WO2024050544A2 - Enhanced targeted knock-in frequency in host genomes through crispr exonuclease processing - Google Patents
Enhanced targeted knock-in frequency in host genomes through crispr exonuclease processing Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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 [CRISPRs]
-
- 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 RNAses, DNAses
Definitions
- compositions and methods to introduce a Cas endonuclease, a gRNA, an exonuclease, and donor DNA to a host cell, e.g. a plant cell.
- a host cell e.g. a plant cell.
- REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0003] The contents of the electronic sequence listing (JRSI_086_01WO_SeqList_ST26.xml; Size: 157,386 bytes; and Date of Creation: September 1, 2023) are herein incorporated by reference in its entirety.
- BACKGROUND [0004] Targeted gene disruption has wide applicability for research, therapeutic, agricultural, and industrial uses.
- DSBs double-strand DNA breaks
- Endonucleases are most often used for targeted gene disruption in organisms that have traditionally been refractive to more conventional gene targeting methods, such as algae, plants, and large animal models, including humans.
- SSA Single-strand annealing
- SSA involving repeats that flank a single DSB causes a deletion rearrangement between the repeats, and hence is relatively mutagenic. Nevertheless, this pathway is conserved, in that SSA events have been found in several organisms.
- the current disclosure solves the aforementioned problem by providing methods and compositions to enhance knock-in frequency of donor DNA in host cell genomes.
- the disclosure relates to the discovery that certain exonucleases enhance Cas endonuclease-mediated knock-in frequency. Knocking down expression of genes involved in the nonhomologous end joining pathway further enhanced Cas mediated knock-in frequency.
- the disclosure provides a method of targeted DNA sequence insertion into a plant genome, comprising: providing host cell; selecting a target site nucleic acid sequence in the host cell genome for DNA sequence insertion; introducing into the host cell a guide RNA (gRNA) with a guide sequence having complementarity to the target site nucleic acid sequence in the host genome and a Cas endonuclease that interacts with the gRNA and is capable of creating a double stranded break in the host genome; introducing into the host cell an exonuclease; and introducing into the host cell a donor DNA sequence template comprising: a DNA sequence of interest and left and right homology arm sequences, wherein the gRNA guide sequence binds to the complementary target site nucleic acid sequence in the host genome and the Cas endonuclease creates a double stranded break in the host genome; and wherein the DNA sequence of interest is inserted into the host genome via a homology dependent repair mechanism.
- gRNA guide RNA
- the disclosure provides a method of targeted DNA sequence insertion into a plant genome, comprising: providing a host cell; selecting a target site nucleic acid sequence in the host cell genome for DNA sequence insertion; introducing into the host cell a guide RNA (gRNA) with a guide sequence having complementarity to the target site nucleic acid sequence in the host genome and a Cas endonuclease that interacts with the gRNA and is capable of creating a double stranded break in the host genome; introducing into the host cell an exonuclease; and introducing into the host cell a donor DNA sequence template comprising: a DNA sequence of interest and left and right homology arm sequences; introducing into the host cell a silencing construct, wherein the silencing construct knocks down expression of a nonhomologous end joining pathway gene, wherein the gRNA guide sequence binds to the complementary target site nucleic acid sequence in the host genome and Page 2 of 61 290326197 v3 the Ca
- gRNA guide
- the exonuclease exhibits 3’ to 5’ exonuclease activity at the double stranded break site.
- the host cell is a plant cell.
- the DNA sequence of interest is inserted into the host cell’s genome with at least a 10% frequency.
- FIG. 1 is a schematic adapted from Rogowsky that describes several applications for CRISPR/Cas9-induced gene targeting.
- FIG.2 is a schematic that describes a knock-in (KI) strategy of donor DNA into either the SSR2 or BAM9 locus in potato protoplasts.
- FIG.3 shows expression of 6 different loci in either CW protoplasts or DE protoplasts.
- FIG.4 is an image of an agarose gel from a PCR amplification assay for PP02g2 with either NLS-DPD1, DPD1, or Trex2.
- FIGs. 5A and 5B shows fluorescent images that show Trex2 significantly increases targeted RFP KI at Bam9 (increased NLS-RFP signal).
- FIG. 6 shows fluorescent images that show Trex2 increases targeted RFP KI with various donor DNA templates.
- FIG.7 shows fluorescent images of either Trex2 with RFP or Trex2 with RFP that has a frame shift mutation.
- FIG. 8 is a bar graph that shows that Trex2 increases targeted RFP KI in potato protoplasts.
- FIG. 9 is a bar graph that shows the percent of RFP(+) FDA cells with various constructs.
- FIG.10A is a schematic that shows the PCR screening strategy to determine 5’ and 3’ integration of RFP at the Bam9 locus.
- FIG.10B is an image of an agarose gel from PCR amplification assay as shown in FIG. 10A with or without Trex2 transient expression.
- FIG. 11 is a bar graph that shows the effect of different homology arm lengths on targeted RFP KI at Bam9 with several negative controls.
- FIG.12A is a schematic of the plasmid used to silence Lig4. Page 3 of 61 290326197 v3
- FIG.12B is a schematic of the plasmid used to silence Ku80.
- FIG. 13 is a bar graph showing that silencing NHEJ pathway can increase Trex2- mediated RFP KI.
- FIG. 14 is a bar graph showing the effect of an exonuclease from Arabidopsis on targeted RFP KI
- FIG.15 is a graph that shows the average percent of RFP(+) cells after treatment with various exonucleases.
- the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
- any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
- the term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%.
- a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
- Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
- substantially complementary refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
- hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
- the hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
- the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these.
- a hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme.
- the term “genomic locus” or “locus” is the specific location of a gene or DNA sequence on a chromosome.
- a “gene” refers to stretches of DNA or RNA that encode a polypeptide or an RNA chain that has functional role to play in an organism and hence is the molecular unit of heredity in living organisms.
- genes include regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences.
- a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
- the term “gene editing” and its grammatical equivalents can refer to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a Page 5 of 61 290326197 v3 genome. Gene editing can be performed using a nuclease (e.g., a natural-existing nuclease or an artificially engineered nuclease).
- the term “knock-in” refers to a gene editing technique that introduces exogenous nucleic acid into the genome of a host cell, e.g. a plant cell.
- the term “function” and its grammatical equivalents can refer to the capability of operating, having, or serving an intended purpose.
- Functional can comprise any percent from baseline to 100% of normal function.
- functional can comprise or comprise about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and/or 100% of normal function.
- the term functional can mean over or over about 100% of normal function, for example, 125, 150, 175, 200, 250, 300% and/or above normal function.
- providing refers to making accessible.
- providing a host cell means the host cell is accessible.
- providing a host cell means the cell is accessible for introducing a nucleic acid to the host cell.
- the term “introducing” refers to transfection or delivery of an expression vector (e.g., plasmids). Transfection or delivery of the expression vectors may be accomplished by any suitable method known in the art including, but not limited to, viral transfection or transduction, microinjection, electroporation, sonoporation, optical transfection, impalefection, hydrodynamic delivery, nucleofection, lipofection, dendrimeric transfection, magnetofection, gene gun transfection, nanoparticle-based transfection, calcium phosphate transfection, viral transfection, and cell squeezing.
- the term “operably linked” refers to an arrangement of elements where the components so described are configured so as to perform their usual function.
- promoter and/or enhancer sequences operably linked to a coding sequence are capable of effecting the transcription, and in some cases, the translation, of a coding sequence.
- the promoter and/or enhancer sequences need not be contiguous with the coding sequence so long as they function to direct the expression of the coding sequence.
- intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
- such sequences need not reside on the same contiguous DNA molecule (i.e. chromosome) and may still have interactions resulting in altered regulation.
- promoter refers to a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a polynucleotide or polypeptide coding sequence such as messenger RNA, ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind of RNA transcribed by any class of any RNA Page 6 of 61 290326197 v3 polymerase I, II or III. Promoters may be constitutive or inducible and, in some embodiments — particularly many embodiments in which selection is employed — the transcription of at least one component of the nucleic acid-guided nuclease editing system is under the control of an inducible promoter. [0042] Sequence identity.
- Sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the number of residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
- sequence identity When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
- Sequences which differ by such conservative substitutions are said to have "sequence similarity" or "similarity.” Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
- NCBI Basic Local Alignment Search Tool (BLAST®) (Altschul et al. 1990 J. Mol. Biol. 215: 403-10), which is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed on the internet via the National Library of Medicine (NLM)'s world-wide-web URL. A description of how to determine sequence identity using this program is available at the NLM's website on BLAST tutorial.
- NLM National Library of Medicine
- the term “host cell” refers to a cell that is targeted with the Cas endonuclease, gRNA, exonuclease, and donor DNA sequence insertion.
- a host cell may comprise a prokaryote or a eukaryote.
- prokaryote refers to non-eukaryotic organisms belonging to the Eubacteria (e.g., Escherichia coli, Thermus thermophilus, etc.) and Archaea (e.g., Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium spp., A. fulgidus, P. firiosus, P. horikoshii, A. pernix, etc.) phylogenetic domains.
- Eubacteria e.g., Escherichia coli, Thermus thermophilus, etc.
- Archaea e.g., Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium spp., A. fulgidus, P. firiosus, P. horikoshii, A. pernix, etc.
- the term “eukaryote” refers to organisms belonging to the phylogenetic domain Eucarya such as animals (e.g., mammals, insects, reptiles, birds, etc.), ciliates, plants, fungi (e.g., yeasts, etc.), flagellates, microsporidia, protists, etc.
- the term “plant” includes the class of higher and lower plants including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.
- CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
- CRISPR-associated protein Cas
- Type I, II, and III There are at least three main CRISPR system types (Type I, II, and III) and at least 10 distinct subtypes (Makarova, K.S., et.al., Nat Rev Microbiol.2011 May 9; 9(6):467-477).
- Type I and III systems use Cas protein complexes and short guide polynucleotide sequences to target selected DNA regions.
- Type II systems rely on a single protein (e.g. Cas9) and the targeting guide polynucleotide, where a portion of the 5’ end of a guide sequence is complementary to a target nucleic acid.
- CRISPR genome editing requires two components, a gRNA and a Cas enzyme. These components associate to form a ribonucleoprotein (RNP) complex, where after the gRNA can base pair with a complementary protospacer sequence (i.e. the target genomic sequence of about 20 bases in length) under the condition that a particular adjacent sequence, called a protospacer-adjacent motif (PAM), is present in the genome.
- RNP ribonucleoprotein
- PAM protospacer-adjacent motif
- the PAM is only a few bases Page 8 of 61 290326197 v3 long, and its sequence depends on the type of Cas enzyme used.
- each Cas enzyme is directed by the gRNA to a user-specified cut site in the genome.
- Cas12a family members contain a RuvC-like endonuclease domain, but lack the second HNH endonuclease domain of Cas9.
- Cas12a cleaves DNA in a staggered pattern in contrast to Cas9 which produces a blunt-end.
- Cas12a requires only one gRNA rather than the two tracrRNA and crRNA needed by Cas9.
- the target sequence of the gRNAs must be next to a PAM sequence.
- the PAM sequence corresponds to NGG, where N is any base.
- the gRNA will recognize and bind to 20 nucleotides on the DNA strand opposite from the NGG PAM site.
- the PAM sequence is TTTV, where V can represent A, C, or G.
- a TTTT PAM sequence may also work. The “V” of the TTTV is immediately adjacent to the base at the 5’ end of the non-targeted strand side of the protospacer element.
- the guide RNA for Cas12a is relatively short and is approximately 40 to 44 bases long.
- the damage caused by the DSB will be repaired in eukaryotic cells, primarily by two pathways: Non-Homologous End-Joining (NHEJ) and Homology Directed Repair (HDR).
