases, acyltransferases, ADP-ribsoyltransferases, methylthiotransferases, N-acetyltransferase 10, tRNA(Met) cytidine acetyltransferase (TmcA), tRNA cytidine acetyltransferase, GCN5-related N-acetyltransferase, lysidine synthase, m 7 G methyltransferase, N6 carbamoyl methyltransferase (Mom), N6-adenosine threonylcarbamoyltransferase, threonylcarbamoyltransferase or threonylcarbamoyltransferase complex, TsaB-TsaE-TsaD (TsaBDE) complex, tRNA N6-adenosine threonylcarbamoyltransferase (Qri7, Tcs4), methyltransferase, ATrm5a, tRNA:m 1The present invention includes an enzyme selected from the group consisting of G / imG2 methyltransferase, tRNA(adenosine(37)-N6)-dimethylallyltransferase, tRNA dimethylallyltransferase (MiaA), and isopentenyltransferase, or a functional fragment, derivative, or variant thereof.

[0019] In some embodiments, the composition comprises at least one guide RNA molecule. In some embodiments, the at least one guide RNA comprises a gRNA, a sgRNA, a crRNA, or any combination thereof. In some embodiments, the at least one guide RNA comprises a handle sequence and a targeting sequence. In some embodiments, the at least one guide RNA is complementary to a DNA target sequence.

[0020] In some embodiments, the composition further comprises at least one gap edit factor accessory factor. In some embodiments, the at least one gap edit factor accessory factor comprises a protein that enhances at least one step in the genome modification process. In some embodiments, the at least one gap edit factor accessory factor is recruited to the gap edit factor complex via an interaction with a DNA modification domain, a DNA recognition domain, and / or at least one guide RNA. In some embodiments, the recruitment of the at least one gap edit factor accessory factor to the gap edit factor complex comprises a peptide tag, a peptide linker, an RNA tag, and any combination thereof. In some embodiments, the at least one gap edit factor accessory factor comprises Rap, DarG, Orf, ExoI, Exonuclease III, PrimPol, RecJ, RecQ1, Rad51, Rad52, CtIP, Rad18, and any combination thereof.

[0021] The present disclosure also includes a kit for targeted genome modification. According to such an embodiment, the kit includes a gap editor complex comprising a DNA recognition domain and a DNA modifying domain, wherein the DNA recognition domain binds to a DNA target sequence in a genome, and the DNA modifying domain induces the formation of a replication blocking moiety at at least one nucleotide in the genome.

[0022] In some embodiments, the kit further comprises a donor nucleic acid template, hi some embodiments, the presence of the donor nucleic acid template facilitates homologous sequence-directed gap repair and / or recombination.

[0023] In some embodiments, the kit further comprises a guide RNA molecule.

[0024] In some embodiments of the kit, the DNA recognition domain comprises at least one Cas protein or fragment thereof that lacks DNase activity. In some embodiments, the DNA recognition domain comprises at least one Cas protein or fragment thereof that has nickase activity. In some embodiments, the Cas protein or Cas protein complex comprises a type I cascade, a type II Cas9, a type IV effector module, a type V Cas12, a Cas9-associated IscB, a Cas9-associated TnpB, and combinations thereof.

[0025] In some embodiments of the kit, the DNA recognition domain and the DNA modifying domain are operably coupled, hi some embodiments, the DNA recognition domain induces a single-stranded break in the DNA target strand and the DNA modifying domain adds a replication-blocking moiety to at least one nucleotide in the DNA strand complementary to the DNA target sequence.

[0026] In some embodiments of the kit, the DNA modifying domain catalyzes the addition of ADP-ribose to thymine or guanine nucleotides. In some embodiments, the DNA modifying domain comprises a DarT enzyme or a functional fragment, derivative or variant thereof. In some embodiments, the DNA modifying domain comprises a Scabin enzyme or a functional fragment, derivative or variant thereof. In some embodiments, the DarT enzyme has been engineered to have reduced DNA binding, increased specificity for single-stranded DNA and / or decreased enzymatic activity.

[0027] In some embodiments of the kit, the DNA-modifying domain catalyzes methylcarbamoylation of adenine nucleotides. In some embodiments, the DNA-modifying domain comprises a Mom enzyme or a functional fragment, derivative or variant thereof. In some embodiments, the Mom enzyme is engineered to have reduced DNA binding, increased specificity for single-stranded DNA and / or reduced enzymatic activity.

[0028] In some embodiments of the kit, the DNA modifying domain catalyzes the addition of a replication blocking moiety selected from the group consisting of glucose, threonylcarbamoyladenosine, acetate, glyceryl, L-ascorbic acid, uridine, adenosine monophosphate, a lipid, an amino acid, agmatine, L-threonylcarbamoyladenylate, L-threonylcarbamoyl, methylthiolate, sulfur, a methyl group, S-adenosyl-L-methionine or a subgroup of S-adenosyl-L-methionine, and dimethylallyl diphosphate or a subgroup thereof.

[0029] In some embodiments of the kit, the DNA modifying enzyme domain is selected from the group consisting of Pierisin, Scabin, cell cycle and apoptosis regulator 1 (CARP-1), SCO5461 protein (ScARP), adenine modifying enzyme, acetyltransferase, amino acid transferase, nucleotidyltransferase, uridyltransferase, acyltransferase, ADP-ribosyltransferase, methylthiotransferase, N-acetyltransferase 10, tRNA(Met) cytidine acetyltransferase (TmcA), tRNA cytidine acetyltransferase, GCN5-related N-acetyltransferase, lysidine synthase, mRNA cytidine acetyltransfer ... 7 G methyltransferase, N6 carbamoyl methyltransferase (Mom), N6-adenosine threonylcarbamoyltransferase, threonylcarbamoyltransferase or threonylcarbamoyltransferase complex, TsaB-TsaE-TsaD (TsaBDE) complex, tRNA N6-adenosine threonylcarbamoyltransferase (Qri7, Tcs4), methyltransferase, ATrm5a, tRNA:m 1 The present invention includes an enzyme selected from the group consisting of G / imG2 methyltransferase, tRNA(adenosine(37)-N6)-dimethylallyltransferase, tRNA dimethylallyltransferase (MiaA), and isopentenyltransferase, or a functional fragment, derivative, or variant thereof.

[0030] In some embodiments of the kit, the at least one guide RNA comprises a gRNA, a sgRNA, a crRNA, or any combination thereof. In some embodiments, the at least one guide RNA comprises a handle sequence and a targeting sequence. In some embodiments, the targeting sequence in the at least one guide RNA is complementary to a DNA target sequence.

[0031] In some embodiments, the kit further comprises at least one gap editing factor accessory factor.

[0032]

[0013] Embodiments of the present disclosure also include methods for targeted genome modification. According to such embodiments, the method includes introducing any of the compositions of the present disclosure into a cell and evaluating the cell for the presence of the desired genome alteration.

[0033] In some embodiments, the gap editor complex and / or at least one guide RNA molecule are introduced into the cell as a polypeptide(s), mRNA(s) and / or DNA expression construct(s). In some embodiments, the gap editor complex and / or guide RNA are introduced into the cell as part of a gene drive system.

[0034] In some embodiments, the cell is a prokaryotic or eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a plant cell.

[0035] In some embodiments, the methods result in a reduced degree of indel formation, chromosomal rearrangements and / or DNA duplications.

[0036] In some embodiments, cell viability is enhanced and / or cytotoxicity is reduced.

[0037] Other aspects and embodiments of the present disclosure will become apparent in light of the following detailed description. [Brief description of the drawings]

[0038] [Figure 1A] Figure 1A presents a representative diagram of the general mechanism of gap editing. Bulky chemical groups appended to one strand of DNA by gap editing factors block DNA replication, resulting in a single-stranded DNA gap. The gap is then repaired by homologous recombination that can incorporate a homologous repair template. The opposite strand can also be nicked or chemically modified to block recombination with sister chromatids and enhance editing. [Figure 1B]FIG. 1B contains representative results of an experiment demonstrating efficient lacZ gene repair with significantly reduced cytotoxic effects using a gap editor complex containing a DNA modifying enzyme (DarT) that has been engineered to have reduced DNA binding. [Diagram 2] FIG. 2 contains representative results of an experiment demonstrating efficient lacZ gene repair with significantly reduced cytotoxic effects using a gap editor complex (DarT_G49D_K56A-ScnCas9 or GE2n) that contains a DNA recognition domain engineered to have nickase activity. [Diagram 3] FIG. 3 contains representative results of an experiment demonstrating attenuation of lacZ gene repair by the gap editor complex when a gap editor accessory factor was used (DarG) to counteract the function of the DNA-modifying domain (DarT) of the gap editor complex. [Figure 4] Figure 4 contains representative results of an experiment demonstrating successful genome modification by increasing the frequency of kanamycin gene repair using a gap editor complex (Scabin) containing a DNA modification domain (Scabin) combined with a Cas9 DNA recognition domain (Scabin-K130A-ScdCas9). [Diagram 5] Figure 5 contains representative results of an experiment demonstrating successful genome modification by increasing the frequency of kanamycin gene repair using a gap editor complex containing a DNA modification domain (Mom) in combination with a Cas9 DNA recognition domain (Mom-D149A-ScdCas9). [Figure 6] Figure 6 includes representative results of experiments demonstrating that successful genome modification using gap editor complexes (e.g., through increased frequency of kanamycin gene repair) relies on a DNA modification domain (DarT) in combination with a Cas9 DNA recognition domain (DarT-G49D-ScdCas9) and active RNA-directed targeting. (ScdCas9 alone did not result in kanamycin gene repair.) [Figure 7]Figure 7 includes representative results of experiments using a gap editor complex (DarT-G49D-R193A-ScdCas9) with a DarT DNA-modifying domain that contains a specific mutation (R193A) that significantly reduces toxicity. [Figure 8] FIG. 8 includes representative results from experiments using gap editor complexes with DarT DNA-modifying domains containing mutations (G49D, R193A, M86L, and R92A) that significantly reduce background editing while maintaining on-target editing, as demonstrated by reduced and maintained frequency of kanamycin gene repair, respectively. [Figure 9] Figure 9 includes representative results of an experiment demonstrating successful genome modification by increasing the frequency of kanamycin gene repair using a gap editor complex containing a DNA modification domain (DarT) with mutations that significantly reduce toxicity (G49D and / or R193A) combined with a Cas9 DNA recognition domain with nickase activity (ScdCas9). Adding the R193A mutation to the G49D mutation further reduced toxicity without compromising modification. Site-specific genome modification was nearly 100% effective. [Figure 10] Figure 10 contains representative results from an experiment demonstrating that genetic knockout of fcy1 confers resistance to 5-fluorocytosine (5-FC). Targeting the fcy1 gene in Saccharomyces cerevisiae with Cas9 nickase (ScnCas9) or a fusion of Cas9 nickase with an engineered DarT gene and providing a repair template resulted in genomic modification in fcy1. For all mutations, DarT fusions resulted in a >10-fold increase in the rate of genome editing, demonstrating the utility of introducing a replication blocker in eukaryotic cells. [Figure 11]FIG. 11 includes representative results of an experiment demonstrating that genetic knockout of fcy1 confers resistance to 5-fluorocytosine (5-FC). Targeting the fcy1 gene in Saccharomyces cerevisiae with Cas9 nickase (ScnCas9) or a fusion of an engineered DarT gene with Cas9 nickase and providing a repair template resulted in genomic modification in fcy1. The repair template encodes six mutations that introduce two or three stop codons in fcy1, resulting in loss of fcy1 function and resistance to 5-FC after genomic modification. Use of an engineered DarT variant containing G49D, R193A, M86L, and R92A mutations improves cell viability up to approximately 50-fold over DarT with G49D and R193A mutations alone. This gap editor complex achieves efficient and low-toxicity genome modification using two separate single guide RNAs targeting fcy1 in yeast and a repair template. [Figure 12] Figure 12 includes representative chromatographs providing confirmation of fcy1 genome modification and gene knockout by Sanger sequencing. Two or three stop codons were introduced by targeting gap editing factor complexes to the fcy1 gene and providing a DNA repair template. Edited nucleotides are highlighted in red. Genome editing of two separate targets within fcy1 is shown. [Figure 13] Figure 13 includes representative results of an experiment demonstrating that gene knockout of lacZ results in white colony color in the presence of lactose analog IPTG and colorimetric indicator X-gal. Targeting the lacZ gene in E. coli with a nuclease-inactive Cas12a protein (dLbCas12a) fused to an engineered DarT gene and providing a repair template resulted in genomic modification at lacZ. No genomic modification was observed without targeting the gap editor complex to the lacZ gene. [Figure 14]FIG. 14 includes representative chromatographs demonstrating successful introduction of one or more stop codons into the lacZ gene, eliminating beta-galactosidase expression, thereby resulting in white colonies when plated in the presence of inducer IPTG and colorimetric indicator X-gal, using DarT(G49D / R193A)-dLbCas12a associated with different crRNAs. [Figure 15] Figure 15 contains representative results of an experiment demonstrating that introduction of the D516G mutation into the rpoB gene confers resistance to the antibiotic rifampicin, thus serving as a readout for genome modification. Targeting of the rpoB gene in E. coli with an engineered DarT variant fused to Cas9 nickase (ScnCas9) and co-expression of an RNA repair template and reverse transcriptase resulted in site-specific RNA-templated genome modification. [Figure 16] Figure 16 contains representative results of an experiment demonstrating that introduction of the D516G mutation into the rpoB gene confers resistance to the antibiotic rifampicin, thus serving as a readout for genome modification. Targeting the rpoB gene in E. coli with an engineered DarT variant fused to Cas9 nickase (ScnCas9) and providing a linear single-stranded DNA repair template resulted in genome modification at rpoB. Targeting of a gap editor complex to rpoB resulted in a 100-6,000-fold increase in genome modification rate, demonstrating the effectiveness of the gap editor. [Figure 17] Figure 17 includes representative chromatograms of RNA-templated mutations in the rpoB gene introduced by targeting a gap editor complex to the rpoB gene, expression of an RNA repair template, and expression of reverse transcriptase Ec86. The mutations include the AC>GT mutation required for D516G-mediated rifampicin resistance. [Figure 18] FIG. 18 includes an image of the consensus sequence for the DarT catalytic domain (SEQ ID NO: 18) of the DNA-modifying domain of the gap editor complex of the present disclosure. [Figure 19]FIG. 19 includes an image of the consensus sequence for the DarT catalytic domain (SEQ ID NO: 19) of the DNA-modifying domain of the gap editor complex of the present disclosure. [Figure 20] FIG. 20 includes an image of the consensus sequence for the DarT catalytic domain (SEQ ID NO: 20) of the DNA-modifying domain of the gap editor complex of the present disclosure. [Figure 21] FIG. 21 includes an image of the consensus sequence for the DarT catalytic domain (SEQ ID NO: 21) of the DNA-modifying domain of the gap editor complex of the present disclosure. [Figure 22] FIG. 22 includes an image of the consensus sequence for the Scabin catalytic domain (SEQ ID NO: 22) of the DNA-modifying domain of the gap editor complex of the present disclosure. [Figure 23] FIG. 23 includes an image of the consensus sequence for the Scabin catalytic domain (SEQ ID NO: 23) of the DNA-modifying domain of the gap editor complex of the present disclosure. [Figure 24] FIG. 24 includes an image of the consensus sequence for the Scabin catalytic domain (SEQ ID NO: 24) of the DNA-modifying domain of the gap editor complex of the present disclosure. [Diagram 25] FIG. 25 includes an image of the consensus sequence for the Mom catalytic domain (SEQ ID NO: 25) of the DNA-modifying domain of the gap editor complex of the present disclosure. [Figure 26] FIG. 26 includes an image of the consensus sequence for the Mom catalytic domain (SEQ ID NO: 26) of the DNA-modifying domain of the gap editor complex of the present disclosure. [Figure 27] FIG. 27 includes an image of the consensus sequence for the Mom catalytic domain (SEQ ID NO: 27) of the DNA-modifying domain of the gap editor complex of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Nucleotide modifications can take the form of functional modifications, such as DNA methylation at certain positions, or damage modifications (DNA lesions), such as cross-links, oxidation, and nitrosylation. These DNA lesions need to be repaired to maintain information fidelity and DNA functionality. Commonly occurring lesions are repaired directly by base excision, mismatch, and nucleotide excision repair processes. However, if these lesions are not repaired before DNA replication, they can become locked in the genome as mutated DNA or completely arrest cell division. To avoid this, replication-dependent repair processes have evolved. One such process, translesion synthesis, can directly bypass some DNA lesions; however, it may introduce DNA mutations across some DNA lesions. Alternatively, replication of DNA near the lesion can be skipped entirely by repriming synthesis downstream of the lesion. This repriming can occur by lagging strand primase or, in higher eukaryotes, by leading strand primase-polymerase, PRIMPOL. This repriming action allows replication to continue but leaves behind an unreplicated region complementary to the DNA lesion and the surrounding DNA. The cell still needs to determine the appropriate sequence complementary to the DNA lesion, and to do this, it uses a mechanism called homology-dependent gap repair (a subset of homologous recombination).

