Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/102—Mutagenizing nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1077—Pentosyltransferases (2.4.2)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

• the present disclosure provides compositions, methods, and systems related to template-mediated genome modification.
• the present disclosure provides novel genome modification technology involving site-specific chemical modification of a nucleotide to introduce a replication-blocking lesion.
• the compositions, methods, and systems described herein facilitate efficient site-specific genome modification of a DNA target, while minimizing the unintended edits and cellular toxicity associated with current genome editing approaches.
• Embodiments of the present disclosure include a composition for targeted genome modification.
• the composition includes a gap editor complex comprising a DNA-recognition domain and a DNA-modifying domain, wherein the DNA-recognition domain binds a DNA target sequence in the genome, and wherein the DNA- modifying domain induces formation of a replication blocking moiety on at least one nucleotide in the genome.
• the composition further comprises a donor nucleic acid template.
• the donor nucleic acid template comprises a polynucleotide from an endogenous homologous sequence corresponding to the DNA target sequence.
• the donor nucleic acid template comprise an exogenous single-stranded DNA (ssDNA) molecule or double-stranded DNA (dsDNA) molecule.
• the donor nucleic acid template is an RNA molecule.
• the presence of the donor nucleic acid template facilitates homology-directed gap repair and/or recombination, wherein the donor nucleic acid template or a fragment thereof is recombined into the genome of the DNA target sequence.
• the DNA-modifying domain comprises a Scabin enzyme or a functional fragment, derivative, or variant thereof.
• the DNA- modifying domain comprises a catalytic domain having at least 70% amino acid sequence identity with any of SEQ ID NOs: 22-24.
• the Scabin enzyme comprises an amino acid substitution that is K130A.
• the DNA-modifying domain catalyzes methylcarbamoylation of an adenine nucleotide.
• the DNA-modifying domain comprises a Mom enzyme or a functional fragment, derivative, or variant thereof.
• the DNA-modifying domain comprises a catalytic domain having at least 70% amino acid sequence identity with SEQ ID NO: 25-27.
• the Mom enzyme comprises an amino acid substitution that is D149A.
• the DNA-modifying enzyme domain comprises an enzyme or functional fragment, derivative, or variant thereof, selected from the group consisting of: Pierisin, Scabin, Cell cycle and apoptosis regulator 1 (CARP-1), SC05461 protein (ScARP), adenine modification enzyme, acetyltransferase, amino acid transferase, nucleotidyl transferase, uridyltransferase, acyltransferase, ADP-ribsoyltransferase, methylthiotransferase, N-acetyl transferase 10, tRNA(Met) cytidine acetyltransferase (TmcA), tRNA cytidine acetyltransferase, GCN5-related N-acetyltransferase, lysidine synthase, m 7 G methyltransferase, N6 carbamoyl
• the at least one gap editor accessory factor comprises Rap, DarG, Orf, Exol, Exonuclease III, Prim Pol , RecJ, RecQl, Rad51, Rad52, CtIP, Radi 8, and any combinations thereof.
• Embodiments of the present disclosure also includes a kit for targeted genome modification.
• the kit includes a gap editor complex comprising a DNA-recognition domain and a DNA-modifying domain, wherein the DNA- recognition domain binds a DNA target sequence in the genome, and wherein the DNA- modifying domain induces formation of a replication blocking moiety on at least one nucleotide in the genome.
• the kit further comprises a donor nucleic acid template. In some embodiments, the presence of the donor nucleic acid template facilitates homology- directed gap repair and/or recombination.
• the DNA-modifying domain catalyzes addition of ADP ribose to a thymine or guanine nucleotide.
• the DNA-modifying domain comprises a DarT enzyme or a functional fragment, derivative, or variant thereof.
• the DNA-modifying domain comprises a Scabin enzyme or a functional fragment, derivative, or variant thereof.
• the DarT enzyme has been engineered to have reduced DNA binding, increased specificity to single-stranded DNA, and/or decreased enzymatic activity.