- NHEJ Non-Homologous End-Joining
- HDR Homology Directed Repair
- the HDR mechanism requires the presence of a donor DNA template containing regions of homology to both sites of the DNA break. This donor DNA can carry specific mutations and has to be delivered simultaneously with a preassembled Cas RNP complex composed of Cas9 or Cas12a and synthetically produced gRNAs.
- a Cas endonuclease relates to a Cas protein encoded by a Cas gene, wherein said Cas protein is capable of introducing a double strand break into a DNA target sequence.
- the Cas endonuclease is guided by the guide polynucleotide to recognize and optionally introduce a double strand break at a specific target site into the genome of a cell.
- the term “guide polynucleotide/Cas endonuclease system” includes a complex of a Cas endonuclease and a guide polynucleotide that is capable of introducing a double strand break into a DNA target sequence.
- the Cas endonuclease unwinds the DNA duplex in close proximity of the genomic target site and cleaves both DNA strands upon recognition of a target sequence by a guide RNA, but only if the correct protospacer-adjacent motif (PAM) is approximately oriented at the 3′ end of the target sequence.
- Page 9 of 61 290326197 v3 the Cas endonuclease gene is plant, maize or soybean optimized Cas9 endonuclease.
- the Cas endonuclease gene is operably linked to a SV40 nuclear targeting signal upstream of the Cas codon region and a bipartite VirD2 nuclear localization signal (Tinland et al.
- the Cas endonuclease gene is a plant codon optimized streptococcus pyogenes Cas9 gene that can recognize any genomic sequence of the form N(12- 30)NGG can in principle be targeted.
- the Cas endonuclease is introduced directly into a cell by any method known in the art, for example, but not limited to transient introduction methods, transfection and/or topical application.
- Cas endonucleases for use in the methods described herein include Class 2 Type V CRISPR/Cas based gene editing systems.
- CRISPR/Cas systems can be used in a wide variety of organisms to add, disrupt, or change the sequence of specific genes.
- Naturally-occurring CRISPR systems are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity.
- CRISPR systems are based on two elements.
- the first element is an endonuclease (e.g., Cas9) that has a binding site for the second element, the guide polynucleotide (e.g., guide RNA, or gRNA).
- endonuclease e.g., Cas9
- the guide polynucleotide e.g., guide RNA, or gRNA
- the gRNA complexed with the Cas endonuclease forms a CRISPR Cas ribonucleoprotein complex, or RNP-complex.
- the guide gRNA directs the endonuclease to double stranded DNA templates based on sequence homology. The endonuclease then cleaves that DNA template.
- the organism By delivering the endonuclease and appropriate guide polynucleotides (e.g., guide RNAs) into a cell, the organism’s genome is cut at a desired location.
- one of the two alternative DNA repair mechanisms described supra can restore chromosomal integrity: 1) non- homologous end joining (NHEJ) which generates insertions and/or deletions of a few base- pairs (bp) of DNA at the gRNA cut site, or 2) homology-directed repair (HDR) which can correct the lesion via an additional “bridging” DNA template that spans the gRNA cut site.
- NHEJ non- homologous end joining
- HDR homology-directed repair
- Naturally occurring CRISPR systems have been modified for use in gene editing (silencing, knock out, knock in, enhancing or changing specific genes) in eukaryotes (Wiedenheft et al. (2012) Nature 482: 331-8). This is accomplished by introducing into the eukaryotic cell a one or more specifically designed guide nucleic acids (gNAs), typically guide RNAs (gRNAs), and an appropriate endonuclease which forms a ribonucleoprotein complex with the gNA.
- the gNA guides the gNA-endonuclease protein complex to a target genomic location, and the endonuclease introduces a double strand break at the target genomic location (locus).
- CRISPR systems can be used to edit a target locus by adding or deleting one or more base pairs, introducing a premature stop codon, or introducing a frame-shift mutation which decreases expression of a target, in part or completely.
- the CRISPR system can alternatively be used like RNA interference, turning off a target gene in a reversible fashion.
- the RNA can guide the endonuclease to a target gene promoter, sterically blocking RNA polymerases.
- the gene editing system comprises a CRISPR system.
- the CRISPR system comprises a Class 2 CRISPR system.
- Class 2 systems currently represent a single protein that is categorized into three distinct types (types II, V and VI). Any class 2 CRISPR system suitable for gene editing, for example a type II, a type V or a type VI system, is envisaged as within the scope of the instant disclosure.
- Exemplary Class 2 type II CRISPR systems include Cas9, Csn2 and Cas4.
- Exemplary Class 2, type V CRISPR systems include, Cas12, Cas12a (Cpf1, MAD7), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i and Cas12k (C2c5).
- Exemplary Class 2 Type VI systems include Cas13, Cas13a (C2c2) Cas13b, Cas13c and Cas13d.
- the endonuclease protein e.g., nucleic acid-directed nuclease
- the endonuclease protein may be derived from any bacterial or archaeal Cas protein.
- the endonuclease protein comprises one or more of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Page 11 of 61 290326197 v3 Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.
- the endonuclease protein is a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, Cas12, modified versions thereof, or combinations or complexes of these.
- the compositions may be transfected into one or more cells using any suitable expression vector which delivers the composition to the cell or cells. Any suitable expression vectors may be used in accordance with the embodiments described herein including, but not limited to, plasmids and recombinant viral vectors.
- Suitable viruses that may be used to design a recombinant viral vector for use in the embodiments described herein include, but are not limited to, adeno-associated virus (AAV), adenovirus, lentivirus, baculovirus, Bean yellow dwarf virus, Wheat dwarf virus (WDV), Cabbage leaf curl virus, Wheat streak mosaic virus (WSMV), Barley stripe mosaic virus (BSMV), Tobacco rattle virus (TRV) (family Virgaviridae), geminiviruses (family Geminiviridae), or Begomoviruses (family Geminiviridae).
- AAV adeno-associated virus
- WDV Wheat dwarf virus
- WSMV Wheat streak mosaic virus
- BSMV Barley stripe mosaic virus
- TRV Tobacco rattle virus
- a vector suitable for Agrobacterium mediated transformation may be used in accordance with the embodiments described herein.
- Donor Nucleic Acid refers to a nucleic acid molecule having a nucleic acid template or insertion sequence for site-directed, targeted insertion or recombination into the genome of a plant cell via repair of a nick or double-stranded DNA break in the genome of a plant cell.
- a “donor template” may be used for site-directed integration of a transgene or suppression construct, or as a template to introduce a mutation, such as an insertion, deletion, substitution, etc., into a target site within the genome of a plant.
- a targeted genome editing technique provided herein may comprise the use of one or more, two or more, three or more, four or more, or five or more donor molecules or templates.
- a “donor template” may be a single-stranded or double-stranded DNA or RNA molecule or plasmid.
- An “insertion sequence” of a donor template is a sequence designed for targeted insertion into the genome of a plant cell, which may be of any suitable length.
- the insertion sequence of a donor template may be about 2 to about 50,000, about 2 to about 10,000, about 2 to about 5000, Page 12 of 61 290326197 v3 about 2 to about 1000, about 2 to about 500, about 2 to about 250, about 2 to about 100, about 2 to about 50, about 2 to about 30, about 15 to about 50, about 15 to about 100, about 15 to about 500, about 15 to about 1000, about 15 to about 5000, about 18 to about 30, about 18 to about 26, about 20 to about 26, about 20 to about 50, about 20 to about 100, about 20 to about 250, about 20 to about 500, about 20 to about 1000, about 20 to about 5000, about 20 to about 10,000, about 50 to about 250, about 50 to about 500, about 50 to about 1000, about 50 to about 5000, about 50 to about 10,000, about 100 to about 250, about 100 to about 500, about 100 to about 1000, about 100 to about 5000, about 100 to about 10,000, about 250 to about 500, about 250 to about 1000, about 250 to about 5000, or about 250 to to about
- a donor template may also have at least one homology sequence or homology arm, such as two homology arms, to direct the integration of a mutation or insertion sequence into a target site within the genome of a plant via homologous recombination, wherein the homology sequence or homology arm(s) are identical or complementary, or have a percent identity or percent complementarity, to a sequence at or near the target site within the genome of the plant.
- the homology arm(s) will flank or surround the insertion sequence of the donor template.
- the homology arm may be about 2 to about 50,000, about 2 to about 10,000, about 2 to about 5000, about 2 to about 1000, about 2 to about 500, about 2 to about 250, about 2 to about 100, about 2 to about 50, about 2 to about 30, about 15 to about 50, about 15 to about 100, about 15 to about 500, about 15 to about 1000, about 15 to about 5000, about 18 to about 30, about 18 to about 26, about 20 to about 26, about 20 to about 50, about 20 to about 100, about 20 to about 250, about 20 to about 500, about 20 to about 1000, about 20 to about 5000, about 20 to about 10,000, about 50 to about 250, about 50 to about 500, about 50 to about 1000, about 50 to about 5000, about 50 to about 10,000, about 100 to about 250, about 100 to about 500, about 100 to about 1000, about 100 to about 5000, about 100 to about 10,000, about 250 to about 500, about 250 to about 1000, about 250 to about 5000, or about 250 to about 10,000 nucleotides or base pairs (bp) in length.
- the donor template only has one homology arm. In some embodiments, the donor template has a left homology arm of at least 100 bp, at least 200 bp, at least 300 bp, or at least 400 bp long. In some embodiments, the donor template has a left homology arm of at least between 100 bp and 800 bp long. In some embodiments, the donor template has a left homology arm of at least between 100 bp and 1000 bp long. Page 13 of 61 290326197 v3 [0069] An insertion sequence of a donor template may comprise one or more genes or sequences that each encode a transcribed non-coding RNA or mRNA sequence and/or a translated protein sequence.
- a transcribed sequence or gene of a donor template may encode a protein or a non-coding RNA molecule.
- An insertion sequence of a donor template may comprise a polynucleotide sequence that does not comprise a functional gene or an entire gene sequence (e.g., the donor template may simply comprise regulatory sequences, such as a promoter sequence, or only a portion of a gene or coding sequence), or may not contain any identifiable gene expression elements or any actively transcribed gene sequence.
- the donor template may be linear or circular, and may be single-stranded or double-stranded.
- a donor template may be delivered to a cell as an RNA molecule expressed from a transgene.
- a donor template may also be delivered to the cell as a naked nucleic acid (e.g., via particle bombardment), as a complex with one or more delivery agents (e.g., liposomes, proteins, poloxamers, T-strand encapsulated with proteins, etc.), or contained in a bacterial or viral delivery vehicle, such as, for example, Agrobacterium tumefaciens or a geminivirus, respectively.
- An insertion sequence of a donor template provided herein may comprise a transcribable DNA sequence that may be transcribed into an RNA molecule, which may be non-coding and may or may not be operably linked to a promoter and/or other regulatory sequence.
- a donor template may not comprise an insertion sequence, and instead comprise one or more homology sequences that include(s) one or more mutations, such as an insertion, deletion, substitution, etc., relative to the genomic sequence at a target site within the genome of a plant, such as at or near a gene within the genome of a plant.
- a donor template may comprise an insertion sequence that does not comprise a coding or transcribable DNA sequence, wherein the insertion sequence is used to introduce one or more mutations into a target site within the genome of a plant, such as at or near a gene within the genome of a plant.
- a donor template provided herein may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten genes or transcribable DNA sequences.
- a donor template may comprise no genes.
- a gene or transcribable DNA sequence of a donor template may include, for example, an insecticidal resistance gene, an herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a yield enhancing gene, a nutritional quality Page 14 of 61 290326197 v3 gene, a DNA binding gene, a selectable marker gene, an RNAi or suppression construct, a site- specific genome modification enzyme gene, a single guide RNA of a CRISPR/Cas9 system, a geminivirus-based expression cassette, or a plant viral expression vector system.
- an insertion sequence of a donor template may comprise a protein encoding sequence or a transcribable DNA sequence that encodes a non-coding RNA molecule, which may target an endogenous gene for suppression.