[0040] Homologous sequence-dependent gap repair (HDGR) is a highly accurate repair process in which a sister chromatid is used as a template to copy complementary DNA onto the lesion-containing strand. As a subset of homologous recombination, experiments were performed as described further herein to investigate whether this pathway can be exploited to use an ectopic repair template instead of (or in addition to) a sister chromatid to generate synthetic genome editing. Previous results have demonstrated that site-specific introduction of abasic DNA can trigger HDGR and be completed using a plasmid-derived DNA template for repair to generate precisely edited genomic DNA. However, in some cases, this approach may be somewhat dependent on the stability of the abasic site. For example, abasic sites can be stabilized by inhibition of cellular AP endonuclease activity, but AP endonuclease inhibition may negatively affect cell viability and genome stability and may not be feasible for some applications. Thus, as described further herein, alternative classes of DNA lesions have been identified that are less sensitive to base excision or similar repair processes than DNA lesions. Embodiments of the present disclosure include a class of lesions that involve the addition of chemical groups to DNA that block DNA replication (replication-blocking moieties) and facilitate HDGR.

[0041] For example, experiments were performed to investigate whether the addition of adenosine diphosphate ribose (ADPr) could be a promising DNA lesion candidate and act as a replication blocking moiety. ADPr transferase, which catalyzes the addition of ADPr to nucleotides, is cytotoxic. Therefore, in an attempt to induce HDGR without loss of cell viability, a method was developed to restrict ADPr activity to the R-loops exposed after CRISPR-Cas binding to the genome. Extracted dsDNA-binding ADPr-transferase was shown to be lethal when electroporated into eukaryotic cells. Separately, dsDNA-binding DNA-modifying enzymes have been fused to DNA-binding proteins to localize their activity, but retain a high rate of off-target modification, which requires additional mitigation steps to control activity. Single-stranded DNA-binding enzymes can have their activity localized to DNA R-loops exposed after target binding by Cas effectors to DNA.

[0042] Previous studies have described a class of single-stranded binding ADPr-transferase enzymes, including DarT and the DarT mutant DarT_G49D, that act as bacterial toxins. DarT expression is lethal in E. coli and appears to be repaired primarily by recombination and, to a lesser extent, by nucleotide excision repair. Experiments were therefore performed to investigate whether DarT could be used to induce site-specific HDGR templated on recombinant rather than genomic DNA sequences. The experiments sought to understand whether DarT could be sufficiently controlled to localize ADPr modification to Cas target sites, avoiding cytotoxicity and allowing efficient genome modification.

[0043] The section headings used in this section, and throughout this disclosure, are for organizational purposes only and are not intended to be limiting.

[0044] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In the event of any conflict, the present document, including definitions, will control. Preferred methods and materials are described below, however, methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. The phrase "in one embodiment" as used herein does not necessarily refer to the same embodiment, but may. Additionally, the phrase "in another embodiment" as used herein does not necessarily refer to different embodiments, but may. Thus, as described below, various embodiments of the present invention can be readily combined without departing from the scope or spirit of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are for illustrative purposes only and are not intended to be limiting.

[0045] The terms "comprise(s)," "include(s)," "having," "having," "can," "containing," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0046] For the recitation of numerical ranges herein, to the same degree of precision, each of the intervening numbers is expressly contemplated. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0047] "Correlated to" as used herein refers to "compared to."

[0048] As used herein, the term "nucleic acid molecule" refers to any nucleic acid-containing molecule, including, but not limited to, DNA or RNA. The term includes 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- Included are sequences that contain any of the known base analogs of DNA and RNA, including, but not limited to, mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0049] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises a coding sequence for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA, sRNA, microRNA, lincRNA). A polypeptide can be encoded by a full-length coding sequence or any portion of a coding sequence, so long as the desired activity or functional properties of the full length or fragment (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) are retained. The term also encompasses the coding region of a structural gene, as well as sequences located adjacent to the coding region at both the 5' and 3' ends for a distance of about 1 kb or more on either side, such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. Genomic forms or clones of a gene contain the coding region interrupted by non-coding sequences, termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns can contain regulatory elements such as enhancers. Introns are removed or "excised" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0050] As used herein, the term "heterologous gene" refers to a gene that is not in its natural environment. For example, a heterologous gene includes a gene from one species that has been introduced into another species. Heterologous genes also include genes native to an organism that have been altered in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc.). Heterologous genes are distinguished from endogenous genes in that heterologous gene sequences are typically linked to DNA sequences that are not found naturally associated with the gene sequence in a chromosome, or are associated with a portion of a chromosome where it is not found in nature (e.g., a gene expressed at a locus where the gene is not normally expressed).

[0051] As used herein, the term "oligonucleotide" refers to a short length, single-stranded polynucleotide chain. Oligonucleotides are typically less than about 300 residues long (e.g., between 15 and 100), however, as used herein, the term is intended to encompass longer polynucleotide chains. Oligonucleotides are often referred to by their length. For example, a 24-residue oligonucleotide is referred to as a "24-mer." Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes.

[0052] The terms "homology" and "homology" refer to the degree of identity. Partial or complete homology can be present. A partially homologous sequence is one that is less than 100% identical to another sequence.

[0053] As used herein, the term "complementary" or "complementarity" is used in reference to a polynucleotide (e.g., a sequence of nucleotides, such as an oligonucleotide or a target nucleic acid) related by the rules of base pairing. For example, the sequence "5'-AGT-3'" is complementary to the sequence "3'-TCA-5'". Complementarity can be "partial", where only a portion of the bases of the nucleic acid match according to the rules of base pairing. Alternatively, there can be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between the nucleic acid strands. This is particularly important in amplification reactions and detection methods that depend on binding between nucleic acids. Either term can also be used in reference to individual nucleotides, particularly within the context of a polynucleotide. For example, a particular nucleotide in an oligonucleotide can be described in terms of its complementarity, or lack thereof, to a nucleotide in another nucleic acid strand, as contrasted or compared to the complementarity between the oligonucleotide and the remainder of the nucleic acid strand.

[0054] In some contexts, the term "complementary" and related terms (e.g., "complementary," "complement") refer to nucleotides of a nucleic acid sequence that can bind to another nucleic acid sequence by hydrogen bonds, e.g., base pairing, e.g., by Watson-Crick base pairing or other base pairing. Nucleotides that can form base pairs, e.g., are complementary to each other: cytosine and guanine, thymine and adenine, adenine and uracil, and guanine and uracil are pairs. Percentage complementarity need not be calculated over the entire length of the nucleic acid sequence. The percentage of complementarity can be limited to a specific region of the nucleic acid sequence that is base paired, e.g., starting from the first base-paired nucleotide and ending with the last base-paired nucleotide. The complement of a nucleic acid sequence, as used herein, refers to an oligonucleotide that "antiparallel associates" when aligned with a nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other. Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention, including, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes can contain mismatched base pairs or non-matching bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically, taking into account a number of variables, including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, ionic strength, and the incidence of mismatched base pairs.

[0055] Thus, in some embodiments, "complementary" refers to a first nucleobase sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second nucleobase sequence 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases, or that the two sequences hybridize under stringent hybridization conditions. "Fully complementary" means that each nucleobase of the first nucleic acid can be paired with each nucleobase at the corresponding position of the second nucleic acid.For example, in certain embodiments, the oligonucleotide in which each nucleobase has complementarity with the nucleic acid has a nucleobase sequence that is identical to the complement of the nucleic acid 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases.

[0056] As used herein, a "double-stranded nucleic acid" can be a portion of a nucleic acid, a region of a longer nucleic acid, or the entire nucleic acid. A "double-stranded nucleic acid" can be, for example, without limitation, double-stranded DNA, double-stranded RNA, double-stranded DNA / RNA hybrids, etc. Single-stranded nucleic acids having secondary structures (e.g., base-paired secondary structures) and / or higher order structures include "double-stranded nucleic acids." For example, triplex structures are considered to be "double-stranded." In some embodiments, any base-paired nucleic acid is a "double-stranded nucleic acid."

[0057] The term "isolated", when used in reference to a nucleic acid, as in "isolated oligonucleotide" or "isolated polynucleotide", refers to a nucleic acid sequence that has been identified and separated from at least one component or contaminant with which it is normally associated in its natural source. An isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, a non-isolated nucleic acid is a nucleic acid, such as DNA and RNA, that is found in the state in which it exists in nature. For example, a given DNA sequence (e.g., a gene) is found on a host cell chromosome near adjacent genes; an RNA sequence, such as a specific mRNA sequence that encodes a specific protein, is found in a cell in a mixture with many other mRNAs that encode many proteins. However, an isolated nucleic acid that encodes a given protein includes, by way of example, such a nucleic acid in a cell that normally expresses the given protein, where the nucleic acid is in a chromosomal location that is different from that of the natural cell location, or is otherwise flanked by nucleic acid sequences that are different from those found in nature. An isolated nucleic acid, oligonucleotide, or polynucleotide can be present in single-stranded or double-stranded form. If the isolated nucleic acid, oligonucleotide or polynucleotide is to be used to express a protein, the oligonucleotide or polynucleotide will at least contain the sense or coding strand (i.e., the oligonucleotide or polynucleotide can be single-stranded), but can contain both the sense and antisense strands (i.e., the oligonucleotide or polynucleotide can be double-stranded).