• the DNA-modifying domain catalyzes addition a replication blocking moiety selected from the group consisting of: glucose, threonyl carbamoyl adenosine, acetate, glyceryl, L-ascorbic acid, uridine, adenosine mono-phosphate, 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-methione, and dimethylallyl diphosphate or a subgroup thereof.
• a replication blocking moiety selected from the group consisting of: glucose, threonyl carbamoyl adenosine, acetate, glyceryl, L-ascorbic acid, uridine, adenosine mono-phosphat
• the at least one guide RNA comprises gRNA, sgRNA, crRNA, or any combinations thereof.
• the at least one guide RNA comprises a handle sequence and a targeting sequence.
• the targeting sequence in the at least one guide RNA is complementary to the DNA target sequence.
• the kit further comprises at least one gap editor accessory factor.
• Embodiments of the present disclosure also include a method for targeted genome modification. In accordance with these embodiments, the method includes introducing any of the compositions of the present disclosure into a cell, and assessing the cell for presence of a desired genome alteration.
• the Mom enzyme has been engineered to have reduced DNA binding, increased specificity to single-stranded DNA, and/or decreased enzymatic activity.
• Mom homologs and any fragments, derivatives, or variants thereof) that can be used in the various embodiments disclosed herein include, but are not limited to, those provided in Table 1 below.
• the DNA-modifying domain/enzyme can include, but is 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), SC05461 protein (ScARP), adenine modification enzyme, acetyltransferase, amino acid transferase, nucleotidyl transferase, uridyltransferase, acyltransferase, ADP-ribsoyltransferase, methylthiotransferase, N-acetyl transferase 10, tRNA(Met) cytidine acetyltransferase (TmcA), tRNA cytidine acetyltransferase, GCN5-related N-acetyltransferase, lysidine synthase, m 7 G methyltrans
• the DNA-modifying domain includes a catalytic domain having at least 92% amino acid sequence identity with SEQ ID NO: 21. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 93% amino acid sequence identity with SEQ ID NO: 21. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 94% amino acid sequence identity with SEQ ID NO: 21. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 95% amino acid sequence identity with SEQ ID NO: 21. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 96% amino acid sequence identity with SEQ ID NO: 21.
• the DNA-modifying domain includes a catalytic domain having at least 92% amino acid sequence identity with SEQ ID NO: 23. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 93% amino acid sequence identity with SEQ ID NO: 23. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 94% amino acid sequence identity with SEQ ID NO: 23. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 95% amino acid sequence identity with SEQ ID NO: 23. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 96% amino acid sequence identity with SEQ ID NO: 23.
• the DNA-modifying domain includes a catalytic domain having at least 92% amino acid sequence identity with SEQ ID NO: 24. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 93% amino acid sequence identity with SEQ ID NO: 24. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 94% amino acid sequence identity with SEQ ID NO: 24. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 95% amino acid sequence identity with SEQ ID NO: 24. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 96% amino acid sequence identity with SEQ ID NO: 24.
• the DNA-modifying domain includes a catalytic domain having at least 90% amino acid sequence identity with SEQ ID NO: 25. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 91% amino acid sequence identity with SEQ ID NO: 25. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 92% amino acid sequence identity with SEQ ID NO: 25. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 93% amino acid sequence identity with SEQ ID NO: 25. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 94% amino acid sequence identity with SEQ ID NO: 25.
• the DNA-modifying domain includes a catalytic domain having at least 92% amino acid sequence identity with SEQ ID NO: 26. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 93% amino acid sequence identity with SEQ ID NO: 26. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 94% amino acid sequence identity with SEQ ID NO: 26. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 95% amino acid sequence identity with SEQ ID NO: 26. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 96% amino acid sequence identity with SEQ ID NO: 26.