- a donor template may comprise a promoter, such as a constitutive promoter, a tissue-specific or tissue-preferred promoter, a developmental stage promoter, or an inducible promoter.
- a donor template may comprise a leader, enhancer, promoter, transcriptional start site, 5'-UTR, one or more exon(s), one or more intron(s), transcriptional termination site, region or sequence, 3'-UTR, and/or polyadenylation signal.
- the leader, enhancer, and/or promoter may be operably linked to a gene or transcribable DNA sequence encoding a non-coding RNA, a guide RNA, an mRNA and/or protein.
- a portion of a recombinant donor template polynucleotide molecule i.e., an insertion sequence
- the insertion sequence of the donor template may comprise a transgene or construct, such as a transgene or transcribable DNA sequence encoding a non-coding RNA molecule that targets an endogenous gene for suppression.
- the donor template may also have one or two homology arms flanking the insertion sequence to promote the targeted insertion event through homologous recombination and/or homology-directed repair.
- Each homology arm may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99% or 100% identical or complementary to at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 2500, or at least 5000 consecutive nucleotides of a target DNA sequence within the genome of a plant cell.
- a plant cell may comprise a recombinant DNA molecule encoding a donor template for site-directed or targeted integration of a transgene or construct, such as a transgene or transcribable DNA sequence encoding a non- coding RNA molecule that targets an endogenous gene for suppression, into the genome of a plant.
- a transgene or construct such as a transgene or transcribable DNA sequence encoding a non- coding RNA molecule that targets an endogenous gene for suppression, into the genome of a plant.
- target site for genome editing or site-directed integration refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by a site-specific nuclease introducing a double stranded break (or single-stranded nick) into the nucleic acid backbone of the polynucleotide sequence and/or its complementary DNA strand.
- a target site may comprise at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29, or at least 30 consecutive nucleotides.
- the term “target site” for an RNA-guided nuclease may comprise the sequence of either complementary strand of a double-stranded nucleic acid (DNA) molecule or chromosome at the target site.
- a site-specific nuclease may bind to a target site, such as via a non-coding guide RNA (e.g., without being limiting, a CRISPR RNA (crRNA) or a single-guide RNA (sgRNA) as described further below).
- a non-coding guide RNA e.g., without being limiting, a CRISPR RNA (crRNA) or a single-guide RNA (sgRNA) as described further below.
- crRNA CRISPR RNA
- sgRNA single-guide RNA
- a non-coding guide RNA provided herein may be complementary to a target site (e.g., complementary to either strand of a double-stranded nucleic acid molecule or chromosome at the target site). It will be appreciated that perfect identity or complementarity may not be required for a non-coding guide RNA to bind or hybridize to a target site.
- target site also refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by another site-specific nuclease that may not be guided by a non-coding RNA molecule, such as a meganuclease, zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN), to introduce a double stranded break (or single-stranded nick) into the polynucleotide sequence and/or its complementary DNA strand.
- ZFN zinc finger nuclease
- TALEN transcription activator-like effector nuclease
- Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. A close relative to exonucleases is the endonuclease, see above, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain.
- Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease, which is a dependent de-capping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease.
- exonucleases are shown below in Table 1 and include, but are not limited to, Exonuclease I, Exonuclease III (E.
- Exonuclease T Exonuclease V (RecBCD), RecQ4, Exonuclease VII, Exonuclease VIII, Lambda Exonuclease, Trex1, Trex2, and Dpd1.
- the exonuclease exhibits 5’ to 3’ exonuclease activity at the double stranded break site.
- the exonuclease is selected from the group consisting of Trex1, Trex2, and DPD1.
- the exonuclease is Trex2.
- the exonuclease is DPD1.
- the exonuclease sequence is a gene selected from Table 1.
- the exonuclease comprises SEQ ID NO: 1 or a sequence at least 75% identical thereto.
- the nucleotide sequence comprises SEQ ID NO: 1 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 1.
- the exonuclease comprises SEQ ID NO: 2 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 2 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 2.
- the exonuclease comprises SEQ ID NO: 3 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 3 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 3.
- the exonuclease comprises SEQ ID NO: 4 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 4 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 4.
- the exonuclease comprises SEQ ID NO: 5 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 5 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 5.
- the exonuclease comprises SEQ ID NO: 6 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 6 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 6.
- the exonuclease comprises SEQ ID NO: 7 or a sequence at least 75% identical thereto.
- the nucleotide sequence comprises SEQ ID NO: 7 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 7.
- the exonuclease comprises SEQ ID NO: 8 or a sequence at least 75% identical thereto.
- the nucleotide sequence comprises SEQ ID NO: 8 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 8.
- the exonuclease comprises SEQ ID NO: 9 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 9 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 9.
- the exonuclease comprises SEQ ID NO: 10 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 10 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 10.
- the exonuclease comprises SEQ ID NO: 11 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 11 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 11.
- the exonuclease comprises SEQ ID NO: 12 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 12 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 12.
- the exonuclease comprises SEQ ID NO: 13 or a sequence at least 75% identical thereto.
- the nucleotide sequence comprises SEQ ID NO: 13 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 13.
- the exonuclease comprises SEQ ID NO: 14 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 14 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 14.
- the exonuclease comprises SEQ ID NO: 15 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 15 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 15.
- the exonuclease comprises SEQ ID NO: 16 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 16 or a sequence at least 85% identical thereto.
- the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 16.
- the methods of enhanced targeted knock-in frequency comprise concurrent silencing of a NHEJ pathway gene.
- Methods of gene silencing are well known in the art (see for example Yan H, et al., New construct approaches for efficient gene silencing in plants. Plant Physiol. 2006 Aug;141(4):1508-18; Wesley SV, et al., Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J.2001 Sep;27(6):581-90; Helliwell CA and Waterhouse PM, Constructs and Methods for Hairpin RNA-Mediated Gene Silencing in Plants.
- the NHEJ gene targeted for silencing is selected from Table 2.
- the NHEJ gene targeted for silencing comprises SEQ ID NO: 17 or a sequence at least 75% identical thereto.
- the NHEJ gene targeted for silencing comprises SEQ ID NO: 17 or a sequence at least 85% identical thereto.
- the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 17.
- the NHEJ gene targeted for silencing comprises SEQ ID NO: 18 or a sequence at least 75% identical thereto. In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 18 or a sequence at least 85% identical thereto.
- the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 18.
- the NHEJ gene targeted for silencing comprises SEQ ID NO: 19 or a sequence at least 75% identical thereto. In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 19 or a sequence at least 85% identical thereto.
- the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 19.
- the NHEJ gene targeted for silencing comprises SEQ ID NO: 20 or a sequence at least 75% identical thereto. In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 20 or a sequence at least 85% identical thereto.
- the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 20.
- the NHEJ gene targeted for silencing comprises SEQ ID NO: 21 or a sequence at least 75% identical thereto. In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 21 or a sequence at least 85% identical thereto.
- the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 21.
- the NHEJ gene targeted for silencing comprises SEQ ID NO: 22 or a sequence at least 75% identical thereto. In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 22 or a sequence at least 85% identical thereto.
- the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 22.
- the expression vector is a plasmid.
- the methods described herein may include a first plasmid that, when delivered to a cell, expresses the CRISPR system components, CAS endonuclease and the guide RNA sequence; a second plasmid that comprises a donor DNA template; and a third plasmid that, when delivered to a cell with the first plasmid, co-expresses an exonuclease.
- the exonuclease Page 28 of 61 290326197 v3 is Trex2.
- the methods disclosed herein include a fourth plasmid comprising a NHEJ pathway gene silencing construct.
- the methods disclosed herein may include a plasmid that comprises all three or all four expression cassettes and donor DNA template, or may include three plasmids, the first of which expresses the CAS endonuclease, the second of which expresses the guide RNA sequence, and the third of which expresses an endonuclease, such as Trex2.
- the methods described herein may be used to knock- in donor DNA to a target genomic nucleotide sequence in a cell.
- Such methods may include transfecting a cell with one or more expression cassettes that, when inserted into an expression vector, express an endonuclease (or endonuclease system) and an exonuclease.
- an endonuclease or endonuclease system
- an exonuclease or endonuclease system
- the endonuclease system is a CRISPR system and the exonuclease is Trex2.
- the method includes providing to a plant cell: (i) a first expression cassette that includes a nucleotide sequence that encodes a CAS9 endonuclease, (ii) a second expression cassette that that includes a nucleotide sequence that encodes a guide RNA sequence designed to be complementary to a target genomic nucleotide sequence in a cell, (iii) a third expression cassette that includes a nucleotide sequence that encodes a Trex2 exonuclease, and (iv) a donor DNA template.
- the method includes providing (i) a first expression cassette that includes a nucleotide sequence that encodes a CAS9 endonuclease, (ii) a second expression cassette that that includes a nucleotide sequence that encodes a guide RNA sequence designed to be complementary to a target genomic nucleotide sequence in a cell, (iii) a third expression cassette that includes a nucleotide sequence that encodes a Trex2 exonuclease, (iv) a fourth expression cassette that includes a gene silencing construct, and (v) a donor DNA template. [0137] In some embodiments, the method provides one expression cassette to a host cell.
- the method provides two expression cassettes to a host cell. In some embodiments, the method provides three expression cassettes to a host cell. In some embodiments, the method provides four expression cassettes to a host cell. [0138] In some embodiments, the method provides one expression vector to a host cell. In some embodiments, the method provides two expression vectors to a host cell. In some embodiments, the method provides three expression vectors to a host cell. In some embodiments, the method provides four expression vectors to a host cell. [0139] In some embodiments, the expression vector(s) comprises Trex2 and a silencing construct for Lig4. In some embodiments, the expression vector(s) comprises Trex2 and a silencing construct for Ku80.
- the expression vector(s) comprises DPD1 and a silencing construct for Lig4. In some embodiments, the expression vector(s) comprises DPD1 and a silencing construct for Ku80. [0141] In some embodiments, the expression vector(s) comprises POLQ and a silencing construct for Lig4. In some embodiments, the expression vector(s) comprises POLQ and a silencing construct for Ku80.
- the methods described herein insert a DNA sequence of interest into a host cell’s genome with at least a 5% frequency, at least a 10% frequency, at least a 20% frequency, at least a 30% frequency, at least a 40% frequency; at least a 50% frequency, at least a 60% frequency, at least a 70% frequency, at least an 80% frequency, at least a 90% frequency, or a 100% frequency.
- the methods described herein insert a DNA sequence of interest into a host cell’s genome with about 5% frequency to about 10% frequency, about a 10% frequency to about 20% frequency, about a 20% frequency to about 30% frequency, about a 30% frequency to about 40% frequency, about a 40% frequency to about 50% frequency; about a 50% frequency to about 60% frequency, about a 60% frequency to about 70% frequency, about a 70% frequency to about 80% frequency, about an 80% frequency to about 90% frequency, or about a 90% frequency to about 100% frequency.
- the methods described herein insert a DNA sequence into a host cell’s genome with at least a 5% frequency, at least a 10% frequency, at least a 20% frequency, at least a 30% frequency, at least a 40% frequency; at least a 50% frequency, at least a 60% frequency, at least a 70% frequency, at least an 80% frequency, at least a 90% frequency, or a 100% frequency.
- the methods described herein insert a DNA sequence of interest into a host cell’s genome with about 5% frequency to about 10% frequency, about a 10% frequency to about 20% frequency, about a 20% frequency to about 30% frequency, about a 30% frequency to about 40% frequency, about a 40% frequency to about 50% frequency; about a 50% frequency to about 60% frequency, about a 60% frequency to about 70% frequency, about a 70% frequency to about 80% frequency, about an 80% frequency to about 90% frequency, or about a 90% frequency to about 100% frequency.