[0058] As used herein, the term "purified" or "purifying" refers to the removal of components (e.g., contaminants) from a sample. For example, antibodies are purified by removal of contaminating non-immunoglobulin proteins; antibodies are also purified by removal of immunoglobulins that do not bind to the target molecule. Removal of non-immunoglobulin proteins and / or removal of immunoglobulins that do not bind to the target molecule results in an increase in the percentage of target-reactive immunoglobulins in the sample. In another example, a recombinant polypeptide is expressed in a bacterial host cell and the polypeptide is purified by removal of host cell proteins; the percentage of recombinant polypeptide is thereby increased in the sample.

[0059] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0060] 2. Gap Editing Factor CRISPR-based genome editing tools have found widespread applications, relying on their easily programmable targeting and robust activity. The initial use of such CRISPR-based tools focused on the ability of Cas nuclease to cleave DNA. In the process of repairing the cleaved DNA, genome editing is introduced. However, DNA cleavage is one of the most toxic events that cells can tolerate. DNA cleavage activates the cell's alarm system, which can lead to mutations, DNA rearrangements, or loss of cell viability. Subsequent CRISPR-Cas genome editing tools have sought to minimize these toxic effects by instead introducing single-stranded nicks or directly modifying DNA with enzymes. Nevertheless, these newer methods show a limited range of edits that can be introduced and can suffer from unwanted insertions, deletions, and mutations.

[0061] The embodiments of the present disclosure demonstrate that efficient non-toxic genome modification can be achieved by the introduction and repair of single-stranded DNA gaps. Previous studies have demonstrated that site-specific introduction of abasic sites into DNA drives homologous sequence-dependent gap recombination. By introducing an ectopic DNA repair template, genome modification can be achieved in the DNA sequence adjacent to the introduced abasic site. However, in some cases, this approach may depend on the stabilization of the abasic site. Thus, the embodiments of the present disclosure include the development of a system to induce homologous sequence-dependent gap repair using the addition of stable chemical groups to DNA. This modified DNA is not recognized or repaired by cellular glycosylases, which increases lesion stability and drives homologous sequence-dependent gap repair. Site-specific DNA targeting is achieved by fusion of the modification enzyme to a Cas effector, and in some cases, the rate of genome modification can be increased using a Cas effector to nick the target DNA strand. As further described herein, the combination of nicking and DNA modification can have a synergistic effect in genome modification because they mutually inhibit sister chromatid repair.

[0062] As recognized by those skilled in the art, the original and most widely used CRISPR-Cas genome editing technology relies on Cas nuclease to introduce double-strand breaks, which are then repaired by homologous recombination via the editing template, similar to gap editing factors.Although widely applied, the toxicity of double-strand breaks and their tendency to drive mutation or chromosomal rearrangement are consistent challenges for therapeutic applications.Such DNA breaks are highly toxic (especially in bacteria) and often lead to error-prone repair by non-homologous end joining pathway.Cutting and repair is potentially the most well-known way to insert large segments of DNA, which is important for many scientific and industrial applications.

[0063] Moreover, base editors can be used in an attempt to avoid toxicity by enzymatically converting one nucleotide into another. For example, cytosine can be converted to thymine, and adenine can be converted to guanine. However, such base editors can only change one or a few nucleotides at a time and need to be carefully targeted to avoid unwanted edits. Furthermore, base editors are mutagenic, which means that non-targeted nucleotides are more likely to be incorrectly replicated when base editors are used. Base editors are also constrained by the availability of target sequences. Compared to other techniques, base editors are relatively efficient and rely only on nicking a single strand of DNA, as opposed to cutting both strands.

[0064] Prime editors have been described only recently. Based on recent publications, Prime editors appear to have the major advantage of being relatively efficient and using a very small repair template encoded in the backbone of the Cas9 single guide RNA. Although touted as a technique that does not use double-stranded breaks, efficient Prime editing still involves nicking both strands of DNA in relatively close proximity (<200bp). This dual nicking is only slightly less toxic than the cut and repair approach. Error-prone insertions and deletions still occur in mammalian cells as a result of dual nicking. It is unclear to what extent Prime editors function in prokaryotes. Although their CRISPR-dependent off-target activity is attenuated, it is also unclear whether any mutagenic side effects may occur in their application.

[0065] Compared to other techniques, gap editing factors have the least amount of data related to their use. Nevertheless, gap editing factors, as described further herein, appear to have minimal toxic effects; some experiments have shown no detectable toxicity. Lack of toxicity can be particularly advantageous for therapeutic applications, since low toxicity typically indicates a low rate of unwanted mutations, DNA insertions or DNA rearrangements. Also, multiplex operations are generally hindered by toxicity (especially in bacteria). For in vivo therapeutics, gap editing factors may suffer from the same DNA and protein delivery problems as all other CRISPR-Cas methods, but there are newer delivery platforms that allow for simultaneous delivery of RNP and repair template.

[0066] The embodiments of the present disclosure include compositions, systems, kits and methods for targeted modification of nucleic acids in genomes. In accordance with such embodiments, the present disclosure provides gap editors and gap editor complexes that generally include a DNA recognition domain and a DNA modification domain. As further described in the examples provided herein, the gap editors and gap editor complexes facilitate programmable DNA targeting by a DNA recognition domain functionally coupled to a DNA modification domain to drive genome modification by homologous sequence-directed gap repair. In some embodiments, the DNA recognition domain binds to a DNA target sequence in a genome, and the DNA modification domain induces the formation of a replication block at at least one nucleotide in the genome. Targeting the gap editor in a specific direction generates a persistent DNA gap, thereby improving gap editor efficiency.

[0067] In some embodiments, the DNA recognition domain and the DNA modification domain are operably coupled. Functionally coupled includes any means for incorporating the DNA recognition domain and the DNA modification domain into a specific target site for the purpose of functioning as a genome editor. In some embodiments, "operably coupled" includes, but is not limited to, a polypeptide fusion, a peptide tag, a peptide linker, an RNA tag, and any combination thereof. For example, a gap editor or gap editor complex can include a DNA recognition domain (e.g., a fusion polypeptide) fused to a DNA modification domain. The DNA recognition domain of the gap editor fusion protein can recognize a specific site in a target nucleic acid (e.g., a nucleic acid sequence in a genome), and then the DNA modification domain can modify one or more nucleic acids in or around the target site to facilitate genome modification.

[0068] As will be appreciated by those of skill in the art based on the present disclosure, the gap editor complexes described herein may be used to modify any portion of the genome of an organism or cell. For example, the gap editor complexes of the present disclosure may be used to target specific sites in a genome to generate desired site-specific modifications, and / or the gap editor complexes of the present disclosure may be used to target one or more specific sites in a genome to generate modifications that result in the addition, replacement, and / or removal of portions of the genome. Moreover, the gap editor complexes of the present disclosure may be used to target any region of a gene, including, but not limited to, open reading frames, introns, exons, intron-exon boundaries, functional non-coding regions, and any upstream and / or downstream DNA / gene regulatory sequences. The terms "DNA / gene regulatory sequence", "control element" and "regulatory element", as used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolytic signals, and the like, that provide and / or regulate the transcription of non-coding or coding sequences and / or regulate the translation of the encoded polypeptide. Thus, the gap editor complexes of the present disclosure can be used to generate modifications in a genome that result in altered gene expression patterns and / or activity (e.g., upregulation or downregulation).

[0069] In some embodiments, the DNA recognition domain and the DNA modification domain do not comprise a fusion polypeptide (e.g., do not form a single fusion polypeptide or protein). In some embodiments, the DNA modification domain is recruited to the gap editor or gap editor complex by the DNA recognition domain. For example, the DNA recognition domain of the gap editor can recruit the DNA modification domain by protein-protein interactions. In some embodiments, this recruitment is facilitated by a tag or linker that serves to recruit and functionally couple the DNA modification domain to the DNA recognition domain at a specific site of the target nucleic acid. Other means for recruiting and functionally coupling the DNA modification domain to the DNA recognition domain based on protein-protein interactions may be used, including but not limited to antigen-antibody interactions (e.g., a DNA modification domain fused to an antigen binding domain and a DNA recognition domain fused to a corresponding antigen), protein tags (e.g., streptavidin-biotin interactions), peptide and single chain variable antibody fragments, split protein systems, or any ligand-receptor interactions. In other embodiments, a DNA-modifying domain can be incorporated into the DNA recognition domain, for example, by replacing the HNH domain of Cas9 with a DNA-modifying domain or by inserting a DNA-modifying domain into the PAM interaction domain.

[0070] In other embodiments, the DNA-modifying domain is recruited to the gap editor or gap editor complex by interaction with the nucleic acid. For example, a guide RNA molecule that interacts with a DNA recognition domain to bind to a site in a target nucleic acid can include a sequence and / or structure (e.g., a scaffolding domain) that binds to the DNA-modifying domain. In some embodiments, the sequence and / or structure in the guide RNA includes a domain that is recognized by an RNA-binding protein. In some embodiments, the DNA-modifying domain is fused to an RNA-binding protein that is recruited to the gap editor or gap editor complex by binding to the domain in the guide RNA. Other means for recruiting and functionally coupling the DNA-modifying domain to the DNA recognition domain based on RNA-binding interactions may be used. In some embodiments, the guide RNA is extended to encode an RNA aptamer that recognizes a different protein or protein domain, such as MS2 coat protein, Tat, or Rev. The recognized protein or protein domain is then fused to the DNA-modifying domain. The guide RNA can encode multiple copies of the same or different protein-binding domains. These protein binding domains can be incorporated into different parts of the gRNA, such as via a loop in the gRNA or sgRNA, or at the 3' end of the sgRNA.

[0071] As further described herein, the gap editor complexes of the present disclosure can be used to generate various modifications in the genome of an organism or cell, such as by mechanisms of homologous sequence-directed repair. In some embodiments, genome modification using the gap editors of the present disclosure can generate specific nucleotide modifications ranging from single nucleotide changes to large insertions or deletions. In some embodiments, the gap editor complexes of the present disclosure can be used to add or remove large sequences of DNA by using two or more guide RNA sequences to target separate sites in the genome (e.g., creating large genomic deletions by removing sequences between two gRNA target sites and / or inserting exogenous DNA sequences). In some embodiments, multiple gRNAs can be used to target multiple sites in the genome (e.g., multiplexing) to generate any number of desired modifications in the genome. As will be recognized by one of skill in the art based on the present disclosure, any type of genetic modification can be achieved using the gap editor complexes of the present disclosure in any cell type and / or organism, regardless of how the gap editor complex is delivered to the cell (e.g., transformation), including in vitro, ex vivo, or in vivo delivery methods. A general review of these methods can be found in Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

[0072] DNA recognition domain. In accordance with such embodiments, the DNA recognition domain of the gap editor or gap editor complex of the present disclosure comprises the use of a sequence-specific nucleic acid binding component (e.g., a molecule, a biomolecule, or a complex of one or more molecules and / or biomolecules) to target a specific nucleic acid target site. In some embodiments, the DNA recognition domain comprises at least one Cas protein or a fragment thereof that lacks nuclease or deoxyribonuclease activity. In some embodiments, the DNA recognition domain comprises a complex of Cas proteins that lack nuclease or deoxyribonuclease activity. In some embodiments, the DNA recognition domain comprises at least one Cas protein or a complex of Cas proteins that exhibit nickase activity, including but not limited to Cas9 or Cas12a that have nickase activity.

[0073] In some embodiments, the Cas protein or Cas protein complex includes Cascade type I, Cas9 type II, effector module type IV, Cas12 type V, IscB associated with Cas9, TnpB associated with Cas9, and combinations thereof. Cascade is a set of Cas proteins that form a stable complex with guide RNA in different ratios. gRNA is usually encoded in CRISPR array, where Cas6 protein of the complex cleaves hairpin in transcribed repeat sequence. Other proteins then form around the released RNA. The fully formed complex binds to target DNA flanked by protospacer adjacent motif (PAM) encoded at the 5' end of non-target strand. After target recognition, the complex then recruits Cas3 type I endonuclease to nick and progressively degrade the non-target strand in 3' to 5' direction, while the complex stably binds to target DNA in the absence of Cas3. The specific number and stoichiometry of proteins in the cascade varies between CRISPR-Cas subtypes, such as Cas8c(1):Cas5c(1):Cas7(7) for IC subtypes, and Cse1(1):Cse2(2):Cas5e(1):Cas7(6):Cas6e(1) for IE subtypes. Furthermore, these proteins may be fused to recreate the complex with fewer expressed polypeptides, and the Cas6 protein is not required when the guide RNA is expressed as a processed CRISPR RNA. Varying the length of the guide sequence within the gRNA can further alter the protein stoichiometry of the cascade, changing the length of the R-loop and the displaced DNA strand. Cas9 is a single effector nuclease that binds to target DNA with a PAM encoded at the 3' end of the non-target strand. The bound DNA is then nicked on the opposite strand by the HNH and RuvC domains of Cas9, resulting in a double-stranded break.The gRNAs utilized by Cas9 are usually encoded by CRISPR arrays, where the transactivating crRNA (tracrRNA) pairs with the transcribed repeats and the RNA duplex is cleaved by the endoribonuclease RNase III. The resulting processed crRNA:tracrRNA duplex is bound by Cas9 and directs DNA targeting. The crRNA:tracrRNA duplex can be fused to form a single guide RNA (sgRNA). Cas12 represents a diverse family of Cas nucleases named by their subtype (e.g., Cas12a, Cas12e) and given alternative names such as Cpf1, C2c1, CasX or Cas14a. Cas12 nuclease targets DNA with a PAM encoded at the 5' end of the non-targeted strand, and the RuvC domain of the nuclease nicks both the target and non-targeted strands to create a staggered double-stranded break with a 5' overhang. The gRNA is encoded within the CRISPR array and can be processed from the transcribed CRISPR array by one of two mechanisms depending on the nuclease: cleavage of a hairpin within the repeat sequence by a riboendonucleolytic domain bearing Cas12 nuclease (e.g., Cas12a) or pairing of the transcribed repeat sequence with the tracrRNA which is then cleaved by RNase III. As a result, if the nuclease alone is responsible for crRNA processing, the gRNA can be easily expressed in its processed form, and if the tracrRNA is involved in crRNA processing, the gRNA can be expressed as a sgRNA.