• the DNA-modifying domain includes a catalytic domain having at least 75% amino acid sequence identity with SEQ ID NO: 27. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 80% amino acid sequence identity with SEQ ID NO: 27. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 85% amino acid sequence identity with SEQ ID NO: 27. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 90% amino acid sequence identity with SEQ ID NO: 27. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 91% amino acid sequence identity with SEQ ID NO: 27.
• the DNA- modifying domain includes a catalytic domain having at least 97% amino acid sequence identity with SEQ ID NO: 27. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 98% amino acid sequence identity with SEQ ID NO: 27. In some embodiments, the DNA-modifying domain includes a catalytic domain having at least 99% amino acid sequence identity with SEQ ID NO: 27. [0118] Replication Blocking Moieties.
• gRNAs can be further expressed using CRISPR arrays that naturally encode the crRNA utilized by the nucleases.
• the gRNAs can also be expressed separately by being operably linked to a promoter and terminator.
• the gRNAs can also be fused in a single transcript by including intervening RNA cleavages sites, such as ribozymes or sites recognized by RNA-cleaving enzymes such as RNase P, RNase Z, RNase III, or Csy4.
• the gRNAs or sgRNAs may include RNA templates for reverse transcription into cDNA repair templates.
• the sgRNAs may include aptamer sequences, for example, RNA-binding protein recognition sites so as to recruit accessory genome editing factors to the gap editor complex or gap editor target site.
• genome modifications using the gap editors of the present disclosure can generate specific nucleotide modifications ranging from a single nucleotide change to large insertions or deletions.
• the gap editor complexes of the present disclosure can be used to add, exchange, and/or remove large sequences of DNA through the use of more than one guide RNA sequence to target distinct sites in the genome.
• large genomic deletions can be generated by removing the sequence between two gRNA target sites and/or inserting an exogenous DNA sequence (e.g., by virtue of the endogenous repair/recombination mechanisms in a cell or organism).
• multiple gRNAs can be used to target multiple sites in a genome to generate any number of desired modifications in a genome (e.g., multiplexing).
• guide RNA molecules are not required in the gap editor complexes of the present disclosure.
• certain embodiments of the compositions and methods described herein do not require guide RNAs to effectuate efficient genome editing and modification.
• these gap editor complexes include, but are not limited to, meganucleases, zinc-fingers (ZFs), and transcription activator-like effectors (TALEs).
• Donor Template In some embodiments, the presence of a donor nucleic acid template facilitates homology-directed gap recombination and/or repair, which includes the donor nucleic acid template or a fragment thereof being recombined into the double- stranded target DNA molecule.
• the donor DNA template can serve 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.
• the donor template is a cDNA that has reversed transcribed from an endogenous, expressed, synthetic, or delivered RNA.
• the donor nucleic acid may be delivered into a cell as plasmid or linear DNA.
• a donor nucleic acid may also be generated in vivo from a template ribonucleic acid by a reverse transcriptase.
• the donor nucleic acid may itself be a ribonucleic acid.
• the donor nucleic acid can also contain chemical modifications.
• the donor nucleic acid may include chemical modifications or sequences specifically recruited to the gap editor complex, or gap editor target site.
• the donor nucleic acid template comprises a polynucleotide from an endogenous homologous sequence corresponding to the DNA target sequence. In some embodiments, the donor nucleic acid template comprises a polynucleotide from an endogenous allele (e.g., to facilitate loss of heterozygosity). In some embodiments, the donor nucleic acid template comprise an exogenous single-stranded DNA (ssDNA) molecule or double-stranded DNA (dsDNA) molecule.
• ssDNA single-stranded DNA