- Expression vectors usually include at least one genetic marker, operably linked to a regulatory element (a promoter, for example) that allows transformed cells containing the marker to be either recovered by negative selection, i.e., inhibiting growth of cells that do not Page 30 of 61 290326197 v3 contain the selectable marker gene, or by positive selection, i.e., screening for the product encoded by the genetic marker.
- a regulatory element a promoter, for example
- positive selection i.e., screening for the product encoded by the genetic marker.
- selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or an herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art.
- Some commonly used selectable marker genes for plant transformation include, but are not limited to, neomycin phosphotransferase II (nptII) (Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983)), aminoglycoside phosphotransferases APH(3')II and APH(3')I (Davies and Smith, 1978; Jimenez and Davies, 1980), kanamycin resistance (KmR) gene ,(Gray and Fitch, 1983), hygromycin phosphotransferase gene (Vanden Elzen et al., Plant Mol.
- Selectable marker genes for plant transformation not of bacterial origin include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet.13:67 (1987), Shah et al., Science 233:478 (1986), Charest et al., Plant Cell Rep.8:643 (1990).
- Another class of marker genes for plant transformation requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic.
- Commonly used genes for screening presumptively transformed cells include beta-glucuronidase (GUS), beta-galactosidase, luciferase and chloramphenicol acetyltransferase.
- GUS beta-glucuronidase
- beta-galactosidase beta-galactosidase
- luciferase luciferase
- chloramphenicol acetyltransferase chloramphenicol acetyltransferase.
- Promoters Genes included in expression vectors are typically driven by a nucleotide sequence comprising a regulatory element, for example, a promoter. Several types of promoters are well Page 31 of 61 290326197 v3 known in the transformation arts as are other regulatory elements that can be used alone or in combination with promoters.
- a “plant promoter” is a promoter capable of initiating transcription in plant cells.
- Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissue-preferred”. Promoters that initiate transcription only in a certain tissue are referred to as “tissue-specific”.
- a “cell- type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
- An “inducible” promoter is a promoter which is under environmental control.
- Terminators As used herein, the term “terminator” or “termination sequence” generally refers to a 3′ flanking region of a gene that contains nucleotide sequences which regulate transcription termination and typically confer RNA stability.
- Terminator sequences that find use in proper transcriptional processing of recombinant nucleic acids in the vectors taught herein are well known in the art. Although terminator sequences do not by themselves initiate gene transcription, their presence can increase accurate processing and termination of the RNA transcript, and result in message stability. The use of recombinant terminator sequences is established in the art. It is appreciated that an understanding of the molecular mechanisms underlying terminator sequence activity are not required to make or use the present disclosure.
- Plants for use with the disclosed methods [0154] The disclosure has use over a broad range of plants, monocots and dicots, including species from the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and Zea.
- Examples include tobacco and Arabidopsis, cereal crops such as maize, wheat, rice, soybean barley, rye, oats, sorghum, alfalfa, clover and the like, oil-producing plants such as canola, Page 32 of 61 290326197 v3 safflower, sunflower, peanut and the like, vegetable crops such as tomato tomatillo, potato, pepper, eggplant, sugar beet, carrot, cucumber, lettuce, pea and the like, horticultural plants such as aster, begonia, chrysanthemum, delphinium, zinnia, lawn and turfgrasses and the like.
- the Solanaceae family contains several well-known cultivated crops such as tomato (Solanum lycopersicum also referred to as Lycopersicon esculentum), eggplant (Solanum melogena), tobacco (Nicotiana tabacum), pepper (Capsicum annuum) and potato (Solanum tuberosum).
- tomato Solanum lycopersicum also referred to as Lycopersicon esculentum
- eggplant Solanum melogena
- tobacco Naturaltiana tabacum
- pepper Capsicum annuum
- Potatoes will not hybridize with non-tuber bearing Solanum (tomato, eggplant, etc.) species including weeds commonly found in and around commercial potato fields (Love 1994).
- the genus Solanum is divided into several subsections, of which the subsection potato contains all tuber-bearing potatoes.
- S. tuberosum is divided into two subspecies: tuberosum and andigena.
- the subspecies tuberosum is the cultivated potato widely in use as a crop plant in, for example, North America and Europe.
- the subspecies andigena is also a cultivated species, but cultivation is restricted to Central and South America (Hanneman 1994).
- Z. Huamán and D. M. Spooner reclassified all cultivated potatoes as a single species, S. tuberosum, with various groups, including a Tuberosum Group (S. tuberosum subsp.
- Tuberosum for the modern cultivars (Huamán et al., Am. J. of Botany 89(6): 947-965.2002).
- Solanum tuberosum subsp. tuberosum is an example of one of the most widely cultivated potato varieties, although there are thousands of potato varieties worldwide.
- Examples of well-known cultivated varieties that may be used with the methods and sequences disclosed herein include, but are not limited to, russets, reds, whites, yellows (also called Yukons) purples, Adirondack Blue, Adirondack Red, Agata, Almond, Amandine, Anya, Arran Victory, Atlantic, Bamberg, Belle de Fontenay, BF-15, Schmtstar, Bintje, Blue Congo, Bonnotte, Cabritas, Camota, Chelina, Chiloé, Cielo, Clavela Blanca, Désirée, Fianna, Fingerling, Flava, Golden Wonder, Innovator, Jersey Royal, Kerr's Pink, Kestrel, King Edward, Kipfler, Lady Balfour, Linda, Marfona, Maris Piper, Marquis, Nicola, Pachaco ⁇ a, Pink Eye, Pink Fir Apple, Primura, Ratte, Red Norland, Red Pontiac, Rooster, Russet Burbank, Russet Norkot
- the host cell is from the Solanum genus. In some embodiments, the host cell is a Solanum tuberosum cell. In some embodiments, the host cell is a potato cell. In some embodiments, the host cell is a Solanum lycopersicum cell. In some embodiments, the host cell is a tomato cell.
- CRISPR/Cas9 can be used to induce targeted DSBs (double-strand breaks) in the genome.
- CRISPR/Cas9-based gene targeting FIG. 1
- targeted KI efficiency has been very low in plants.
- the RFP coding sequences without both promoter and terminator are used to target the endogenous locus (Bam9 or SSR2) (FIG.2). If NLS-RFP KI is successful, the potato protoplasts will express NLS-RFP signals.
- NLS stands for nuclear localization signal, which can be used to concentrate RFP protein to nucleus.
- the Bam9 loci had the highest expression in either CW protoplasts or DE protoplasts, and thus was used in most RFP KI experiments (FIG.3).
- NLS-DPD1, DPD1, and Trex2 three different exonucleases were co-expressed: NLS-DPD1, DPD1, and Trex2.
- DPD1 is an exonuclease from Arabidopsis. As shown in FIG. 4, smaller PCR products flanking PPO2 cleavage site suggest that DNA resection occurs at DSB cleavage site when NLS-DPD1, DPD1 or Trex2 is co-expressed with CRISPR/Cas9.
- FIG.8 shows that NLS-RFP KI efficiency can reach 40% among transfected protoplasts (GFP positive) with Trex2 co-expression.
- NLS-RFP signals are observed among 1% of GFP- positive protoplasts.
- Table 3 RFP donor constructs with various homology arm lengths [0168] When the homology arm length reduces from 400-bp to 50-bp, observed NLS-RFP KI efficiency also decreases (FIG. 11). The presence of a left homology arm resulted in more targeted RFP KI than a right homology arm (data from p6081/p3899, p6082/p3899 and p6083/p3899, FIG. 11). Very few protoplasts with NLS-RFP signals were observed for the two negative controls (p6084/p3899 and p6085/p3899). Page 35 of 61 290326197 v3 Example 3.
- Lig4 and Ku80 are two important genes involved in the canonical nonhomologous end joining (NHEJ) pathway.
- NHEJ canonical nonhomologous end joining
- FIG.12A and 12B silencing constructs comprising the inverted sequence of Lig4 (FIG.12A) and Ku80 (FIG.12B) were co-delivered to protoplasts together with RFP donor template (p6076) and Trex2 (p3899).
- the RFP KI efficiency can reach 40% with Trex2, however when Lig4 or Ku80 are also suppressed, the RFP KI efficiency increased to between 50% and 60%.
- Example 4 The effect of an exonuclease from Arabidopsis on targeted RFP knock-in [0170] Since Trex2 exonuclease is from a mammalian system, exonucleases from Arabidopsis were tested to determine if they too could be used for enhancing targeted RFP knock-in in potato protoplasts.
- FIG. 14 shows that the co-expression of p5762 (AtExonuclease, AT1G02270) can enhance targeted RFP knock-in at Bam9 to 30% compared to only 2% for the control.
- exonucleases can be used to enhance targeted KI in potato protoplasts.
- FIG.15 To determine the which exonucleases are most effective at increasing targeting knock- in frequency, multiple exonucleases were tested (FIG.15). The 3’-5’ exonucleases yielded the highest percent positive RFP cells: Trex2 ⁇ 43% RFP(+) cells and AtDPD1 ⁇ 23% RFP(+) cells. Both the 5’-3’ exonuclease and GFP control yielded less than 5% RFP(+) cells. [0172] The study demonstrates that inclusion of exonucleases can significantly increase the knock-in frequency of donor DNA.
- Embodiment 1 A method of targeted DNA sequence insertion into a host cell genome, comprising: a) providing a host cell; b) selecting a target site nucleic acid sequence in the host cell genome for DNA sequence insertion; c) introducing into the host cell a guide RNA (gRNA) with a guide sequence having complementarity to the target site nucleic acid sequence in the host genome, and a Cas endonuclease that interacts with the gRNA and is capable of creating a double stranded break in the host genome; d) introducing into the host cell an exonuclease; and e) introducing into the host cell a donor DNA sequence template comprising: a DNA sequence of interest and left and right homology arm sequences, wherein the gRNA guide sequence binds to a complementary target site nucle
- Embodiment 2 The method of embodiment 1, wherein the target site nucleic acid sequence is within a coding sequence.
- Embodiment 3. The method of any one of embodiments 1 to 2, wherein the target site nucleic acid sequence is within a gene.
- Embodiment 4. The method of any one of embodiments 1 to 3, wherein the target site nucleic acid sequence is within a non-coding sequence.
- Embodiment 5. The method of any one of embodiments 1 to 4, wherein the target site nucleic acid sequence is within a non-coding regulatory sequence.
- Embodiment 6. The method of any one of embodiments 1 to 5, wherein the target site nucleic acid sequence is within a promoter sequence region.
- Embodiment 7. The method of any one of embodiments 1 to 6, wherein the host cell is a eukaryotic cell.
- Embodiment 8. The method of any one of embodiments 1 to 6, wherein the host cell is a prokaryotic cell. Page 37 of 61 290326197 v3
- Embodiment 9. The method of any one of embodiments 1 to 6, wherein the host cell is a mammalian cell.
- Embodiment 9.1 The method of any one of embodiments 1 to 6, wherein the host cell is a plant cell.
- Embodiment 10. The method of embodiment 9.1, wherein the host cell is a plant protoplast cell.
- Embodiment 12 The method of any one of embodiments 1-6, wherein the host cell is an Angiosperm cell.
- Embodiment 12. The method of any one of embodiments 1-6, wherein the host cell is a dicot cell.
- Embodiment 12.1 The method of any one of embodiments 1-6, wherein the host cell is a monocot cell.
- Embodiment 13 The method of any one of embodiments 1-6, wherein the host cell is from the Solanum genus.
- Embodiment 14 The method of any one of embodiments 1-6, wherein the host cell is a Solanum tuberosum cell.
- Embodiment 15. The method of any one of embodiments 1-6, wherein the host cell is a potato cell.
- Embodiment 16 The method of any one of embodiments 1-6, wherein the host cell is a Solanum lycopersicum cell.
- Embodiment 17. The method of any one of embodiments 1-6, wherein the host cell is a tomato cell.
- Embodiment 18 The method of any one of embodiments 1-17, wherein the gRNA is a single gRNA.
- Embodiment 19 The method of any one of embodiments 1-18, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex).