[0074] In some embodiments, the DNA recognition domain comprises a deoxyribonuclease-inactivated Cas9 ("dCas9"), which can be generated by introducing a deactivating mutation in the HNH and RuvC domains of the protein. In some embodiments, the DNA recognition domain comprises a deoxyribonuclease-inactivated Cas12a ("dCas12a"), which can be generated by introducing a deactivating mutation in at least one of the RuvC domains, such as RuvC-I. Alternatively, a guide RNA that is truncated or contains a mismatch with the target at the PAM distal end can allow DNA binding but not DNA nicking or cleavage by an otherwise catalytically active Cas nuclease.

[0075] In some embodiments, various other DNA recognition domains may be used in the gap editor complexes of the present disclosure. For example, certain embodiments of the compositions and methods described herein do not require guide RNA to achieve efficient genome editing and modification. As described above, such gap editor complexes include, but are not limited to, meganucleases, zinc fingers (ZFs), and transcription activator-like effectors (TALEs). In some embodiments, the DNA recognition domains of the present disclosure may include meganucleases. Meganucleases may be used to replace, eliminate, or modify sequences in a targeted manner, and their recognition target sequences may be altered by protein engineering. Meganucleases may be used to modify any genome type, be it bacterial, plant, or animal, and are amenable to in vivo delivery due to their relatively small size. The high degree of target specificity of meganucleases allows for a concomitant high degree of precision and much lower cytotoxicity. However, targeting novel sequences is difficult due to the limited number of meganucleases available.

[0076] In some embodiments, the DNA recognition domain of the present disclosure can include a zinc finger (ZF). A ZF is a fusion of a non-specific DNA cleavage domain derived from a restriction endonuclease with a zinc finger protein. ZFNs can target specific DNA sequences, which allows them to target and precisely alter unique sequences inside the target organism. A single zinc finger is composed of around 30 amino acids in a conserved ββα shape. Several amino acids on the surface of the α-helix usually select three base pairs within the smooth groove of DNA. Zinc finger proteins have become an important framework for the design of custom DNA binding proteins, as the development of non-natural arrays with more than three domains has become available, along with the development of highly conserved linker sequences that allow synthetic zinc finger proteins to recognize DNA sequences of 9-18 bp in length.

[0077] In some embodiments, the DNA recognition domain of the present disclosure can comprise a transcription activator-like effector (TALE). TALEs are highly versatile and can be combined with a number of effector domains, including nucleases, transcription activators and repressors, recombinases, transposases, DNA and histone methyltransferases and histone acetyltransferases, to affect genome structure and function. TALENs are transcription activator-like effector nucleases that are a fusion of a Fokl cleavage domain and a DNA binding domain. TALEs are naturally occurring proteins from bacteria of the genus Xanthomonas that contain a DNA binding domain composed of a series of 33-35 amino acid repeat domains that each recognize a single base pair. TALE specificity is determined by two hypervariable amino acids known as repeat variable 2 (di) residues (RVD). A number of effector domains, including nucleases, transcription activators and site-specific recombinases, are now available for fusion to TALE repeats for targeted genetic modification. Although the single-base recognition of TALE-DNA binding repeats offers greater design flexibility than triplet confined zinc fingers, cloning of repeat TALE arrays presents increased technical challenges due to extensive identical repeat sequences.

[0078] A DNA modifying domain. In some embodiments, the DNA modifying domain catalyzes the formation or addition of at least one replication blocking moiety to at least one nucleotide in a DNA target sequence. In some embodiments, the DNA modifying domain blocks DNA replication by adding a replication blocking moiety to at least one nucleotide in a DNA strand complementary to the DNA target sequence. In some embodiments, the DNA modifying domain blocks DNA replication by adding a replication blocking moiety to at least one nucleotide in a DNA strand containing the DNA target sequence. In some embodiments, the DNA modifying domain blocks DNA replication by adding a replication blocking moiety to both a nucleotide in a DNA strand complementary to the DNA target sequence and a nucleotide in a DNA strand containing the DNA target sequence.

[0079] In some embodiments, the DNA recognition domain induces a single-stranded break in the DNA target strand (by way of nickase activity) and the DNA modifying domain adds a replication blocking moiety to at least one nucleotide in the DNA strand complementary to the DNA target sequence. In some embodiments, the DNA modifying domain catalyzes the addition of ADP-ribose to a thymine or guanine nucleotide. In some embodiments, the DNA modifying domain comprises a DarT enzyme or a functional fragment, derivative or variant thereof. In some embodiments, the DarT enzyme is engineered to have reduced DNA binding, increased specificity for single-stranded DNA and / or reduced enzymatic activity. DarT homologs (and any fragment, derivative or variant thereof) that may be used in various embodiments disclosed herein include, but are not limited to, those presented in Table 1 below. In some embodiments, the DNA modifying domain comprises a Scabin enzyme or a functional fragment, derivative or variant thereof. In some embodiments, the Scabin enzyme is engineered to have reduced DNA binding, increased specificity for single-stranded DNA and / or reduced enzymatic activity. Scabin homologs (and any fragments, derivatives or variants thereof) that may be used in the various embodiments disclosed herein include, but are not limited to, those presented in Table 1 below. In some embodiments, the Mom enzyme is engineered to have reduced DNA binding, increased specificity for single-stranded DNA and / or decreased enzymatic activity. Mom homologs (and any fragments, derivatives or variants thereof) that may be used in the various embodiments disclosed herein include, but are not limited to, those presented in Table 1 below.

[0080] [Table 1] TIFF2024506375000002.tif235164TIFF2024506375000003.tif234163TIFF2024506375 000004.tif235164TIFF2024506375000005.tif234164TIFF2024506375000006.tif77164

[0081] As will be appreciated by those of skill in the art based on the present disclosure, other DNA-modifying domains / enzymes may be used in the gap editors and gap editor complexes of the present disclosure to induce the formation of a replication block at a given target site. For example, in some embodiments, the DNA-modifying domains / enzymes may include, but are not limited to, any of the following enzymes (or functional fragments, derivatives, or variants thereof): Pierisin, Scabin, Cell Cycle and Apoptosis Regulator 1 (CARP-1), SCO5461 protein (ScARP), adenine-modifying enzymes, acetyltransferases, amino acid transferases, nucleotidyltransferases, uridyltransferases, acyltransferases, ADP-ribosyltransferases, methylthiotransferases, N-acetyltransferase 10, tRNA(Met) cytidine acetyltransferase (TmcA), tRNA cytidine acetyltransferase, GCN5-related N-acetyltransferase, lysidine synthase, m 7 G methyltransferase, N6C carbamoylmethyltransferase (Mom), N6-adenosine threonylcarbamoyltransferase, threonylcarbamoyltransferase or threonylcarbamoyltransferase complex, TsaB-TsaE-TsaD (TsaBDE) complex, tRNA N6-adenosine threonylcarbamoyltransferase (Qri7, Tcs4), methyltransferase, ATrm5a, tRNA:m 1G / imG2 methyltransferase, tRNA(adenosine(37)-N6)-dimethylallyltransferase, tRNA dimethylallyltransferase (MiaA), and isopentenyltransferase.

[0082] In some embodiments, the DNA-modifying domain used in the gap editor complexes of the present disclosure comprises a catalytic domain (or a functional fragment, derivative, or variant thereof) that induces the formation of a replication block at at least one nucleotide in a genome. In some embodiments, the catalytic domain comprises a portion of a DarT enzyme sufficient to carry out ADP-ribosylation of a target nucleic acid, as further described herein. In some embodiments, the catalytic domain comprises a portion of a Scabin enzyme sufficient to carry out ADP-ribosylation of a target nucleic acid, as further described herein.

[0083] For example, catalytic domains of DNA-modifying domains that may be used in the gap editing factor complexes of the present disclosure include, but are not limited to, any sequence having at least 70% amino acid identity to any of SEQ ID NOs: 18-21. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 75% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 80% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 90% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 91% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 92% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 93% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 94% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 95% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 96% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 97% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 98% amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 99% amino acid sequence identity to SEQ ID NO: 18.

[0084] In some embodiments, the DNA modified domain comprises a catalytic domain having at least 75% amino acid sequence identity to SEQ ID NO:19. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 80% amino acid sequence identity to SEQ ID NO:19. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO:19. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 90% amino acid sequence identity to SEQ ID NO:19. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 91% amino acid sequence identity to SEQ ID NO:19. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 92% amino acid sequence identity to SEQ ID NO:19. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 93% amino acid sequence identity to SEQ ID NO:19. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 94% amino acid sequence identity to SEQ ID NO:19. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 95% amino acid sequence identity to SEQ ID NO:19. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 96% amino acid sequence identity to SEQ ID NO: 19. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 97% amino acid sequence identity to SEQ ID NO: 19. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 98% amino acid sequence identity to SEQ ID NO: 19. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 99% amino acid sequence identity to SEQ ID NO: 19.

[0085] In some embodiments, the DNA modified domain comprises a catalytic domain having at least 75% amino acid sequence identity to SEQ ID NO:20. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 80% amino acid sequence identity to SEQ ID NO:20. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO:20. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 90% amino acid sequence identity to SEQ ID NO:20. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 91% amino acid sequence identity to SEQ ID NO:20. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 92% amino acid sequence identity to SEQ ID NO:20. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 93% amino acid sequence identity to SEQ ID NO:20. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 94% amino acid sequence identity to SEQ ID NO:20. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 95% amino acid sequence identity to SEQ ID NO:20. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 96% amino acid sequence identity to SEQ ID NO: 20. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 97% amino acid sequence identity to SEQ ID NO: 20. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 98% amino acid sequence identity to SEQ ID NO: 20. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 99% amino acid sequence identity to SEQ ID NO: 20.

[0086] In some embodiments, the DNA modified domain comprises a catalytic domain having at least 75% amino acid sequence identity to SEQ ID NO:21. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 80% amino acid sequence identity to SEQ ID NO:21. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO:21. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 90% amino acid sequence identity to SEQ ID NO:21. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 91% amino acid sequence identity to SEQ ID NO:21. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 92% amino acid sequence identity to SEQ ID NO:21. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 93% amino acid sequence identity to SEQ ID NO:21. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 94% amino acid sequence identity to SEQ ID NO:21. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 95% amino acid sequence identity to SEQ ID NO:21. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 96% amino acid sequence identity to SEQ ID NO: 21. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 97% amino acid sequence identity to SEQ ID NO: 21. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 98% amino acid sequence identity to SEQ ID NO: 21. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 99% amino acid sequence identity to SEQ ID NO: 21.

[0087] In some embodiments, the catalytic domain of the DNA-modifying domain that may be used in the gap editing factor complexes of the present disclosure includes, but is not limited to, any sequence having at least 70% amino acid identity to any of SEQ ID NOs:22-24. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 75% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 80% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 90% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 91% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 92% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 93% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 94% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 95% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 96% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 97% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 98% amino acid sequence identity to SEQ ID NO:22. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 99% amino acid sequence identity to SEQ ID NO:22.

[0088] In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 75% amino acid sequence identity to SEQ ID NO:23. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 80% amino acid sequence identity to SEQ ID NO:23. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO:23. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 90% amino acid sequence identity to SEQ ID NO:23. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 91% amino acid sequence identity to SEQ ID NO:23. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 92% amino acid sequence identity to SEQ ID NO:23. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 93% amino acid sequence identity to SEQ ID NO:23. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 94% amino acid sequence identity to SEQ ID NO:23. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 95% amino acid sequence identity to SEQ ID NO:23. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 96% amino acid sequence identity to SEQ ID NO: 23. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 97% amino acid sequence identity to SEQ ID NO: 23. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 98% amino acid sequence identity to SEQ ID NO: 23. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 99% amino acid sequence identity to SEQ ID NO: 23.