• dsDNA double-stranded DNA
• compositions and systems of the present disclosure further comprise a one gap editor accessory factor.
• the composition further comprises at least one gap editor accessory factor.
• the at least one gap editor accessory factor comprises a protein that augments at least one step in a genome modification process.
• the present disclosure can include gap editor complexes in which the DNA-modifying domain comprises DarT.
• the DNA-modifying domain comprises DarT.
• DarG, TARG1, or another glycohydolase domain can be included as a gap editor accessory factor by modulating off-target editing (e.g., attenuating DarT activity) or removing the added ADPr after HDGR occurs.
• the delivery system can include mechanical and/or electrical devices and methods for delivering the gap editors and gap editor complexes of the present disclosure as polynucleotides and/or as polypeptides/proteins (or any combinations thereof).
• gap editors and gap editor complexes are delivered using a gene gun (e.g., bombardment and Agrobacterium transformation as used for plant cells), and electroporation- based methods, as well as any other physical methods (e.g., mechanical, electrical, thermal, optical, chemical stimulation, and the like) that use membrane disruption as a means for delivering polynucleotides and polypeptides/proteins (see, e.g., Sun et al., Recent advances in micro/nanoscale intracellular delivery, Nanotechnology and Precision Engineering s, 18 (2020)).
• kits and systems for targeted modification of a nucleic acid also include kits and systems for targeted modification of a nucleic acid.
• the kit includes a gap editor complex comprising a DNA-recognition domain and a DNA-modifying domain.
• the kit also includes at least one guide RNA molecule.
• the DNA-recognition domain binds a DNA target sequence in the genome, and the DNA- modifying domain induces formation of a replication blocking moiety on at least one nucleotide in the genome.
• the kits and systems can also include one or more of the other components of the gene modification compositions described herein (e.g., gap editor accessory factors).
• the composition further comprises a donor nucleic acid template.
• the presence of the donor nucleic acid template facilitates homology-directed gap repair and/or recombination.
• the DNA-recognition domain comprises at least one Cas protein or fragment thereof lacking deoxyribonuclease activity. In some embodiments of the kit, the DNA-recognition domain comprises at least one Cas protein or fragment thereof having 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 Cas 12, a Cas9-related IscB, a Cas9-related TnpB, and combinations thereof.
• the DNA-modifying domain catalyzes addition of a replication blocking moiety selected from the group consisting of: glucose, threonyl carbamoyl adenosine, acetate, glyceryl, L-ascorbic acid, uridine, adenosine mono-phosphate, a lipid, an amino acid, agmatine, L-threonylcarbamoyladenylate, L-threonylcarbamoyl, methyl thiolate, sulfur, a methyl group, S-adenosyl-L-methione or a subgroup of S-adenosyl-L- methione, and dimethylallyl diphosphate or a subgroup thereof.
• a replication blocking moiety selected from the group consisting of: glucose, threonyl carbamoyl adenosine, acetate, glyceryl, L-ascorbic acid, uridine, adenosine mono
• compositions, systems, and methods of the present disclosure include one or more components that enhance or improve one or more aspects of gene modification.
• improving or enhancing one or more aspects of genome modification includes the use of a gap editor accessory factor(s), as described above.
• methods that enhance or improve one or more aspects of genome modification include reducing or attenuating nuclease activity in a cell in which genome modification is desired. Reducing nuclease activity in a cell can lead to enhanced or improved modification frequency and/or efficiency.
• suitable prokaryotic cells include, but are not limited to, cells from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Clostridia (such as Clostridium difficile or Clostridium autoethanogenum), Escherichia (such as E. coli ), Lactobacilli, Klebsiella, Myxobacteria, Pseudomonas, Streptomyces, Salmonella, Vibrio (such as Vibrio cholerae or Vibrio nutrifaciens ) and Envinia.
• Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells.