- Embodiment 20 The method of any one of embodiments 1-6, wherein the host cell is a Solanum lycopersicum cell.
- Embodiment 17 The method of any one of embodiments 1-6, wherein the host cell is a tomato cell.
- Embodiment 18 The method of any one of embodiments 1-17, wherein the gRNA is a single
- Embodiment 21 The method of any one of embodiments 1-20, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro.
- Embodiment 21 The method of any one of embodiments 1-20, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro and the exonuclease is expressed on a transient vector.
- Embodiment 22 The method of any one of embodiments 1-21, wherein the gRNA, Cas, and/or exonuclease are expressed on the same vector.
- Embodiment 23 The method of any one of embodiments 1-22, wherein the gRNA, Cas, and/or exonuclease are expressed on a different vector.
- Embodiment 24 The method of any one of embodiments 1-23, wherein the gRNA, Cas, and/or exonuclease are expressed from a nucleotide sequence integrated into the host cell’s genome.
- Embodiment 25 Embodiment 25.
- Embodiment 26 The method of any one of embodiments 1-25, wherein the gRNA and exonuclease are expressed on a vector and the Cas is expressed from a nucleotide sequence integrated into the host cell’s genome.
- Embodiment 27 The method of any one of embodiments 1-26, wherein the Cas is a Class II endonuclease.
- Embodiment 28 The method of any one of embodiments 1-27, wherein the Cas is a Type II endonuclease.
- Embodiment 29 The method of any one of embodiments 1-28, wherein the Cas is a Cas9.
- Embodiment 30 The method of any one of embodiments 1-29, wherein the Cas is a Type V endonuclease.
- Embodiment 31 The method of any one of embodiments 1-30, wherein the Cas is a Cas12a.
- Embodiment 32 The method of any one of embodiments 1-30, wherein the Cas is a Cas12a.
- Embodiment 32.1 The method of any one of embodiments 1-31, wherein the exonuclease exhibits 5’ to 3’ exonuclease activity at the double stranded break site.
- Embodiment 32.2 The method of any one of embodiments 1-31, wherein the exonuclease is selected from the group consisting of: Trex1, Trex2, and DPD1.
- Embodiment 33 The method of any one of embodiments 1-32.2, wherein the exonuclease is Trex2.
- Embodiment 34 The method of any one of embodiments 1-32.2, wherein the exonuclease is DPD1. Page 39 of 61 290326197 v3
- Embodiment 34.1 The method of any one of embodiments 1-32.2, wherein the exonuclease is POLQ.
- Embodiment 34.2 The method of any one of embodiments 1-31, wherein the exonuclease is a gene selected from Table 1.
- Embodiment 34.3 The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 1 or a sequence at least 75% identical thereto.
- Embodiment 34.4 The method of any one of embodiments 1-32.2, wherein the exonuclease is DPD1. Page 39 of 61 290326197 v3
- Embodiment 34.2 The method of any one of embodiments 1-31, wherein the exonuclease is a gene selected from Table 1.
- Embodiment 34.3
- Embodiment 34.11 The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 8 or a sequence at least 75% identical thereto.
- Embodiment 34.12. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 10 or a sequence at least 75% identical thereto.
- Embodiment 34.13 The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 10 or a sequence at least 75% identical thereto.
- Embodiment 34.14 The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 12 or a sequence at least 75% identical thereto.
- Embodiment 34.15 The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 13 or a sequence at least 75% identical thereto.
- Embodiment 34.17 The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 15 or a sequence at least 75% identical thereto. Page 40 of 61 290326197 v3
- Embodiment 34.18 The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 16 or a sequence at least 75% identical thereto.
- Embodiment 35 Embodiment 35.
- each of the left and right homology arms are at least 100-bp long.
- Embodiment 36 The method of any one of embodiments 1-34, wherein each of the left and right homology arms are at least 200-bp long.
- Embodiment 37 The method of any one of embodiments 1-34, wherein each of the left and right homology arms are at least 300-bp long.
- Embodiment 38 The method of any one of embodiments 1-34, wherein each of the left and right homology arms are at least 400-bp long.
- Embodiment 39 Embodiment 39.
- Embodiment 40 The method of any one of embodiments 1-34, wherein each of the left and right homology arms are at least between 100-bp and 800-bp long.
- Embodiment 40 The method of any one of embodiments 1-34, wherein each of the left and right homology arms are at least between 100-bp and 1000-bp long.
- Embodiment 40.1 The method of any one of embodiments 1-34, wherein the left homology arm is at least 100-bp long.
- Embodiment 40.2. The method of any one of embodiments 1-34, wherein the left homology arm is at least 200-bp long.
- Embodiment 40.4 The method of any one of embodiments 1-34, wherein the left homology arm is at least 400-bp long.
- Embodiment 40.5 The method of any one of embodiments 1-34, wherein the left homology arm is at least between 100-bp and 800-bp long.
- Embodiment 40.6 The method of any one of embodiments 1-34, wherein the left homology arm is at least between 100-bp and 1000-bp long.
- Embodiment 41 The method of any one of embodiments 1-40.6, wherein the DNA sequence of interest is a coding sequence.
- Embodiment 42 The method of any one of embodiments 1-41, wherein the DNA sequence of interest is a gene.
- Embodiment 43 The method of any one of embodiments 1-42, wherein the DNA sequence of interest is a heterologous gene.
- Embodiment 44 The method of any one of embodiments 1-43, wherein the DNA sequence of interest is a non-heterologous gene. Page 41 of 61 290326197 v3
- Embodiment 45 The method of any one of embodiments 1-44, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome.
- Embodiment 46 The method of any one of embodiments 1-44, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome.
- Embodiment 47 The method of any one of embodiments 1-46, wherein the DNA sequence of interest causes a point mutation upon insertion into the host cell’s genome.
- Embodiment 48 The method of any one of embodiments 1-47, wherein the DNA sequence of interest is a non-coding sequence.
- Embodiment 49 The method of any one of embodiments 1-48, wherein the DNA sequence of interest is a non-coding regulatory sequence.
- Embodiment 50 Embodiment 50.
- Embodiment 51 The method of any one of embodiments 1-50, wherein the DNA sequence of interest is inserted into the host cell’s genome via single strand annealing.
- Embodiment 52 The method of any one of embodiments 1-51, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 5% frequency.
- Embodiment 53 The method of any one of embodiments 1-52, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 10% frequency.
- Embodiment 54 Embodiment 54.
- Embodiment 55 The method of any one of embodiments 1-54, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 30% frequency.
- Embodiment 56 The method of any one of embodiments 1-55, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 40% frequency.
- Embodiment 57 The method of any one of embodiments 1-56, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 50% frequency.
- Embodiment 58 Embodiment 58.
- Embodiment 59 A method of targeted DNA sequence insertion into a host cell genome, comprising: Page 42 of 61 290326197 v3 a) determining a target site nucleic acid sequence in the host cell genome for DNA sequence insertion; b) providing a host cell; c) introducing into the host cell a guide RNA (gRNA) with a guide sequence having complementarity to the target site nucleic acid sequence in the host genome and a Cas endonuclease that interacts with the gRNA and is capable of creating a double stranded break in the host genome; d) introducing into the host cell an exonuclease; e) introducing into the host cell a donor DNA sequence template comprising: a DNA sequence of interest and left and right homology arm sequences, and f) introducing into the host cell a sile
- Embodiment 60 The method of embodiment 59, wherein the target site nucleic acid sequence is within a coding sequence.
- Embodiment 61 The method of any one of embodiments 59-60, wherein the target site nucleic acid sequence is within a gene.
- Embodiment 62 The method of any one of embodiments 59-61, wherein the target site nucleic acid sequence is within a non-coding sequence.
- Embodiment 63 The method of any one of embodiments 59-62, wherein the target site nucleic acid sequence is within a non-coding regulatory sequence.
- Embodiment 64 Embodiment 64.
- Embodiment 65 The method of any one of embodiments 59-64, wherein the host cell is a eukaryotic cell.
- Embodiment 66 The method of any one of embodiments 59-64, wherein the host cell is a prokaryotic cell.
- Embodiment 67 The method of any one of embodiments 59-64, wherein the host cell is a mammalian cell. Page 43 of 61 290326197 v3
- Embodiment 68 The method of any one of embodiments 59-64, wherein the host cell is a plant cell.
- Embodiment 69 The method of embodiment 68, wherein the host cell is a plant protoplast cell.
- Embodiment 70 The method of any one of embodiments 59-64, wherein the host cell is an Angiosperm cell.
- Embodiment 71 The method of any one of embodiments 59-64, wherein the host cell is a dicot cell.
- Embodiment 72 The method of any one of embodiments 59-64, wherein the host cell is a monocot cell.
- Embodiment 73 The method of any one of embodiments 59-64, wherein the host cell is from the Solanum genus.
- Embodiment 74 The method of any one of embodiments 59-64, wherein the host cell is from the Solanum genus.
- Embodiment 75 The method of any one of embodiments 59-64, wherein the host cell is a potato cell.
- Embodiment 76 The method of any one of embodiments 59-64, wherein the host cell is a Solanum lycopersicum cell.
- Embodiment 77 The method of any one of embodiments 59-64, wherein the host cell is a tomato cell.
- Embodiment 78 The method of any one of embodiments 59-77, wherein the gRNA is a single gRNA. [0280] Embodiment 79.
- Embodiment 80 The method of any one of embodiments 59-79, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro.
- Embodiment 81 Embodiment 81.
- Embodiment 82 The method of any one of embodiments 59-80, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro and the exonuclease is expressed on a transient vector.
- Embodiment 82 The method of any one of embodiments 59-81, wherein the gRNA, Cas, and/or exonuclease are expressed on the same vector. Page 44 of 61 290326197 v3 [0284] Embodiment 83.
- Embodiment 85 The method of any one of embodiments 59-82, wherein the gRNA, Cas, and/or exonuclease are expressed on a different vector.
- Embodiment 84 The method of any one of embodiments 59-83, wherein the gRNA, Cas, and/or exonuclease are expressed from a nucleotide sequence integrated into the host cell’s genome.
- Embodiment 85 Embodiment 85.
- Embodiment 86 The method of any one of embodiments 59-84, wherein at least one of the gRNA, Cas, and/or exonuclease are expressed on a vector, and wherein at least one of the gRNA, Cas, and/or exonuclease are expressed from a nucleotide sequence integrated into the host cell’s genome.
- Embodiment 86 The method of any one of embodiments 59-85, wherein the gRNA and exonuclease are expressed on a vector and the Cas is expressed from a nucleotide sequence integrated into the host cell’s genome.
- Embodiment 87 Embodiment 87.
- Embodiment 91 The method of any one of embodiments 59-86, wherein the Cas is a Class II endonuclease.
- Embodiment 88 The method of any one of embodiments 59-87, wherein the Cas is a Type II endonuclease.
- Embodiment 89 The method of any one of embodiments 59-88, wherein the Cas is a Cas9.
- Embodiment 90 The method of any one of embodiments 59-89, wherein the Cas is a Type V endonuclease.
- Embodiment 91 The method of any one of embodiments 59-90, wherein the Cas is a Cas12a.
- Embodiment 92 The method of any one of embodiments 59-91, wherein the exonuclease exhibits 3’ to 5’ exonuclease activity at the double stranded break site.
- Embodiment 93 The method of any one of embodiments 59-91, wherein the exonuclease exhibits 5’ to 3’ exonuclease activity at the double stranded break site.
- Embodiment 94 The method of any one of embodiments 59-91, wherein the exonuclease is selected from the group consisting of: Trex1, Trex2, and DPD1.
- Embodiment 95 Embodiment 95.
- Embodiment 96.1 The method of any one of embodiments 59-94, wherein the exonuclease is POLQ. Page 45 of 61 290326197 v3
- Embodiment 96.2 The method of any one of embodiments 59-93, wherein the exonuclease is a gene selected from Table 1. [0300] Embodiment 96.3.