[0089] In some embodiments, the DNA modified domain comprises a catalytic domain having at least 75% amino acid sequence identity to SEQ ID NO:24. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 80% amino acid sequence identity to SEQ ID NO:24. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO:24. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 90% amino acid sequence identity to SEQ ID NO:24. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 91% amino acid sequence identity to SEQ ID NO:24. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 92% amino acid sequence identity to SEQ ID NO:24. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 93% amino acid sequence identity to SEQ ID NO:24. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 94% amino acid sequence identity to SEQ ID NO:24. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 95% amino acid sequence identity to SEQ ID NO:24. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 96% amino acid sequence identity to SEQ ID NO: 24. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 97% amino acid sequence identity to SEQ ID NO: 24. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 98% amino acid sequence identity to SEQ ID NO: 24. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 99% amino acid sequence identity to SEQ ID NO: 24.

[0090] In some embodiments, the DNA-modifying domain used in the gap editor complexes of the present disclosure comprises a catalytic domain of Mom (also referred to as methylcarbamoyltransferase, methylcarbamoylase, or acetyltransferase) (or a functional fragment, derivative, or variant thereof). The catalytic domain can comprise a portion of a methylcarbamoylase enzyme sufficient to carry out methylcarbamoylation of adenine using acetyl-CoA as a donor substrate that is transferred to a target nucleic acid, as further described herein. For example, catalytic domains of Mom that may be used as DNA-modifying domains in the gap editor complexes of the present disclosure include, but are not limited to, any sequence having at least 70% amino acid identity to any of SEQ ID NOs: 25-27. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 75% amino acid sequence identity to SEQ ID NO: 25. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 80% amino acid sequence identity to SEQ ID NO: 25. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO: 25. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 90% amino acid sequence identity to SEQ ID NO:25. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 91% amino acid sequence identity to SEQ ID NO:25. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 92% amino acid sequence identity to SEQ ID NO:25. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 93% amino acid sequence identity to SEQ ID NO:25. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 94% amino acid sequence identity to SEQ ID NO:25. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 95% amino acid sequence identity to SEQ ID NO:25. In some embodiments, the DNA modifying domain comprises a catalytic domain having at least 96% amino acid sequence identity to SEQ ID NO:25.In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 97% amino acid sequence identity to SEQ ID NO: 25. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 98% amino acid sequence identity to SEQ ID NO: 25. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 99% amino acid sequence identity to SEQ ID NO: 25.

[0091] In some embodiments, the DNA modified domain comprises a catalytic domain having at least 75% amino acid sequence identity to SEQ ID NO:26. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 80% amino acid sequence identity to SEQ ID NO:26. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO:26. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 90% amino acid sequence identity to SEQ ID NO:26. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 91% amino acid sequence identity to SEQ ID NO:26. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 92% amino acid sequence identity to SEQ ID NO:26. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 93% amino acid sequence identity to SEQ ID NO:26. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 94% amino acid sequence identity to SEQ ID NO:26. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 95% amino acid sequence identity to SEQ ID NO:26. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 96% amino acid sequence identity to SEQ ID NO: 26. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 97% amino acid sequence identity to SEQ ID NO: 26. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 98% amino acid sequence identity to SEQ ID NO: 26. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 99% amino acid sequence identity to SEQ ID NO: 26.

[0092] In some embodiments, the DNA modified domain comprises a catalytic domain having at least 75% amino acid sequence identity to SEQ ID NO:27. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 80% amino acid sequence identity to SEQ ID NO:27. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 85% amino acid sequence identity to SEQ ID NO:27. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 90% amino acid sequence identity to SEQ ID NO:27. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 91% amino acid sequence identity to SEQ ID NO:27. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 92% amino acid sequence identity to SEQ ID NO:27. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 93% amino acid sequence identity to SEQ ID NO:27. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 94% amino acid sequence identity to SEQ ID NO:27. In some embodiments, the DNA modified domain comprises a catalytic domain having at least 95% amino acid sequence identity to SEQ ID NO:27. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 96% amino acid sequence identity to SEQ ID NO: 27. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 97% amino acid sequence identity to SEQ ID NO: 27. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 98% amino acid sequence identity to SEQ ID NO: 27. In some embodiments, the DNA-modifying domain comprises a catalytic domain having at least 99% amino acid sequence identity to SEQ ID NO: 27.

[0093] Replication blocking moiety. Based on the present disclosure, those skilled in the art will recognize that replication blocking moieties can include, but are not limited to, glucose, threonylcarbamoyl adenosine, acetate, glyceryl, L-ascorbic acid, uridine, adenosine monophosphate, adenosine diphosphate ribose, methylcarbamoyl, lipids, amino acids, agmatine, L-threonylcarbamoyl adenylate, L-threonylcarbamoyl, methylthiolate, sulfur, methyl group, S-adenosyl-L-methionine or a subgroup of S-adenosyl-L-methionine, and dimethylallyl diphosphate or a subgroup thereof. The above and other replication blocking moieties have the common feature that they are capable of functionalizing nucleotides in a target sequence such that DNA replication is blocked and homologous sequence-directed gap repair is induced. This can occur by enzymatic or enzyme-independent means.

[0094] Guide RNA. The embodiments of the present disclosure also include gap editors and gap editor complexes that can include at least one guide RNA molecule. According to such embodiments, the guide RNA molecule includes a handle sequence and a targeting sequence. The targeting sequence interacts with a sequence in the target nucleic acid, and the handle sequence facilitates the binding of the gap editor or the gap editor complex. As will be recognized by those skilled in the art based on the present disclosure, a single chimeric guide RNA (sgRNA) can mimic the structure of the annealed crRNA / tracrRNA; this type of guide RNA has become more widely used than crRNA / tracrRNA because the gRNA approach provides a simplified system with only two components (e.g., Cas9 and sgRNA). Thus, sequence-specific binding to nucleic acid targets can be guided by natural dual RNA complexes (e.g., including crRNA, tracrRNA, and Cas9) or chimeric single guide RNAs (e.g., sgRNA and Cas9) (see, e.g., Jinek et al. (2012) "A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity," Science 337:816-821). Multiple gRNAs can further be expressed using CRISPR arrays that naturally encode the crRNAs utilized by the nuclease. gRNAs can also be expressed separately by being operably linked to a promoter and terminator. gRNAs can also be fused in a single transcript by including an intervening RNA cleavage site, such as a ribozyme, or a site recognized by an RNA cleavage enzyme, such as RNase P, RNase Z, RNase III, or Csy4. The gRNA or sgRNA can include an RNA template for reverse transcription into a cDNA repair template. The sgRNA can include an aptamer sequence, e.g., an RNA-binding protein recognition site, so that it can recruit accessory genome editing factors to the gap editor complex or to the gap editor target site.

[0095] As further described herein, genome modification using the gap editors of the present disclosure can generate specific nucleotide modifications ranging from single nucleotide changes to large insertions or deletions. In some embodiments, the gap editor complexes of the present disclosure can be used to add, replace, and / or remove large sequences of DNA by using two or more guide RNA sequences to target separate sites in the genome. For example, large genome deletions can be generated by removing sequences between two gRNA target sites and / or inserting exogenous DNA sequences (e.g., based on endogenous repair / recombination mechanisms in cells or organisms). In some embodiments, multiple gRNAs can be used to target multiple sites in the genome (e.g., multiplexing) to generate any number of desired modifications in the genome.

[0096] In some embodiments, guide RNA molecules are not required in the gap editor complexes of the present disclosure. For example, certain embodiments of the compositions and methods described herein do not require guide RNA to achieve efficient genome editing and modification. As described above, such gap editor complexes include, but are not limited to, meganucleases, zinc fingers (ZFs) and transcription activator-like effectors (TALEs).

[0097] Donor template. In some embodiments, the presence of the donor nucleic acid template facilitates homologous sequence-directed gap recombination and / or repair, including the donor nucleic acid template or a fragment thereof being recombined into the double-stranded target DNA molecule. In some embodiments, the donor DNA template can function as a replication template resulting in the sequence encoded by the exogenous DNA or RNA being copied into the genome, but the exogenous DNA or RNA polynucleotide molecule itself is not directly transferred into the genome. The donor nucleic acid template can be single-stranded or double-stranded. In some embodiments, the donor template is a cDNA reverse transcribed from endogenous, expressed, synthetic, or delivered RNA. The donor nucleic acid can be delivered into the cell as a plasmid or linear DNA. The donor nucleic acid can also be generated in vivo from a template ribonucleic acid by reverse transcriptase. In other embodiments, the donor nucleic acid itself can be a ribonucleic acid. The donor nucleic acid can also contain chemical modifications. The donor nucleic acid can include chemical modifications or sequences that are specifically recruited to a gap edit factor complex or a gap edit factor target site.

[0098] In some embodiments, the donor nucleic acid template comprises a polynucleotide derived from an endogenous homologous sequence corresponding to the DNA target sequence. In some embodiments, the donor nucleic acid template comprises a polynucleotide derived from an endogenous allele (e.g., to facilitate loss of heterozygosity). In some embodiments, the donor nucleic acid template comprises an exogenous single-stranded DNA (ssDNA) molecule or a double-stranded DNA (dsDNA) molecule. In some embodiments, the presence of the donor nucleic acid template facilitates homologous sequence-directed gap repair and / or recombination, and the donor nucleic acid template or a fragment thereof is recombined into the genome of the DNA target sequence. In accordance with such embodiments, the gap editors of the present disclosure may be particularly advantageous for inserting large donor DNA sequences, replacing large segments of DNA, and / or removing large DNA sequences in a genome. In some embodiments, the gap editor complexes of the present disclosure may be used to add, replace, and / or remove large sequences of DNA by using two or more guide RNA sequences to target separate sites in a genome. For example, large genomic deletions can be generated by removing sequences between two gRNA target sites and / or inserting exogenous DNA sequences (e.g., based on endogenous repair / recombination mechanisms in a cell or organism). In some embodiments, multiple gRNAs can be used to target multiple sites in a genome (e.g., multiplexing) to generate any number of desired modifications in the genome.

[0099] Accessory factors. In some embodiments, the compositions and systems of the present disclosure further comprise a gap editor accessory factor. In some embodiments, the compositions further comprise at least one gap editor accessory factor. In some embodiments, the at least one gap editor accessory factor comprises a protein that enhances at least one step in the genome modification process. In some embodiments, the at least one gap editor accessory factor is recruited to the gap editor complex via an interaction with a DNA-modifying domain, a DNA recognition domain, and / or at least one guide RNA. In some embodiments, the recruitment of the at least one gap editor accessory factor to the gap editor complex comprises a peptide tag, a peptide linker, an RNA tag, and any combination thereof. In some embodiments, the at least one gap editor accessory factor comprises Rap, DarG, Orf, ExoI, Exonuclease III, PrimPol, RecJ, RecQ1, Rad51, Rad52, CtIP, Rad18, and any combination thereof. In some embodiments, and as further described herein, the present disclosure can comprise a gap editor complex, wherein the DNA-modifying domain comprises DarT. In accordance with such embodiments, DarG, TARG1 or another glycohydolase domain can be included as a gap edit factor accessory factor by modulating off-target editing (e.g., attenuating DarT activity) or removing the added ADPr after HDGR has occurred.

[0100] As will be appreciated by those of skill in the art based on the present disclosure, methods for delivering gap edit factors and gap edit factor complexes into cells include any currently known methods and systems for delivering polynucleotides and / or polypeptides / proteins. For example, gap edit factors and gap edit factor complexes may be delivered using plasmid DNA, ssDNA, RNA, or other means for delivering polynucleotide molecules, including, but not limited to, lipid-based delivery systems (e.g., using cationic lipids), conjugation from donor cells, virus / bacteriophage-based delivery systems, and chemical-based systems (e.g., calcium phosphate precipitation, DEAE-dextran, polybrene). In some embodiments, the delivery system may include mechanical and / or electrical devices and methods for delivering the gap edit factors and gap edit factor complexes of the present disclosure as polynucleotides and / or polypeptides / proteins (or any combination thereof). In some embodiments, the gap editing factors and gap editing factor complexes are delivered using gene guns (e.g., bombardment and Agrobacterium transformation used on plant cells) and electroporation-based methods, as well as any other physical method (e.g., mechanical, electrical, thermal, optical, chemical stimulation, etc.) that uses membrane disruption as a means to deliver polynucleotides and polypeptides / proteins (see, e.g., Sun et al., Recent advances in micro / nanoscale intracellular delivery, Nanotechnology and Precision Engineering 3, 18 (2020)).

[0101] 3. Kits, Systems and Methods The embodiments of the present disclosure also include kits and systems for targeted modification of nucleic acids. According to such embodiments, the kits include a gap editor complex comprising a DNA recognition domain and a DNA modification domain. In some embodiments, the kits also include at least one guide RNA molecule. In some embodiments, the DNA recognition domain binds to a DNA target sequence in a genome, and the DNA modification domain induces the formation of a replication blocker at at least one nucleotide in the genome. As will be recognized by those skilled in the art based on the present disclosure, the kits and systems can also include one or more of the other components of the genetic modification compositions described herein (e.g., gap editor accessory factors). In some embodiments of the kits, the composition further includes a donor nucleic acid template. In some embodiments of the kits, the presence of the donor nucleic acid template facilitates homologous sequence-directed gap repair and / or recombination.