• a bacterial cell e.g., a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like, a fungal cell (e.g., a yeast cell), an animal cell, a cell from an invertebrate animal (e.g.
• the cell can also be a cell that is used for therapeutic purposes.
• the cell can be a mammalian cell, and in some embodiments, the cell is a human cell.
• suitable mammalian and human cells are known in the art, and many 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.
• CHO Chinese hamster ovary cells
• CHO DHFR-cells Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)
• HEK human embryonic kidney
• Measurement of gap editing in E. coli by a colorimetric assay was performed by cotransforming the DNA modifying domain fused to a DNA binding domain such as Cas9 (e.g. DarT-ScdCas9) and an sgRNA and nucleic acid donor into E. coli by electroporation and plated on LB agar plus the appropriate antibiotic(s). The resulting colonies were picked and inoculated into 750 mL of liquid LB media in a deep well plate shaking at 900 rpm and 37°C for 12 to 16 hours overnight.
• Cas9 e.g. DarT-ScdCas9
• sgRNA and nucleic acid donor e.g.
• the resulting colonies were picked and inoculated into 750 mL of liquid LB media in a deep well plate shaking at 900 rpm and 37°C for 12 to 16 hours overnight.
• One plate contained antibiotics to selected only for the gap editor, sgRNA, and repair template (typically chloramphenicol and ampicillin) and the other plate also included either rifampicin or kanamycin to select for edited cells. The next day colonies were counted. Genome editing efficiency was tabulated as being the number of colonies on the plates with rifampicin or kanamycin divided by the number of colonies on plates without rifampicin or kanamycin.
• cultures were back-diluted 1:500 into LB Chloramphenicol with glucose to maintain gap editor repression, or arabinose to induce expression of the gap editor. Cultures were incubated shaking at 900 rpm in a deep well plate at 37°C for 5 hours. Cultures were then spot plated on LB Chloramphenicol. The next day, colonies were counted to assess the final cell density, and therefore the rate of off-target DNA modification.
• Measurement of ssDNA-templated gap editing in E. coli by rifampicin resistance was performed by first co-transforming the strand annealing beta recombinase plasmid and a DNA modifying domain fused to a DNA binding domain such as Cas9. The resulting clones were inoculated into LB, antibiotics, and anhydrotetracycline for induction of beta recombinase expression. These cultures were prepared for electroporation and transformed with the sgRNA plasmid, and cultured for 3 hours in a rich media at 37 °C and shaking at 250 RPM prior to spot plating on two separate LB agar plates.
• One plate contained antibiotics to selected only for the gap editor, sgRNA, and recombinase.
• the other plate additionally included rifampicin to select for edited cells. The next day colonies were counted. Genome editing efficiency was tabulated as being the number of colonies on the plates with rifampicin divided by the number of colonies on plates without rifampicin.
• Table 2 Strain information corresponding to gap editors and gap editor complexes used in the present disclosure. 5. Examples
• a plasmid encoding an arabinose inducible DarT-ScdCas9 was co-transformed with a plasmid containing a 1.5 kb repair template encoding mutations to block ScdCas9 re-targeting while repairing the lacZ stop codon. After culturing these colonies overnight, the cells were back-diluted into inducing medium, cultured for 8 hours, and then plated onto selective media with the b-galactosidase (lacZ gene product) indicator dye X-gal with the inducer IPTG. [0147] When targeting only one site, the lacZ gene was efficiently repaired, as demonstrated by the results of in FIG. 1.
• either the N-terminal or C-terminal domains of DarG can be used to counteract DarT activity.
• the N-terminal domain can remove ADP ribose, reverting the nucleotide to its original state.
• the C-terminal domain can directly inhibit DarT activity.
• single domains of DarG can be expressed at a low level, and in some cases, randomly distributed through the cell, to help counteract off- target effects of the DarT-Cas protein.
• a single DarT domain can be used to reduce off-target effects without affecting on-target genome modification activity.