- Embodiment 96.4 The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 2 or a sequence at least 75% identical thereto.
- Embodiment 96.5 The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 3 or a sequence at least 75% identical thereto.
- Embodiment 96.7 The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 5 or a sequence at least 75% identical thereto.
- Embodiment 96.8 The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 6 or a sequence at least 75% identical thereto.
- Embodiment 96.10 The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 8 or a sequence at least 75% identical thereto.
- Embodiment 96.11 The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 9 or a sequence at least 75% identical thereto.
- Embodiment 96.13 The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 11 or a sequence at least 75% identical thereto.
- Embodiment 96.14 The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 12 or a sequence at least 75% identical thereto.
- Embodiment 96.15 Embodiment 96.15.
- Embodiment 96.16 The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 14 or a sequence at least 75% identical thereto.
- Embodiment 96.17 The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 15 or a sequence at least 75% identical thereto.
- Embodiment 96.18 Embodiment 96.18.
- Embodiment 100 The method of any one of embodiments 59-96, wherein each of the left and right homology arms are at least 400-bp long.
- Embodiment 101 The method of any one of embodiments 59-96, wherein each of the left and right homology arms are at least between 100-bp and 800-bp long.
- Embodiment 102 The method of any one of embodiments 59-96, wherein each of the left and right homology arms are at least between 100-bp and 1000-bp long.
- Embodiment 103 The method of any one of embodiments 59-96, wherein the left homology arm is at least 100-bp long.
- Embodiment 104 The method of any one of embodiments 59-96, wherein the left homology arm is at least 100-bp long.
- Embodiment 105 The method of any one of embodiments 59-96, wherein the left homology arm is at least 300-bp long.
- Embodiment 106 The method of any one of embodiments 59-96, wherein the left homology arm is at least 400-bp long.
- Embodiment 107 The method of any one of embodiments 59-96, wherein the left homology arm is at least between 100-bp and 800-bp long.
- Embodiment 108 Embodiment 108.
- Embodiment 109 The method of any one of embodiments 59-108, wherein the DNA sequence of interest is a coding sequence.
- Embodiment 110 The method of any one of embodiments 59-109, wherein the DNA sequence of interest is a gene.
- Embodiment 111 The method of any one of embodiments 59-110, wherein the DNA sequence of interest is a heterologous gene.
- Embodiment 112. The method of any one of embodiments 59-111, wherein the DNA sequence of interest is a non-heterologous gene.
- Embodiment 113 The method of any one of embodiments 59-112, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome.
- Embodiment 114 The method of any one of embodiments 59-113, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome, which upon insertion causes a single base pair edit.
- Embodiment 115 Embodiment 115.
- Embodiment 116 The method of any one of embodiments 59-114, wherein the DNA sequence of interest causes a point mutation upon insertion into the host cell’s genome.
- Embodiment 116 The method of any one of embodiments 59-115, wherein the DNA sequence of interest is a non-coding sequence.
- Embodiment 117 The method of any one of embodiments 59-116, wherein the DNA sequence of interest is a non-coding regulatory sequence.
- Embodiment 118 The method of any one of embodiments 59-117, wherein the DNA sequence of interest is a promoter.
- Embodiment 119 Embodiment 119.
- Embodiment 119.1. The method of any one of embodiments 59-119, wherein the nonhomologous end joining pathway gene is a gene selected from Table 2.
- Embodiment 119.2. The method of any one of embodiments 59-119, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 17 or a sequence at least 75% identical thereto.
- Embodiment 122 The method of any one of embodiments 59-121, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 20% frequency.
- Embodiment 123 The method of any one of embodiments 59-122, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 30% frequency.
- Embodiment 124 The method of any one of embodiments 59-123, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 40% frequency.
- Embodiment 125 The method of any one of embodiments 59-124, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 50% frequency.
- Embodiment 126 The method of any one of embodiments 59-125, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 60% frequency. INCORPORATION BY REFERENCE [0353] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes.
- Patent Citations 1 US 2019/0093104 2) US 10,745,690 3) EP2633040B1 Non-Patent Citations 1) Rogowsky P. CRISPR-Cas Technology in Plant Science. Potato Research.2017.60:353-360. 2) Cejka P. DNA End Resection: Nucleases Team Up with the Right Partners to Initiate Homologous Recombination. J Biol Chem.2015 Sep 18;290(38):22931-8.
Abstract
The present disclosure relates to methods of increasing the efficiency of targeted DNA sequence insertion into host cell genomes using exonucleases and/or silencing of nonhomologous end joining gene(s).
Description
ENHANCED TARGETED KNOCK-IN FREQUENCY IN HOST GENOMES THROUGH CRISPR EXONUCLEASE PROCESSING CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63/403,200, filed September 1, 2022, the entire contents of which is incorporated herein by reference. FIELD [0002] The field of the disclosure is directed to increasing the efficiency of targeted DNA sequence insertion into host cell genomes. In certain aspects, the disclosure provides for increased knock-in frequency of a DNA sequence of interest into a plant cell genome. To these ends, the disclosure provides compositions and methods to introduce a Cas endonuclease, a gRNA, an exonuclease, and donor DNA to a host cell, e.g. a plant cell. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0003] The contents of the electronic sequence listing (JRSI_086_01WO_SeqList_ST26.xml; Size: 157,386 bytes; and Date of Creation: September 1, 2023) are herein incorporated by reference in its entirety. BACKGROUND [0004] Targeted gene disruption has wide applicability for research, therapeutic, agricultural, and industrial uses. One strategy for producing targeted gene disruption is through the generation of double-strand DNA breaks (DSBs) caused by site-specific endonucleases. Endonucleases are most often used for targeted gene disruption in organisms that have traditionally been refractive to more conventional gene targeting methods, such as algae, plants, and large animal models, including humans. [0005] Single-strand annealing (SSA) is a DSB repair pathway that uses homologous repeats to bridge DSB ends. SSA involving repeats that flank a single DSB causes a deletion rearrangement between the repeats, and hence is relatively mutagenic. Nevertheless, this pathway is conserved, in that SSA events have been found in several organisms. [0006] The factors and pathways that influence DSB repair may be exploited for the purpose of developing experimental and therapeutic systems in a wide range of species. These systems may be used for various functions, such as to study gene function in plants and animals, to engineer transgenic cells and organisms; however, these pathways are prone to cause different Page 1 of 61 290326197 v3
mutagenic outcomes. Moreover, low frequencies of certain targeted gene disruptions, such as knock-in of donor DNA, make the exploitation of DSB repair mechanisms nearly impossible. Consequently, there is a gap in the art for methods and compositions that enhance the knock- in frequency of donor DNA. SUMMARY [0007] The current disclosure solves the aforementioned problem by providing methods and compositions to enhance knock-in frequency of donor DNA in host cell genomes. In some aspects, the disclosure relates to the discovery that certain exonucleases enhance Cas endonuclease-mediated knock-in frequency. Knocking down expression of genes involved in the nonhomologous end joining pathway further enhanced Cas mediated knock-in frequency. [0008] In one embodiment, the disclosure provides a method of targeted DNA sequence insertion into a plant genome, comprising: providing host cell; selecting a target site nucleic acid sequence in the host cell genome for DNA sequence insertion; introducing into the host cell a guide RNA (gRNA) with a guide sequence having complementarity to the target site nucleic acid sequence in the host genome and a Cas endonuclease that interacts with the gRNA and is capable of creating a double stranded break in the host genome; introducing into the host cell an exonuclease; and introducing into the host cell a donor DNA sequence template comprising: a DNA sequence of interest and left and right homology arm sequences, wherein the gRNA guide sequence binds to the complementary target site nucleic acid sequence in the host genome and the Cas endonuclease creates a double stranded break in the host genome; and wherein the DNA sequence of interest is inserted into the host genome via a homology dependent repair mechanism. [0009] In another embodiment, the disclosure provides a method of targeted DNA sequence insertion into a plant genome, comprising: providing a host cell; selecting a target site nucleic acid sequence in the host cell genome for DNA sequence insertion; introducing into the host cell a guide RNA (gRNA) with a guide sequence having complementarity to the target site nucleic acid sequence in the host genome and a Cas endonuclease that interacts with the gRNA and is capable of creating a double stranded break in the host genome; introducing into the host cell an exonuclease; and introducing into the host cell a donor DNA sequence template comprising: a DNA sequence of interest and left and right homology arm sequences; introducing into the host cell a silencing construct, wherein the silencing construct knocks down expression of a nonhomologous end joining pathway gene, wherein the gRNA guide sequence binds to the complementary target site nucleic acid sequence in the host genome and Page 2 of 61 290326197 v3
the Cas endonuclease creates a double stranded break in the host genome; and wherein the DNA sequence of interest is inserted into the host genome via a homology dependent repair mechanism. [0010] In some embodiments, the exonuclease exhibits 3’ to 5’ exonuclease activity at the double stranded break site. In some embodiments, the host cell is a plant cell. In some embodiments, the DNA sequence of interest is inserted into the host cell’s genome with at least a 10% frequency. BRIEF DESCRIPTION OF THE FIGURES [0011] FIG. 1 is a schematic adapted from Rogowsky that describes several applications for CRISPR/Cas9-induced gene targeting. [0012] FIG.2 is a schematic that describes a knock-in (KI) strategy of donor DNA into either the SSR2 or BAM9 locus in potato protoplasts. [0013] FIG.3 shows expression of 6 different loci in either CW protoplasts or DE protoplasts. [0014] FIG.4 is an image of an agarose gel from a PCR amplification assay for PP02g2 with either NLS-DPD1, DPD1, or Trex2. [0015] FIGs. 5A and 5B shows fluorescent images that show Trex2 significantly increases targeted RFP KI at Bam9 (increased NLS-RFP signal). [0016] FIG. 6 shows fluorescent images that show Trex2 increases targeted RFP KI with various donor DNA templates. [0017] FIG.7 shows fluorescent images of either Trex2 with RFP or Trex2 with RFP that has a frame shift mutation. [0018] FIG. 8 is a bar graph that shows that Trex2 increases targeted RFP KI in potato protoplasts. [0019] FIG. 9 is a bar graph that shows the percent of RFP(+) FDA cells with various constructs. [0020] FIG.10A is a schematic that shows the PCR screening strategy to determine 5’ and 3’ integration of RFP at the Bam9 locus. [0021] FIG.10B is an image of an agarose gel from PCR amplification assay as shown in FIG. 10A with or without Trex2 transient expression. [0022] FIG. 11 is a bar graph that shows the effect of different homology arm lengths on targeted RFP KI at Bam9 with several negative controls. [0023] FIG.12A is a schematic of the plasmid used to silence Lig4. Page 3 of 61 290326197 v3
[0024] FIG.12B is a schematic of the plasmid used to silence Ku80. [0025] FIG. 13 is a bar graph showing that silencing NHEJ pathway can increase Trex2- mediated RFP KI. [0026] FIG. 14 is a bar graph showing the effect of an exonuclease from Arabidopsis on targeted RFP KI [0027] FIG.15 is a graph that shows the average percent of RFP(+) cells after treatment with various exonucleases. DETAILED DESCRIPTION Definitions [0028] Unless defined otherwise herein, all technical and scientific terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art to which the present disclosure belongs. [0029] The term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In various embodiments, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. [0030] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. [0031] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%. The terms “a” and “an” refers to “one or more” of the enumerated components unless otherwise indicated. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and/or” should be understood to mean either one, or both of the alternatives. The terms “include” and “comprise” are used synonymously. Page 4 of 61 290326197 v3
[0032] Complementary sequences are based upon complementarity. The term “complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. The term “substantially complementary” refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. [0033] The term “hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. [0034] The term “genomic locus” or “locus” (plural loci) is the specific location of a gene or DNA sequence on a chromosome. A “gene” refers to stretches of DNA or RNA that encode a polypeptide or an RNA chain that has functional role to play in an organism and hence is the molecular unit of heredity in living organisms. For the purpose of this disclosure, it may be considered that genes include regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. [0035] The term “gene editing” and its grammatical equivalents can refer to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a Page 5 of 61 290326197 v3