[0102] In some embodiments of the kit, the DNA recognition domain comprises at least one Cas protein or fragment thereof that lacks deoxyribonuclease activity. In some embodiments of the kit, the DNA recognition domain comprises at least one Cas protein or fragment thereof that has nickase activity. In some embodiments, the Cas protein or Cas protein complex comprises a type I cascade, a type II Cas9, a type IV effector module, a type V Cas12, a Cas9 associated IscB, a Cas9 associated TnpB, and combinations thereof.

[0103] In some embodiments of the kit, the DNA recognition domain and the DNA modifying domain are functionally coupled. In some embodiments of the kit, the DNA recognition domain induces a single-stranded break in the DNA target strand, and the DNA modifying domain adds a replication blocking moiety to at least one nucleotide in the DNA strand complementary to the DNA target sequence. In some embodiments of the kit, the DNA modifying domain catalyzes the addition of ADP-ribose to a thymine or guanine nucleotide. In some embodiments, the DNA modifying domain comprises a DarT enzyme or a functional fragment, derivative or variant thereof. In some embodiments of the kit, the DarT enzyme is engineered to have reduced DNA binding, increased specificity for single-stranded DNA and / or reduced enzymatic activity.

[0104] In some embodiments of the kit, the DNA modifying domain catalyzes the addition of a replication blocking moiety selected from the group consisting of glucose, threonylcarbamoyladenosine, acetate, glyceryl, L-ascorbic acid, uridine, adenosine monophosphate, a lipid, an amino acid, agmatine, L-threonylcarbamoyladenylate, L-threonylcarbamoyl, methylthiolate, sulfur, a methyl group, S-adenosyl-L-methionine or a subgroup of S-adenosyl-L-methionine, and dimethylallyl diphosphate or a subgroup thereof. In some embodiments of the kit, the DNA modifying enzyme domain is selected from the group consisting of Pierisin, Scabin, cell cycle and apoptosis regulator 1 (CARP-1), SCO5461 protein (ScARP), adenine modifying enzyme, acetyltransferase, amino acid transferase, nucleotidyltransferase, uridyltransferase, acyltransferase, ADP-ribosyltransferase, methylthiotransferase, N-acetyltransferase 10, tRNA(Met) cytidine acetyltransferase (TmcA), tRNA cytidine acetyltransferase, GCN5-related N-acetyltransferase, lysidine synthase, mRNA cytidine acetyltransfer ... 7G methyltransferase, N6 carbamoyl methyltransferase (Mom), N6-adenosine threonylcarbamoyltransferase, threonylcarbamoyltransferase or threonylcarbamoyltransferase complex, TsaB-TsaE-TsaD (TsaBDE) complex, tRNA N6-adenosine threonylcarbamoyltransferase (Qri7, Tcs4), methyltransferase, ATrm5a, tRNA:m 1 The present invention includes an enzyme selected from the group consisting of G / imG2 methyltransferase, tRNA(adenosine(37)-N6)-dimethylallyltransferase, tRNA dimethylallyltransferase (MiaA), and isopentenyltransferase, or a functional fragment, derivative, or variant thereof.

[0105] In some embodiments of the kit, at least one guide RNA comprises a gRNA, sgRNA, crRNA, or any combination thereof. In some embodiments of the kit, at least one guide RNA comprises a handle sequence and a targeting sequence. In some embodiments of the kit, the targeting sequence in at least one guide RNA is complementary to a DNA target sequence. In some embodiments, the gap editor complexes of the present disclosure can be used to add, replace, and / or remove large sequences of DNA by using two or more guide RNA sequences to target separate sites in a genome. For example, large genomic deletions can be generated by removing sequences between two gRNA target sites and / or inserting exogenous DNA sequences (e.g., based on endogenous repair / recombination mechanisms in a cell or organism). In some embodiments, multiple gRNAs can be used to target multiple sites in a genome (e.g., multiplexing) to generate any number of desired modifications in the genome.

[0106] The embodiments of the present disclosure also include methods for targeted modification of nucleic acids. In accordance with such embodiments, the methods include introducing any of the components of the genome modification compositions described herein and evaluating the cell for the presence of the desired genetic alteration using techniques known in the art. In some embodiments of the methods, the components include a gap editor and a gap editor complex comprising a DNA recognition domain and a DNA modification domain, at least one guide RNA molecule, and a donor nucleic acid template. In some embodiments, one or more gap editor accessory factors may also be included. One or more of such factors may be introduced into a cell or organism as a polypeptide(s), mRNA(s) and / or DNA expression construct(s), or any combination thereof, by means known in the art. As will be recognized by those of skill in the art based on the present disclosure, the gap editor compositions, systems, and methods may be used to facilitate the modification of whole organisms, including, but not limited to, humans, plants, livestock, and others.

[0107] In some embodiments of the method, at least one of these components is introduced into the cell as part of a gene drive system. In a gene drive system, all or part of the genome modification components, such as DNA recognition domain, DNA modification domain, gRNA and accessory factors, are encoded in a donor nucleic acid sequence present in one copy of a chromosome. The gRNA directs the DNA modification domain to the sister chromosome in the region where the donor nucleic acid sequence is present. After targeting by a gap editor protein or complex, the donor nucleic acid (which also encodes the gap editor system) is copied into a new chromosome. Thus, the gap editor system becomes self-propagating, effectively forming a homozygously edited organism. Examples of organisms in which gene drives can be implemented include fungi, flatworms, mosquitoes and mice.

[0108] In some embodiments, the compositions, systems and methods of the present disclosure include one or more components that enhance or improve one or more aspects of genetic modification. In some embodiments, the improvement or enhancement of one or more aspects of genome modification includes the use of a gap editing factor accessory factor(s) as described above. In some embodiments, the method of enhancing or improving one or more aspects of genome modification includes reducing or attenuating nuclease activity in a cell in which genome modification is desired. Reducing nuclease activity in a cell can result in enhanced or improved modification frequency and / or efficiency. In some embodiments, reducing nuclease activity in a cell includes reducing the activity of an endogenous AP endonuclease (e.g., encoded by xthA) by any means known in the art. In some embodiments, nuclease activity in cells may be reduced by genetic and / or pharmacological means (e.g., treatment with endonuclease inhibitors including, but not limited to, AJAY-4, CRT0044876, aurintricarboxylic acid, 6-hydroxy-DL-DOPA, Reactive Blue 2, myricetin, mitoxantrone, methyl-3,4-dephostatin, thiolactomycin, and (2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2-propenoic acid (E3330)).

[0109] The compositions, systems and methods embodiments provided herein can be used to edit the genome of a cell. The cell can be a prokaryotic cell, a eukaryotic cell, or a plant cell. In some embodiments, the cell is a mammalian cell. The present disclosure also provides an isolated cell comprising any of the components or systems described herein. Exemplary cells include those that can be easily and reliably cultivated, have a reasonably fast growth rate, have a well-characterized expression system, and can include cells that can be easily and efficiently transformed or transfected. Examples of suitable prokaryotic cells include those from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Clostridium (such as Clostridium difficile or Clostridium autoethanogenum), Escherichia (such as E. coli), Lactobacilli, Klebsiella, Myxobacteria, Pseudomonas, Streptomyces, Salmonella, Vibrio (Vibrio cholerae), and the like. Suitable eukaryotic cells include, but are not limited to, cells from the genera Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include yeast cells from the genera Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces.Exemplary insect cells include Sf-9 and HIS (Invitrogen, Carlsbad, Calif.), as described, e.g., in Kitts et al., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al., J. Virol., 67:4566-4579 (1993).

[0110] In some embodiments, the compositions and methods of the disclosure can be used to induce DNA modifications and / or transcriptional modulation in mitotic or post-mitotic cells in vivo and / or ex vivo and / or in vitro (e.g., to produce genetically modified cells that can be reintroduced into an individual). Because the gap editing factors of the disclosure involve site-specific DNA targeting, the mitotic and / or post-mitotic cells of interest can be cells from any organism (e.g., bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, plant cells, algal cells, such as Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agarwal, and others). C. Agardh), as well as fungal cells (e.g., yeast cells), animal cells, cells from invertebrates (e.g., Drosophila, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals, cells from rodents, cells from humans, etc. Any type of cell may be of interest (e.g., stem cells, e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, germ cells; somatic cells, e.g., fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells; in vitro or in vivo embryonic cells of any stage of embryo, e.g., zebrafish embryos at the 1-cell, 2-cell, 4-cell, 8-cell, etc. stage; etc.). The cells may be derived from an established cell line or may be primary cells, where "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein to refer to cells and cell cultures that are derived from a subject and grown in vitro for a limited number of culture passages. Target cells can include any unicellular organism, multicellular organism, or any cell grown in culture.

[0111] In some embodiments, the cell may be a cell used for therapeutic purposes. The cell may be a mammalian cell, and in some embodiments, the cell is a human cell. Numerous suitable mammalian and human cells are known in the art, many of which are available from the American Type Culture Collection (ATCC, Manassas, Va.). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR- cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651) and CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian cells include primate, rodent and human cell lines, including transformed cell lines. Cell lines derived from normal diploid cells, in vitro culture of primary tissues or primary explants are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, HEK, A549, HepG2, mouse L-929 cells and BHK or HaK hamster cell lines. Methods for selecting suitable cells, as well as methods for transforming, culturing, amplifying, screening and purifying cells are known in the art. Examples of suitable plant cell lines are derived from plants such as Arabidopsis (such as the Landsberg erecta cell line), sugarcane, tomato, pea, rice, wheat, tobacco (such as the BY-2 cell line).

[0112] According to the methods described in the above embodiments, the compositions and systems disclosed herein can be used to edit the genome of a cell in a manner that reduces the extent of indel formation, chromosomal rearrangements, or DNA duplications. In some embodiments, the compositions, systems, and methods described herein reduce cytotoxicity compared to currently available methods, at least in part due to the lack of double-stranded breaks in the target nucleic acid.

[0113] 4. Materials and Methods Measurement of gap editing in E. coli by colorimetric assay was performed by co-transforming a DNA modification domain (e.g., DarT-ScdCas9) and sgRNA fused to a DNA binding domain such as Cas9 and a nucleic acid donor into E. coli by electroporation and plating on LB agar plus appropriate antibiotic(s). Resulting colonies were picked and inoculated into 750 mL of liquid LB medium in deep-well plates and shaken at 900 rpm overnight for 12-16 hours at 37 °C. Gap editing factor expression was induced by diluting the overnight culture 1:500 into 750 mL of liquid LB medium with antibiotics, 1 mM IPTG and 33 mM arabinose and shaking at 900 rpm for 8 hours. After 8 hours, samples were removed for spot plating on LB agar with antibiotics, IPTG and X-gal. The next day, white and blue colonies were counted to determine the frequency of lacZ recombination and repair. Repair was confirmed by Sanger sequencing.

[0114] Gap editing in E. coli was measured by antibiotic resistance assay by co-transforming the nucleic acid donor with a DNA modification domain fused to a DNA binding domain such as Cas9 or Cas12a and sgRNA by electroporation. The transformation mixture was plated on LB agar plus the appropriate antibiotic. The resulting colonies were picked and inoculated into 750 mL of liquid LB medium in deep-well plates and shaken at 900 rpm overnight at 30°C for 12-16 hours. The gap editing factor culture was first back-dilute 1:100 into liquid LB medium with antibiotic and shaken at 37°C for 1 hour. Gap editing factor expression was then induced by further diluting the culture 1:100 into 750 mL of liquid LB medium with antibiotic and 33 mM arabinose and shaken at 900 rpm for 5 hours. After 5 hours of induction, samples were removed for spot plating on two separate LB agar plates. One plate contained antibiotics (typically chloramphenicol and ampicillin) to select for only the gap editing factors, sgRNA and repair template, and the other plate also contained either rifampicin or kanamycin to select for edited cells. The next day, colonies were counted. Genome editing efficiency was tabulated as the number of colonies on plates containing rifampicin or kanamycin divided by the number of colonies on plates not containing rifampicin or kanamycin.

[0115] Measurement of gap editor toxicity in FIG. 7 was performed by co-transforming the DarT-ScdCas9 gap editor into an E. coli strain lacking recA, a key factor in homologous recombination. Such bacteria lack the ability for lesion bypass by homologous recombination and are therefore highly sensitive to replication-blocking lesions in DNA. Thus, the DNA-modifying domain is expected to be particularly toxic in such strains unless its potential DNA-binding activity is contained. In this manner, applicants can more easily evaluate the gap editor complex for undesired off-target DNA modifications. After transformation and plating, single colonies were selected and inoculated into 750 mL of LB chloramphenicol in deep-well plates and shaken overnight at 37° C. The next day, the culture was diluted 1:500 back into LB chloramphenicol with glucose to maintain gap editor repression or 1:500 back into LB chloramphenicol with arabinose to induce gap editor expression. Cultures were incubated at 37° C. for 5 hours in deep-well plates with shaking at 900 rpm. Cultures were then spot-plated onto LB chloramphenicol. Colonies were counted the next day to assess the final cell density and therefore the rate of off-target DNA modification.