• Scabin is known to modify guanine within single and double-stranded DNA with an adenosine diphosphate ribose group, but it is structurally and evolutionarily divergent from DarT outside of a single shared catalytic motif. Recombination between the plasmid repair template and the targeted defective kanamycin gene in the E. coli genome results in repair of the targeted gene, and consequently, kanamycin resistance. Therefore, the fraction of kanamycin resistance serves as a readout for the rate of genome modification.
• the K130A mutation in Scabin attenuated Scabin’s activity, which is otherwise toxic to the cells.
• the E160A mutation catalytically inactivates Scabin, removing all DNA modification activity (negative control). As shown in FIG. 4, the Scabin-K130A- ScdCas9 gap editor complex resulted in successful genome modification through increased frequency of kanamycin gene repair.
• a gap editing complex comprising a Mom DNA-modifying domain in combination with a 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 tested. Fusion of the Mom to dCas9 and targeting a defective kanamycin gene resulted in recombination, genome modification, and thereby kanamycin resistant cells.
• the Mom protein is known to modify adenine with a methylcarbamoyl group, which is known to block DNA replication, triggering gap repair recombination.
• the D149A mutation in Mom attenuated the catalytic activity, which is otherwise lethal to the cells.
• the MOM-D149A-ScdCas9 gap editor complex resulted in successful genome modification through increased frequency of kanamycin gene repair.
• FIG. 6 includes representative results of experiments demonstrating that successful genome modification (e.g., though increased frequency of kanamycin gene repair) using gap editor complexes reliant on a DNA-modifying domain (DarT) in combination with a Cas9 DNA-recognition domain (DarT-G49D-ScdCas9).
• DarT DNA-modifying domain
• DarT-G49D-ScdCas9 Cas9 DNA-recognition domain
• DarT was used as an exemplary DNA- modifying domain in these experiments.
• the repair template encoded the D516G mutation conferring 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 to 6,000 fold increase in genome modification rates, demonstrating the effect of the gap editors.
• DarT catalytic domain motif X 1 X 2 X 3 X 3 R (SEQ ID NO: 18), wherein Xi is L, I, V, or A; X 2 is I, Q, K, T, or N; and X 3 is any amino acid (FIG. 18).
• DarT catalytic domain motif (SEQ ID NO: 19), wherein Xi is any amino acid; X 2 is L, V, or I; X 3 is H, G, N, S, or A; X 4 is D or E; X 5 is Y or F; X 6 is V, I, or A; X 7 is T, A, G, K, N, or W; X 8 is S, T, N, M, or K; and X 9 is P, V, M, I, A; X 10 is L, M or F (FIG. 19).
• DarT catalytic domain motif X1X2X3X4X5X6X7X8 (SEQ ID NO: 20), wherein Xi is F, Y, W, V, or C; X 2 is V, L, I, A, C, or F; X 3 is F, Y, or A; X 4 is T, S, Y, or F; X 5 is D, N, or S; X 6 is G, R, S, A, M or Q; X 7 is H, N, S, or Q; and X 8 is A, G, C, H or K (FIG. 20).
• Scabin catalytic domain motif: 22 wherein Xi is and amino acid; X 2 is Q, E, or R; X 3 is V or I; X 4 is A, L, V, S, or T; X 5 is F, I, V, or L; Xe is P, A, or I; and X 7 is I, V, or L (FIG. 22).
• DarT catalytic motif of SEQ ID NO: 21 and Scabin catalytic motif of SEQ ID NO: 22 are structural and functional analogs, with the conserved glutamate (E) being the catalytic residue.
• Scabin catalytic domain motif wherein Xi is S, T, or G; X 2 is any amino acid; X 3 is F, Y, or L; X 4 is V, I, A, or L; X 5 is S, G, or A; Xe is T or A; and X 7 is T, S, or A (FIG. 23).
• Xi is F, W, Y, or M
• X 2 is A or S
• X 3 is E, G, P, A, or T
• X 4 is any amino acid
• X5 is G, C, or Q
• C 6 is T, V, Y, or I
• X7 is V or I
• X 8 is Q, K, or R
• X9 is A, S, C, T, or N
• Xio is N, G, or A
• X 11 is F, W, or Y (FIG. 27).

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)
• Crystallography & Structural Chemistry (AREA)
• Enzymes And Modification Thereof (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (1)

Family

ID=82837338

Family Applications (1)

Country Status (5)

Cited By (5)

Families Citing this family (1)

Citations (4)

Family Cites Families (1)

• 2022
  • 2022-02-14 EP EP22753488.0A patent/EP4291664A4/en active Pending
  • 2022-02-14 CN CN202280027473.3A patent/CN117222741A/zh active Pending
  • 2022-02-14 WO PCT/US2022/016313 patent/WO2022174144A1/en not_active Ceased
  • 2022-02-14 JP JP2023548848A patent/JP2024506375A/ja active Pending
  • 2022-02-14 US US18/546,378 patent/US20240229012A9/en active Pending

Patent Citations (4)

Non-Patent Citations (9)

Also Published As

Similar Documents

Legal Events