[0116] Measurement of ssDNA template gap editing in E. coli by rifampicin resistance was performed by first co-transforming a strand annealing beta recombinase plasmid and a DNA modification domain fused to a DNA binding domain such as Cas9. The resulting clones were plated on LB, antibiotics and anhydrotetracycline for induction of beta recombinase expression. Such cultures were prepared for electroporation, transformed with sgRNA plasmids, and grown in rich medium at 37°C with shaking at 250 RPM for 3 hours, and then spot plated on two separate LB agar plates. One plate contained antibiotics to select for gap editors, sgRNA and recombinase only. The other plate also contained rifampicin to select for edited cells. The next day, colonies were counted. Genome editing efficiency was tabulated as the number of colonies on the plate containing rifampicin divided by the number of colonies on the plate not containing rifampicin.

[0117] [Table 2] TIFF2024506375000008.tif230164TIFF2024506375000009.tif229164TIFF2024506375000010.tif143164 EXAMPLES

[0118] 5. Working Example It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the disclosed methods described herein can be readily applied and recognized, and can be made using suitable equivalents, without departing from the scope of the disclosure or the aspects and embodiments disclosed herein. Having described the present disclosure in detail, it will be more clearly understood by reference to the following examples, which are intended merely to illustrate certain aspects and embodiments of the present disclosure, and should not be considered as limiting the scope of the present disclosure. The disclosures of all journal references, U.S. patents and publications referenced herein are hereby incorporated by reference in their entirety.

[0119] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

[0120] [Example 1] Experiments were carried out to evaluate the efficiency and toxicity of the gap editor complex of the present disclosure. In one set of experiments, the DarT enzyme from E. coli EPEC with the attenuating mutation G49D was fused to the N-terminus of a fully or partially catalytically inactive version of ScCas9 (ScdCas9 or ScCas9 D10A, also known as ScnCas9). It was hypothesized that chemical modifications, if any, would be made on the non-target strand exposed by ScdCas9 binding to its DNA target. Previous studies have indicated that DarT likely modifies thymines within sequence motifs as extensive as TYTN. Thus, such gap editor complexes were used to evaluate genome editing in E. coli.

[0121] DarT-ScdCas9 fusion protein (gap editor complex) was targeted to four sites containing NGG or NAG PAM and TTTC motifs in the non-targeted strand. The four sites surrounded a premature stop codon in the lacZ gene, the desired site of genome modification. The targets were chosen such that when a replication-blocking lesion was introduced, a DNA gap overlapping the premature stop codon would be formed. The four sites included two lagging strand targets and two leading strand targets. A plasmid encoding arabinose-inducible DarT-ScdCas9 was co-transformed with a plasmid containing a 1.5 kb repair template encoding the mutation to repair the lacZ stop codon while blocking ScdCas9 retargeting. After growing these colonies overnight, the cells were diluted back into induction medium, grown for 8 hours, and then plated on selection medium with the β-galactosidase (lacZ gene product) indicator dye X-gal and the inducer IPTG.

[0122] When only one site was targeted, the lacZ gene was efficiently repaired as demonstrated by the results in FIG. 1. However, targeting this site included a 10-fold drop in CFUs compared to non-targeted conditions and a 50-fold drop in CFUs compared to ScdCas9 controls. This observed cytotoxicity may be due to ScdCas9-independent binding of DarT to ssDNA, which introduced a widespread DNA replication block. It was hypothesized that by attenuating DNA binding within DarT, DarT may become more dependent on ScdCas9 for DNA binding. A computational prediction tool was used to identify potential DNA binding sites. To improve prediction accuracy, a set of DarT homologs with some sequence divergence was identified, and DNA binding sites were predicted for all of these homologs. By aligning protein and DNA predictions, some DNA binding site predictions were found to be conserved across these DarT homologs. Based on this, alanine mutations were introduced into these predicted sites. In one example, the K56A mutation substantially reduced the cytotoxic effect of DarT-ScdCas9 while maintaining efficient genome modification activity (Figure 1). This new DarT-ScdCas9 fusion protein was named gap editing factor 2 (GE2).

[0123] [Example 2] Since a single replication block was introduced into the DNA, it was expected that the dominant repair template would be the sister chromatid and not the ectopic repair template. Previous studies have demonstrated that targeting two sites on either side of the DNA sequence of interest can boost genome modification, presumably by creating overlapping DNA gaps and interfering with sister chromatid repair. Therefore, it was hypothesized that the combination of DNA nicking and DNA modification / gap formation could similarly prevent sister chromatid repair, leaving the plasmid repair template as the preferred template for repair.

[0124] Cas9 nicking can drive low rates of genome editing in prokaryotes and eukaryotes. Such nicks form single-end double-strand breaks (seDSBs) when they encounter the replisome, which typically involves replisome dissociation. Such single-end breaks are most frequently repaired by homologous recombination with sister chromatids. Importantly, in eukaryotic cells, Cas9 nicking can generate precise edits while minimizing indels, likely caused by non-homologous end joining (NHEJ) mechanisms. Because there is no natural end-joining partner at the seDSB, NHEJ is inhibited at this break.

[0125] According to an embodiment of the present disclosure, it was hypothesized that overlapping DNA gaps and seDSBs can mutually exclude (e.g., synergize) sister chromatid repair. Where seDSB ends typically seek homology in sister chromatids, there is instead a ssDNA gap. Similarly, where DNA gaps typically find homologous DNA templates, there is a seDSB that is likely to be excised to ssDNA. Thus, the H848A mutation in ScdCas9 was reactivated to create the target strand nickase ScnCas9.

[0126] This nicking DarT-ScnCas9 fusion was tested in the lacZ repair assay described above using the most efficient target. As shown in Figure 2, nickase alone produced low levels of gene repair and a substantial drop in CFU when expressed with the targeting sgRNA. DarT-ScdCas9 and the engineered DarT_K56A-ScdCas9 (GE2) produced moderate levels of gene repair. After reactivating nicking ability, DarT-ScnCas9 was found to be cytotoxic, whereas DarT_K56A-ScnCas9 showed no cytotoxicity and successfully edited almost 80% of cells after 8 hours of induction. This nicking version of GE2 was designated GE2n.

[0127] Experiments were also performed to investigate the use of DarT's antitoxin partner, DarG, to determine whether this would eliminate the genome modification ability of GE2. The N-terminal domain of DarG contains a glycohydrolase that can directly repair ADPr-modified thymines. The C-terminal domain of DarG contains a DarT inhibitor. GE2 and GE2n were each co-expressed with full-length DarG, the C-terminal domain of DarG, or the N-terminal domain of DarG in an operon in a lacZ gene repair assay (Figure 3). As shown in Figure 3, the genome modification ability of GE2 and GE2n was attenuated when both the N- and C-terminal domains of DarG were expressed. This provides a means to reduce potential off-target modification effects and toxicity without compromising on-target modification.

[0128] Moreover, as will be appreciated by those skilled in the art based on the present disclosure, either the N-terminal or C-terminal domain of DarG can be used to counteract DarT activity. The N-terminal domain can remove ADP-ribose and restore nucleotides to their original state. The C-terminal domain can directly inhibit DarT activity. Thus, a single domain of DarG can be expressed at low levels and, in some cases, randomly distributed throughout the cell to help counteract the off-target effects of DarT-Cas protein. In some embodiments, a single DarT domain can be used to reduce off-target effects without affecting on-target genome modification activity.

[0129] [Example 3] Experiments were performed to test the ability of a gap editing complex containing a Scabin DNA modification domain combined with a Cas9 DNA recognition domain (Scabin-K130A-ScdCas9) to induce successful genome modification measured based on the frequency of kanamycin gene repair in E. coli. In this exemplary set of experiments, expression of a Scabin-dCas9 fusion protein increased the frequency of kanamycin gene repair, which depends on Scabin's DNA modification catalytic activity. Scabin is known to modify guanines in single-stranded and double-stranded DNA with adenosine diphosphate ribose groups, which is structurally and evolutionarily divergent from DarT outside of a single shared catalytic motif. Recombination between a plasmid repair template and a targeted defective kanamycin gene in the E. coli genome results in repair of the targeted gene and consequent kanamycin resistance. Thus, the rate of kanamycin resistance serves as a readout for the rate of genome modification. The K130A mutation in Scabin attenuates the activity of Scabin, which is otherwise toxic to cells. The E160A mutation catalytically inactivates Scabin and eliminates all DNA modification activity (negative control). As shown in Figure 4, the Scabin-K130A-ScdCas9 gap editor complex led to successful genome modification with an increased frequency of kanamycin gene repair.

[0130] In another set of exemplary experiments, the ability of a gap editing complex containing the Mom DNA modification domain combined with the Cas9 DNA recognition domain (Mom-D149A-ScdCas9) to induce successful genome modification measured based on the frequency of kanamycin gene repair in E. coli was also examined. Fusion of Mom to dCas9 and targeting of the defective kanamycin gene resulted in recombination, genome modification, and thus kanamycin-resistant cells. The Mom protein is known to modify adenines with methylcarbamoyl groups, which are known to block DNA replication and induce gap repair recombination. The D149A mutation in Mom attenuates catalytic activity, which is otherwise lethal to cells. As shown in Figure 5, the MOM-D149A-ScdCas9 gap editor complex resulted in successful genome modification with an increased frequency of kanamycin gene repair.

[0131] [Example 4] Experiments were also carried out to evaluate the DNA modification domain in the gap editing complex of the present disclosure.First, Figure 6 includes representative results of experiments that demonstrate that the success of genome modification using gap editing factor complex (e.g., through increased frequency of kanamycin gene repair) is dependent on the DNA modification domain (DarT) (DarT-G49D-ScdCas9) combined with Cas9 DNA recognition domain (ScdCas9 alone did not result in kanamycin gene repair).DarT was used as an exemplary DNA modification domain in these experiments.

[0132] Moreover, experiments were performed to investigate whether DarT could be improved by reducing its toxic effects in cells. As shown in FIG. 7, the introduction of the R193A mutation into DarT (DarT-G49D-R193A-ScdCas9) significantly reduced the toxicity of DarT when expression was induced by the addition of arabinose to the culture medium. As shown in FIG. 8, the M86L and R92A mutations further reduced the toxicity of DarT and also reduced CRISPR-independent off-target modification over and above the R193A mutation (FIG. 7). Furthermore, FIG. 9 shows the successful genome modification using a gap editor complex containing a DarT DNA modification domain with mutations that significantly reduced toxicity (G49D and / or R193A) combined with a Cas9 DNA recognition domain with nickase activity (ScnCas9). Site-specific genome modification was nearly 100% effective.

[0133] Thus, these results demonstrate the novel CRISPR-based genome modification technology disclosed herein, which facilitates efficient site-specific genome modification while minimizing unintended modifications and cellular toxicity associated with current genome editing approaches.

[0134] [Example 5] As shown in FIG. 10, experiments were performed to evaluate the efficacy of genome modification in eukaryotic cells using the gap editor complex of the present disclosure by assessing whether genetic knockout of fcy1 can confer resistance to 5-fluorocytosine (5-FC). The fcy1 gene was targeted in Saccharomyces cerevisiae with Cas9 nickase (ScnCas9) or a fusion of Cas9 nickase with an engineered DarT gene, and a repair template was provided. As shown, this resulted in successful genome modification in fcy1. The repair templates encoded six mutations that introduced two or three stop codons in fcy1, which resulted in loss of fcy1 function after genome modification and resistance to 5-FC. Moreover, as shown, one single guide RNA is combined with five different repair templates. For all mutations, the fusion of DarT resulted in a >10-fold increase in the rate of genome modification, demonstrating the utility of introducing a replication blocker moiety in eukaryotic cells.

[0135] As shown in FIG. 11, experiments were performed to evaluate the efficacy of genome modification using the gap editor complex of the present disclosure by assessing whether genetic knockout of fcy1 could confer resistance to 5-fluorocytosine (5-FC). The fcy1 gene was targeted in Saccharomyces cerevisiae with Cas9 nickase (ScnCas9) or a fusion of an engineered DarT gene with Cas9 nickase and a repair template was provided. As shown, this resulted in successful genome modification in fcy1. The repair template encoded six mutations that introduced two or three stop codons in fcy1, which resulted in loss of fcy1 function after genome modification and resistance to 5-FC. The use of an engineered DarT variant containing G49D, R193A, M86L and R92A mutations improved cell viability by up to approximately 50-fold over DarT with G49D and R193A mutations alone. This gap editor complex achieves efficient and low-toxicity genome modification using two separate single-guide RNAs targeting fcy1 and a repair template in yeast.

[0136] Figure 12 includes representative chromatographs providing confirmation of fcy1 genome modification and gene knockout by Sanger sequencing. Two or three stop codons were introduced by targeting gap editing factor complexes to the fcy1 gene and providing a DNA repair template. Edited nucleotides are highlighted in red. Genome editing of two separate targets within fcy1 is shown.

[0137] [Example 6] As shown in FIG. 13, an experiment was performed to evaluate the efficacy of genome modification using the gap editor complex of the present disclosure by evaluating gene knockout of lacZ. Gene knockout of lacZ results in a white colony color in the presence of lactose analog IPTG and colorimetric indicator X-gal. The lacZ gene was targeted in E. coli by a nuclease-inactive Cas12a protein (dLbCas12a) fused to an engineered DarT gene and a repair template was provided. As shown, this resulted in genome modification in lacZ. The repair template encoded lacZ DNA with a stop codon, which resulted in loss of lacZ function and a white colony color after genome modification. No genome modification was observed without targeting the gap editor complex to the lacZ gene.

[0138] Figure 14 includes representative chromatographs demonstrating successful introduction of one or more stop codons into the lacZ gene using DarT(G49D / R193A)-dLbCas12a associated with different crRNAs. The lacZ genes from white colonies were amplified and sent for Sanger sequencing. Mutations that introduce one or more stop codons into the lacZ gene and eliminate beta-galactosidase expression, resulting in white colonies when plated in the presence of inducer IPTG and colorimetric indicator X-gal, are highlighted in red.

[0139] [Example 7] As shown in Figure 15, an experiment was performed to evaluate the efficacy of genome modification using the gap editor complex of the present disclosure by evaluating whether the introduction of the D516G mutation into the rpoB gene can confer resistance to the antibiotic rifampicin. The rpoB gene was targeted in E. coli by an engineered DarT variant fused to Cas9 nickase (ScnCas9), and an RNA repair template and reverse transcriptase were co-expressed. This resulted in successful site-specific RNA-templated genome modification. A recT-type recombinase was co-expressed to accelerate strand annealing. The RNA repair template encoded the D516G mutation and was successfully integrated into the genome after targeting by the gap editor complex.

[0140] As shown in FIG. 16, experiments were performed to evaluate the efficacy of genome modification using the gap editor complex of the present disclosure by assessing whether introduction of the D516G mutation into the rpoB gene could confer resistance to the antibiotic rifampicin. The rpoB gene was targeted in E. coli by an engineered DarT variant fused to Cas9 nickase (ScnCas9) to provide a linear single-stranded DNA repair template. As shown, this resulted in successful genome modification in rpoB. A recT-type recombinase was co-expressed to accelerate annealing of the single-stranded DNA repair template. The repair template encoded the D516G mutation that confers rifampicin resistance. Two guides and repair templates were tested, targeting opposite DNA strands at the rpoB D516 genomic locus. Targeting of the gap editor complex to rpoB resulted in a 100-6,000-fold increase in genome modification rate, demonstrating the efficacy of the gap editor.

[0141] Figure 17 includes representative chromatograms of RNA-templated mutations in the rpoB gene introduced by targeting a gap editor complex to the rpoB gene, expression of an RNA repair template, and expression of reverse transcriptase Ec86. The mutations include the AC>GT mutation required for D516G-mediated rifampicin resistance.

[0142] Sequences. The sequences of exemplary gap editors described herein are provided below. SPC1879 darT G49D-ScdCas9 pBAD araC CmR p15a:

[0143] [ka] TIFF2024506375000012.tif102166

[0144] SPC1881 GE2 darT G49D-K56A-ScdCas9 pBAD araC CmR p15a:

[0145] [ka] TIFF2024506375000014.tif74166

[0146] SPC1883 darT G49D-ScnCas9 pBAD araC CmR p15a:

[0147] [ka] TIFF2024506375000016.tif47166

[0148] SPC1884 GE2n darT G49D-K56A-ScnCas9 pBAD araC CmR p15a:

[0149] [ka] TIFF2024506375000018.tif22166

[0150] DarG:

[0151] [ka]

[0152] DarG_C-terminus:

[0153]

change

[0154] DarG N-terminus:

[0155]

change

[0156] Mom:

[0157]

change

[0158] Mom_D149A:

[0159]

change

[0160] Mom_D149A-ScdCas9:

[0161]

change

[0162] Scabin:

[0163]

change

[0164] Scabin_K130A:

[0165]

change

[0166] Scabin_K130A-ScdCas9:

[0167]

Chem.

[0168] DarT_G49D_R193A:

[0169]

Chem.

[0170] DarT_G49D_R193A-ScdCas9:

[0171]

Chem.

[0172] DarT_G49D_R193A_M86L_R92A:

[0173]

Chem.

[0174] DarT_G49D_R193A_M86L_R92A-ScdCas9

[0175]

Chem.

[0176] DarT catalytic domain motif: X1X2X3X3R (SEQ ID NO: 18) (wherein X1 is L, I, V or A; X2 is I, Q, K, T or N; and X3 is any amino acid) (Figure 18).

[0177] DarT catalytic domain motif: X1X1X1X1X2X3X4X5X6PFYFX7X1X1X8X9MX 10 X1 (SEQ ID NO: 19), where X1 is any amino acid; X2 is L, V, or I; X3 is H, G, N, S, or A; X4 is D or E; X5 is Y or F; X6 is V, I, or A; X7 is T, A, G, K, N, or W; X8 is S, T, N, M, or K; X9 is P, V, M, I, A; 10 is L, M or F) (Figure 19).

[0178] DarT catalytic domain motif: X1X2X3X4X5X6X7X8 (SEQ ID NO: 20) (wherein X1 is F, Y, W, V or C; X2 is V, L, I, A, C or F; X3 is F, Y or A; X4 is T, S, Y or F; X5 is D, N or S; X6 is G, R, S, A, M or Q; X7 is H, N, S or Q; and X8 is A, G, C, H or K) (Figure 20).

[0179] DarT catalytic domain motif: X1X2X3X4X5X6X7X8X9 (SEQ ID NO: 21), where X1 is any amino acid; X2 is R, K, H, E, F, L, T or M; X3 is Y, R, K, D, E or H; X4 is Q, M, E, Y, A, R or H; X5 is A, Q, S or Y; X6 is E, A or Q; X7 is F, A, L, E, V or C; X8 is L, A, E or M; and X9 is V, I, L or A (Figure 21).

[0180] Scabin catalytic domain motif: X1X1X1X1X2X1EX3X4X5X6GGX7 (SEQ ID NO: 22), where X1 is any amino acid; X2 is Q, E or R; X3 is V or I; X4 is A, L, V, S or T; X5 is F, I, V or L; X6 is P, A or I; and X7 is I, V or L (Figure 22). The DarT catalytic motif of SEQ ID NO: 21 and the Scabin catalytic motif of SEQ ID NO: 22 are structural and functional analogs, with the conserved glutamic acid (E) being the catalytic residue.

[0181] Scabin catalytic domain motif: X1X2X3X4X5X6X7 (SEQ ID NO: 23) (wherein X1 is S, T or G; X2 is any amino acid; X3 is F, Y or L; X4 is V, I, A or L; X5 is S, G or A; X6 is T or A; and X7 is T, S or A) (Figure 23).

[0182] Scabin catalytic domain motif: X1X2X3X2X4X2X5 (SEQ ID NO: 24) (wherein X1 is L or V; X2 is any amino acid; X3 is R, H or K; X4 is D, S or A; and X5 is R or D) (Figure 24).

[0183] Mom catalytic domain motif: X1HYX2X3 (SEQ ID NO: 25) (wherein X1 is any amino acid; X2 is S or L; X3 is H, G, K, R, N, D or A) (Figure 25).

[0184] Mom catalytic domain motif: EX1X2X3X4X5X6X7X8X7X9X 10 X 11 X 12 X 13 EX 14(SEQ ID NO:26) (wherein X1 is L, I, or F; X2 is N, G, S, or T; X3 is R or K; X4 is M, L, or A; X5 is W, A, C, V, F, or Y; X6 is L, I, F, M, V, C, or T; X7 is any amino acid; X8 is D or E; X9 is L, A, M, C, V, Q, or T; X 10 is P, G, A or L; X 11 is R, K, H, T or M; X 12 is N or F; X 13 is S, A, T or G; X 14 is S or T) (Figure 26).

[0185] Mom catalytic domain motif: X1X2DX3X4X4X5X4X4GX6X7YX8AX9X 10 X 11 (SEQ ID NO:27) (wherein X1 is F, W, Y or M; X2 is A or S; X3 is E, G, P, A or T; X4 is any amino acid; X5 is G, C or Q; X6 is T, V, Y or I; X7 is V or I; X8 is Q, K or R; X9 is A, S, C, T or N; X 10 is N, G or A; X 11 is F, W or Y) (Figure 27).

[0186] It is understood that the foregoing detailed description and accompanying examples are for illustrative purposes only and are not to be taken as limitations on the scope of the present disclosure, which is defined solely by the appended claims and equivalents thereof.

[0187] All publications and patents mentioned in the above specification are incorporated herein by reference as if expressly set forth herein. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and can be made without departing from the spirit and scope thereof.

Claims

1. 1. A composition for targeted genome modification, comprising a gap editor complex comprising a DNA recognition domain and a DNA-modifying domain, wherein the DNA recognition domain binds to a DNA target sequence in a genome, and the DNA-modifying domain induces the formation of a replication blocking portion at at least one nucleotide in the genome.

2. The composition of claim 1 , further comprising a donor nucleic acid template.

3. 3. The composition of claim 1 or claim 2, comprising at least one guide RNA molecule.

4. The composition of any one of claims 1 to 3, wherein the DNA recognition domain comprises at least one Cas protein or a fragment thereof that lacks deoxyribonuclease activity.

5. The composition of any one of claims 1 to 4, wherein the DNA recognition domain comprises a Cas protein or a fragment thereof having nickase activity.

6. The composition according to any one of claims 1 to 5, wherein the DNA recognition domain and the DNA modification domain are functionally coupled.

7. The DNA modifying domain is (i) at least one nucleotide in a DNA strand that is complementary to a DNA target sequence; (ii) at least one nucleotide in the DNA strand containing the DNA target sequence; or (iii) both at least one nucleotide in the DNA strand that is complementary to the DNA target sequence and at least one nucleotide in the DNA strand that contains the DNA target sequence; The composition of any one of claims 1 to 6, which blocks DNA replication by adding a replication blocking moiety to

8. 8. The composition of any one of claims 1 to 7, wherein the DNA recognition domain induces a single-stranded break in the DNA target strand and the DNA modifying domain adds a replication blocking moiety to at least one nucleotide in the DNA strand complementary to the DNA target sequence.

9. The composition of any one of claims 1 to 8, wherein the DNA-modifying domain comprises a DarT enzyme or a functional fragment, derivative or variant thereof.

10. The composition of claim 9, wherein the DNA-modifying domain comprises a catalytic domain having at least 70% amino acid sequence identity to any of SEQ ID NOs: 18-21.

11. 11. The composition of claim 9 or claim 10, wherein the DarT enzyme comprises one or more of the following amino acid substitutions: G49D, K56A, M86L, R92A and / or R193A.

12. The composition of any one of claims 1 to 8, wherein the DNA-modifying domain comprises a Scabin enzyme or a functional fragment, derivative or variant thereof.

13. The composition of claim 12, wherein the DNA-modifying domain comprises a catalytic domain having at least 70% amino acid sequence identity to any of SEQ ID NOs: 22-24.

14. 14. The composition of claim 12 or claim 13, wherein the Scabin enzyme comprises the amino acid substitution K130A.

15. The composition of any one of claims 1 to 8, wherein the DNA-modifying domain catalyzes methylcarbamoylation of an adenine nucleotide.

16. The composition of claim 15 , wherein the DNA-modifying domain comprises a Mom enzyme or a functional fragment, derivative or variant thereof.

17. The composition of claim 15 or 16, wherein the DNA-modifying domain comprises a catalytic domain having at least 70% amino acid sequence identity with SEQ ID NOs: 25-27.

18. 18. The composition of claim 16 or claim 17, wherein the Mom enzyme comprises an amino acid substitution that is D149A.

19. A kit for targeted genome modification, comprising: A kit comprising a gap editor complex comprising a DNA recognition domain and a DNA modifying domain, wherein the DNA recognition domain binds to a DNA target sequence in a genome and the DNA modifying domain induces the formation of a replication blocking portion at least one nucleotide in the genome.

20. 1. A method for targeted genome modification, comprising: Introducing any of the compositions according to claims 1 to 18 into a cell; and Evaluating the cells for the presence of the desired genomic alteration The method includes:

21. 21. The method of claim 20, wherein the gap editor complex and / or the at least one guide RNA molecule are introduced into the cell as a polypeptide, an mRNA and / or a DNA expression construct.

22. 22. The method of claim 20 or 21, wherein the gap editor complex and / or the guide RNA are introduced into the cell as part of a gene drive system.

23. 21. The method of claim 20, wherein the cell is a prokaryotic or eukaryotic cell.

24. 21. The method of claim 20, wherein the cell is a mammalian cell.

25. 21. The method of claim 20, wherein the cell is a plant cell.

26. 26. The method of any one of claims 22 to 25, which results in a reduced degree of indel formation, chromosomal rearrangements and / or DNA duplications.

27. The method of any one of claims 22 to 26, wherein cell viability is enhanced and / or cell toxicity is reduced.

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