genome. Gene editing can be performed using a nuclease (e.g., a natural-existing nuclease or an artificially engineered nuclease). [0036] The term “knock-in” refers to a gene editing technique that introduces exogenous nucleic acid into the genome of a host cell, e.g. a plant cell. [0037] The term “function” and its grammatical equivalents can refer to the capability of operating, having, or serving an intended purpose. Functional can comprise any percent from baseline to 100% of normal function. For example, functional can comprise or comprise about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and/or 100% of normal function. In some cases, the term functional can mean over or over about 100% of normal function, for example, 125, 150, 175, 200, 250, 300% and/or above normal function. [0038] The term “providing” refers to making accessible. For example, providing a host cell means the host cell is accessible. In some embodiments, providing a host cell means the cell is accessible for introducing a nucleic acid to the host cell. [0039] The term “introducing” refers to transfection or delivery of an expression vector (e.g., plasmids). Transfection or delivery of the expression vectors may be accomplished by any suitable method known in the art including, but not limited to, viral transfection or transduction, microinjection, electroporation, sonoporation, optical transfection, impalefection, hydrodynamic delivery, nucleofection, lipofection, dendrimeric transfection, magnetofection, gene gun transfection, nanoparticle-based transfection, calcium phosphate transfection, viral transfection, and cell squeezing. [0040] The term “operably linked” refers to an arrangement of elements where the components so described are configured so as to perform their usual function. Thus, promoter and/or enhancer sequences operably linked to a coding sequence are capable of effecting the transcription, and in some cases, the translation, of a coding sequence. The promoter and/or enhancer sequences need not be contiguous with the coding sequence so long as they function to direct the expression of the coding sequence. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence. In fact, such sequences need not reside on the same contiguous DNA molecule (i.e. chromosome) and may still have interactions resulting in altered regulation. [0041] The term “promoter” or “promoter sequence” refers to a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a polynucleotide or polypeptide coding sequence such as messenger RNA, ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind of RNA transcribed by any class of any RNA Page 6 of 61 290326197 v3
polymerase I, II or III. Promoters may be constitutive or inducible and, in some embodiments — particularly many embodiments in which selection is employed — the transcription of at least one component of the nucleic acid-guided nuclease editing system is under the control of an inducible promoter. [0042] Sequence identity. “Sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the number of residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. An example of a local alignment algorithm utilized for the comparison of sequences is the NCBI Basic Local Alignment Search Tool (BLAST®) (Altschul et al. 1990 J. Mol. Biol. 215: 403-10), which is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed on the internet via the National Library of Medicine (NLM)'s world-wide-web URL. A description of how to determine sequence identity using this program is available at the NLM's website on BLAST tutorial. Another example of a mathematical algorithm utilized for the global comparison of sequences is the Clustal W and Clustal X (Larkin et al.2007 Bioinformatics, 23, 2947-294, Clustal W and Clustal X version 2.0) as well as Clustal omega. Unless otherwise stated, references to sequence identity used herein refer to the NCBI Basic Local Alignment Search Tool (BLAST®). Page 7 of 61 290326197 v3
[0043] The term “host cell” refers to a cell that is targeted with the Cas endonuclease, gRNA, exonuclease, and donor DNA sequence insertion. A host cell may comprise a prokaryote or a eukaryote. [0044] The term “prokaryote” refers to non-eukaryotic organisms belonging to the Eubacteria (e.g., Escherichia coli, Thermus thermophilus, etc.) and Archaea (e.g., Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium spp., A. fulgidus, P. firiosus, P. horikoshii, A. pernix, etc.) phylogenetic domains. [0045] The term “eukaryote” refers to organisms belonging to the phylogenetic domain Eucarya such as animals (e.g., mammals, insects, reptiles, birds, etc.), ciliates, plants, fungi (e.g., yeasts, etc.), flagellates, microsporidia, protists, etc. [0046] The term “plant” includes the class of higher and lower plants including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous. CRISPR [0047] Genome editing by CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is based on a natural immune process used by bacteria to defend themselves against invading viruses. Indeed, in bacteria the invading viral DNA will be cut through use of a guide RNA (gRNA), or piece of RNA, and a CRISPR-associated protein (Cas). The last step of the bacterial immune process, when the gRNA is combined with Cas and cleaves the target DNA, has been adopted for genome editing in laboratories. [0048] There are at least three main CRISPR system types (Type I, II, and III) and at least 10 distinct subtypes (Makarova, K.S., et.al., Nat Rev Microbiol.2011 May 9; 9(6):467-477). Type I and III systems use Cas protein complexes and short guide polynucleotide sequences to target selected DNA regions. Type II systems rely on a single protein (e.g. Cas9) and the targeting guide polynucleotide, where a portion of the 5’ end of a guide sequence is complementary to a target nucleic acid. For more information on the CRISPR gene editing compositions and methods of the present disclosure, see US Patent Nos. 8,697,359; 8,889,418; 8,771,945; and 8,871,445, each of which is hereby incorporated in its entirety for all purposes. [0049] CRISPR genome editing requires two components, a gRNA and a Cas enzyme. These components associate to form a ribonucleoprotein (RNP) complex, where after the gRNA can base pair with a complementary protospacer sequence (i.e. the target genomic sequence of about 20 bases in length) under the condition that a particular adjacent sequence, called a protospacer-adjacent motif (PAM), is present in the genome. The PAM is only a few bases Page 8 of 61 290326197 v3
long, and its sequence depends on the type of Cas enzyme used. Once the gRNA binds to the target DNA (protospacer), the Cas enzyme recognizes this complex and makes a precise cut at the target site, resulting in a double strand break (DSB). [0050] Each Cas enzyme is directed by the gRNA to a user-specified cut site in the genome. Like Cas9 nucleases, Cas12a family members contain a RuvC-like endonuclease domain, but lack the second HNH endonuclease domain of Cas9. Cas12a cleaves DNA in a staggered pattern in contrast to Cas9 which produces a blunt-end. Moreover, for cleavage Cas12a requires only one gRNA rather than the two tracrRNA and crRNA needed by Cas9. For Cas9 as well as Cas12a, the target sequence of the gRNAs must be next to a PAM sequence. In the case of Cas9, the PAM sequence corresponds to NGG, where N is any base. The gRNA will recognize and bind to 20 nucleotides on the DNA strand opposite from the NGG PAM site. For Cas12a, the PAM sequence is TTTV, where V can represent A, C, or G. Using Alt-R Cas12a Ultra from Integrated DNA Technologies, a TTTT PAM sequence may also work. The “V” of the TTTV is immediately adjacent to the base at the 5’ end of the non-targeted strand side of the protospacer element. The guide RNA for Cas12a is relatively short and is approximately 40 to 44 bases long. [0051] The damage caused by the DSB will be repaired in eukaryotic cells, primarily by two pathways: Non-Homologous End-Joining (NHEJ) and Homology Directed Repair (HDR). The HDR mechanism requires the presence of a donor DNA template containing regions of homology to both sites of the DNA break. This donor DNA can carry specific mutations and has to be delivered simultaneously with a preassembled Cas RNP complex composed of Cas9 or Cas12a and synthetically produced gRNAs. Endonucleases [0052] A Cas endonuclease relates to a Cas protein encoded by a Cas gene, wherein said Cas protein is capable of introducing a double strand break into a DNA target sequence. The Cas endonuclease is guided by the guide polynucleotide to recognize and optionally introduce a double strand break at a specific target site into the genome of a cell. The term “guide polynucleotide/Cas endonuclease system” includes a complex of a Cas endonuclease and a guide polynucleotide that is capable of introducing a double strand break into a DNA target sequence. The Cas endonuclease unwinds the DNA duplex in close proximity of the genomic target site and cleaves both DNA strands upon recognition of a target sequence by a guide RNA, but only if the correct protospacer-adjacent motif (PAM) is approximately oriented at the 3′ end of the target sequence. Page 9 of 61 290326197 v3
[0053] In some embodiments, the Cas endonuclease gene is plant, maize or soybean optimized Cas9 endonuclease. In another embodiment, the Cas endonuclease gene is operably linked to a SV40 nuclear targeting signal upstream of the Cas codon region and a bipartite VirD2 nuclear localization signal (Tinland et al. (1992) Proc. Natl. Acad. Sci. USA 89:7442-6) downstream of the Cas codon region. [0054] The terms “functional fragment”, “fragment that is functionally equivalent” and “functionally equivalent fragment” are used interchangeably herein. These terms refer to a portion or subsequence of the Cas endonuclease sequence of the present disclosure in which the ability to create a double-strand break is retained. [0055] The terms “functional variant”, “variant that is functionally equivalent” and “functionally equivalent variant” are used interchangeably herein. These terms refer to a variant of the Cas endonuclease of the present disclosure in which the ability create a double-strand break is retained. Fragments and variants can be obtained via methods such as site-directed mutagenesis and synthetic construction. [0056] In some embodiments, the Cas endonuclease gene is a plant codon optimized streptococcus pyogenes Cas9 gene that can recognize any genomic sequence of the form N(12- 30)NGG can in principle be targeted. [0057] In some embodiments, the Cas endonuclease is introduced directly into a cell by any method known in the art, for example, but not limited to transient introduction methods, transfection and/or topical application. [0058] Cas endonucleases for use in the methods described herein include Class 2 Type V CRISPR/Cas based gene editing systems. CRISPR/Cas systems can be used in a wide variety of organisms to add, disrupt, or change the sequence of specific genes. Naturally-occurring CRISPR systems are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. [0059] CRISPR systems are based on two elements. The first element is an endonuclease (e.g., Cas9) that has a binding site for the second element, the guide polynucleotide (e.g., guide RNA, or gRNA). The gRNA complexed with the Cas endonuclease forms a CRISPR Cas ribonucleoprotein complex, or RNP-complex. The guide gRNA directs the endonuclease to double stranded DNA templates based on sequence homology. The endonuclease then cleaves that DNA template. By delivering the endonuclease and appropriate guide polynucleotides (e.g., guide RNAs) into a cell, the organism’s genome is cut at a desired location. Following Page 10 of 61 290326197 v3
cleavage of a targeted genomic sequence by an endonuclease/gRNA complex, one of the two alternative DNA repair mechanisms described supra can restore chromosomal integrity: 1) non- homologous end joining (NHEJ) which generates insertions and/or deletions of a few base- pairs (bp) of DNA at the gRNA cut site, or 2) homology-directed repair (HDR) which can correct the lesion via an additional “bridging” DNA template that spans the gRNA cut site. CRISPR systems are classified by class and by type. [0060] Naturally occurring CRISPR systems have been modified for use in gene editing (silencing, knock out, knock in, enhancing or changing specific genes) in eukaryotes (Wiedenheft et al. (2012) Nature 482: 331-8). This is accomplished by introducing into the eukaryotic cell a one or more specifically designed guide nucleic acids (gNAs), typically guide RNAs (gRNAs), and an appropriate endonuclease which forms a ribonucleoprotein complex with the gNA. The gNA guides the gNA-endonuclease protein complex to a target genomic location, and the endonuclease introduces a double strand break at the target genomic location (locus). As outlined above, this double strand break can be repaired by cellular mechanisms, such as NHEJ or homology directed repair. [0061] Thus, CRISPR systems can be used to edit a target locus by adding or deleting one or more base pairs, introducing a premature stop codon, or introducing a frame-shift mutation which decreases expression of a target, in part or completely. Alternatively, the CRISPR system can alternatively be used like RNA interference, turning off a target gene in a reversible fashion. In a mammalian cell, for example, the RNA can guide the endonuclease to a target gene promoter, sterically blocking RNA polymerases. [0062] In some embodiments, the gene editing system comprises a CRISPR system. In some embodiments, the CRISPR system comprises a Class 2 CRISPR system. Class 2 systems currently represent a single protein that is categorized into three distinct types (types II, V and VI). Any class 2 CRISPR system suitable for gene editing, for example a type II, a type V or a type VI system, is envisaged as within the scope of the instant disclosure. Exemplary Class 2 type II CRISPR systems include Cas9, Csn2 and Cas4. Exemplary Class 2, type V CRISPR systems include, Cas12, Cas12a (Cpf1, MAD7), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i and Cas12k (C2c5). Exemplary Class 2 Type VI systems include Cas13, Cas13a (C2c2) Cas13b, Cas13c and Cas13d. [0063] The endonuclease protein (e.g., nucleic acid-directed nuclease) may be derived from any bacterial or archaeal Cas protein. Any suitable CRISPR system is contemplated as within the scope of the instant disclosure. In some embodiments, the endonuclease protein comprises one or more of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Page 11 of 61 290326197 v3
Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. In some embodiments, the endonuclease protein is a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, Cas12, modified versions thereof, or combinations or complexes of these. [0064] In certain embodiments, the compositions may be transfected into one or more cells using any suitable expression vector which delivers the composition to the cell or cells. Any suitable expression vectors may be used in accordance with the embodiments described herein including, but not limited to, plasmids and recombinant viral vectors. Suitable viruses that may be used to design a recombinant viral vector for use in the embodiments described herein include, but are not limited to, adeno-associated virus (AAV), adenovirus, lentivirus, baculovirus, Bean yellow dwarf virus, Wheat dwarf virus (WDV), Cabbage leaf curl virus, Wheat streak mosaic virus (WSMV), Barley stripe mosaic virus (BSMV), Tobacco rattle virus (TRV) (family Virgaviridae), geminiviruses (family Geminiviridae), or Begomoviruses (family Geminiviridae). In some embodiments, a vector suitable for Agrobacterium mediated transformation may be used in accordance with the embodiments described herein. In some embodiments, a vector suitable for particle bombardment may be used in accordance with the embodiments described herein. Donor Nucleic Acid [0065] The terms “donor molecule”, “donor template”, or “donor template molecule” (collectively a “donor template”), which may be a recombinant polynucleotide, DNA or RNA donor template, refer to a nucleic acid molecule having a nucleic acid template or insertion sequence for site-directed, targeted insertion or recombination into the genome of a plant cell via repair of a nick or double-stranded DNA break in the genome of a plant cell. [0066] For example, a “donor template” may be used for site-directed integration of a transgene or suppression construct, or as a template to introduce a mutation, such as an insertion, deletion, substitution, etc., into a target site within the genome of a plant. A targeted genome editing technique provided herein may comprise the use of one or more, two or more, three or more, four or more, or five or more donor molecules or templates. A “donor template” may be a single-stranded or double-stranded DNA or RNA molecule or plasmid. An “insertion sequence” of a donor template is a sequence designed for targeted insertion into the genome of a plant cell, which may be of any suitable length. For example, the insertion sequence of a donor template may be about 2 to about 50,000, about 2 to about 10,000, about 2 to about 5000, Page 12 of 61 290326197 v3
about 2 to about 1000, about 2 to about 500, about 2 to about 250, about 2 to about 100, about 2 to about 50, about 2 to about 30, about 15 to about 50, about 15 to about 100, about 15 to about 500, about 15 to about 1000, about 15 to about 5000, about 18 to about 30, about 18 to about 26, about 20 to about 26, about 20 to about 50, about 20 to about 100, about 20 to about 250, about 20 to about 500, about 20 to about 1000, about 20 to about 5000, about 20 to about 10,000, about 50 to about 250, about 50 to about 500, about 50 to about 1000, about 50 to about 5000, about 50 to about 10,000, about 100 to about 250, about 100 to about 500, about 100 to about 1000, about 100 to about 5000, about 100 to about 10,000, about 250 to about 500, about 250 to about 1000, about 250 to about 5000, or about 250 to about 10,000 nucleotides or base pairs in length. [0067] A donor template may also have at least one homology sequence or homology arm, such as two homology arms, to direct the integration of a mutation or insertion sequence into a target site within the genome of a plant via homologous recombination, wherein the homology sequence or homology arm(s) are identical or complementary, or have a percent identity or percent complementarity, to a sequence at or near the target site within the genome of the plant. When a donor template comprises homology arm(s) and an insertion sequence, the homology arm(s) will flank or surround the insertion sequence of the donor template. The homology arm may be about 2 to about 50,000, about 2 to about 10,000, about 2 to about 5000, about 2 to about 1000, about 2 to about 500, about 2 to about 250, about 2 to about 100, about 2 to about 50, about 2 to about 30, about 15 to about 50, about 15 to about 100, about 15 to about 500, about 15 to about 1000, about 15 to about 5000, about 18 to about 30, about 18 to about 26, about 20 to about 26, about 20 to about 50, about 20 to about 100, about 20 to about 250, about 20 to about 500, about 20 to about 1000, about 20 to about 5000, about 20 to about 10,000, about 50 to about 250, about 50 to about 500, about 50 to about 1000, about 50 to about 5000, about 50 to about 10,000, about 100 to about 250, about 100 to about 500, about 100 to about 1000, about 100 to about 5000, about 100 to about 10,000, about 250 to about 500, about 250 to about 1000, about 250 to about 5000, or about 250 to about 10,000 nucleotides or base pairs (bp) in length. [0068] In some embodiments, the donor template only has one homology arm. In some embodiments, the donor template has a left homology arm of at least 100 bp, at least 200 bp, at least 300 bp, or at least 400 bp long. In some embodiments, the donor template has a left homology arm of at least between 100 bp and 800 bp long. In some embodiments, the donor template has a left homology arm of at least between 100 bp and 1000 bp long. Page 13 of 61 290326197 v3
[0069] An insertion sequence of a donor template may comprise one or more genes or sequences that each encode a transcribed non-coding RNA or mRNA sequence and/or a translated protein sequence. A transcribed sequence or gene of a donor template may encode a protein or a non-coding RNA molecule. An insertion sequence of a donor template may comprise a polynucleotide sequence that does not comprise a functional gene or an entire gene sequence (e.g., the donor template may simply comprise regulatory sequences, such as a promoter sequence, or only a portion of a gene or coding sequence), or may not contain any identifiable gene expression elements or any actively transcribed gene sequence. Further, the donor template may be linear or circular, and may be single-stranded or double-stranded. [0070] A donor template may be delivered to a cell as an RNA molecule expressed from a transgene. A donor template may also be delivered to the cell as a naked nucleic acid (e.g., via particle bombardment), as a complex with one or more delivery agents (e.g., liposomes, proteins, poloxamers, T-strand encapsulated with proteins, etc.), or contained in a bacterial or viral delivery vehicle, such as, for example, Agrobacterium tumefaciens or a geminivirus, respectively. An insertion sequence of a donor template provided herein may comprise a transcribable DNA sequence that may be transcribed into an RNA molecule, which may be non-coding and may or may not be operably linked to a promoter and/or other regulatory sequence. [0071] According to some embodiments, a donor template may not comprise an insertion sequence, and instead comprise one or more homology sequences that include(s) one or more mutations, such as an insertion, deletion, substitution, etc., relative to the genomic sequence at a target site within the genome of a plant, such as at or near a gene within the genome of a plant. [0072] Alternatively, a donor template may comprise an insertion sequence that does not comprise a coding or transcribable DNA sequence, wherein the insertion sequence is used to introduce one or more mutations into a target site within the genome of a plant, such as at or near a gene within the genome of a plant. [0073] A donor template provided herein may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten genes or transcribable DNA sequences. Alternatively, a donor template may comprise no genes. [0074] Without being limiting, a gene or transcribable DNA sequence of a donor template may include, for example, an insecticidal resistance gene, an herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a yield enhancing gene, a nutritional quality Page 14 of 61 290326197 v3
gene, a DNA binding gene, a selectable marker gene, an RNAi or suppression construct, a site- specific genome modification enzyme gene, a single guide RNA of a CRISPR/Cas9 system, a geminivirus-based expression cassette, or a plant viral expression vector system. According to other embodiments, an insertion sequence of a donor template may comprise a protein encoding sequence or a transcribable DNA sequence that encodes a non-coding RNA molecule, which may target an endogenous gene for suppression. A donor template may comprise a promoter, such as a constitutive promoter, a tissue-specific or tissue-preferred promoter, a developmental stage promoter, or an inducible promoter. A donor template may comprise a leader, enhancer, promoter, transcriptional start site, 5'-UTR, one or more exon(s), one or more intron(s), transcriptional termination site, region or sequence, 3'-UTR, and/or polyadenylation signal. The leader, enhancer, and/or promoter may be operably linked to a gene or transcribable DNA sequence encoding a non-coding RNA, a guide RNA, an mRNA and/or protein. [0075] According to present embodiments, a portion of a recombinant donor template polynucleotide molecule (i.e., an insertion sequence) may be inserted or integrated at a desired site or locus within the plant genome. The insertion sequence of the donor template may comprise a transgene or construct, such as a transgene or transcribable DNA sequence encoding a non-coding RNA molecule that targets an endogenous gene for suppression. The donor template may also have one or two homology arms flanking the insertion sequence to promote the targeted insertion event through homologous recombination and/or homology-directed repair. [0076] Each homology arm may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99% or 100% identical or complementary to at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 2500, or at least 5000 consecutive nucleotides of a target DNA sequence within the genome of a plant cell. Thus, a plant cell may comprise a recombinant DNA molecule encoding a donor template for site-directed or targeted integration of a transgene or construct, such as a transgene or transcribable DNA sequence encoding a non- coding RNA molecule that targets an endogenous gene for suppression, into the genome of a plant. [0077] The term “target site” for genome editing or site-directed integration refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by a site-specific nuclease introducing a double stranded break (or single-stranded nick) into the nucleic acid backbone of the polynucleotide sequence and/or its complementary DNA strand. Page 15 of 61 290326197 v3
A target site may comprise at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29, or at least 30 consecutive nucleotides. [0078] The term “target site” for an RNA-guided nuclease may comprise the sequence of either complementary strand of a double-stranded nucleic acid (DNA) molecule or chromosome at the target site. [0079] A site-specific nuclease may bind to a target site, such as via a non-coding guide RNA (e.g., without being limiting, a CRISPR RNA (crRNA) or a single-guide RNA (sgRNA) as described further below). [0080] A non-coding guide RNA provided herein may be complementary to a target site (e.g., complementary to either strand of a double-stranded nucleic acid molecule or chromosome at the target site). It will be appreciated that perfect identity or complementarity may not be required for a non-coding guide RNA to bind or hybridize to a target site. For example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 mismatches (or more) between a target site and a non-coding RNA may be tolerated. A “target site” also refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by another site-specific nuclease that may not be guided by a non-coding RNA molecule, such as a meganuclease, zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN), to introduce a double stranded break (or single-stranded nick) into the polynucleotide sequence and/or its complementary DNA strand. Exonucleases [0081] Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. A close relative to exonucleases is the endonuclease, see above, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease, which is a dependent de-capping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease. [0082] Examples of exonucleases are shown below in Table 1 and include, but are not limited to, Exonuclease I, Exonuclease III (E. coli), Exonuclease T, Exonuclease V (RecBCD), RecQ4, Exonuclease VII, Exonuclease VIII, Lambda Exonuclease, Trex1, Trex2, and Dpd1. Page 16 of 61 290326197 v3
2 6 6 2 Q : -7 E O 7 5 7 1 3 O W 1 0 / 6 8 0-I S R J
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2 6 6 2-7 7 5 7 1 3 O W 1 0 / 6 8 0-I S R J
A c i G E 7 6 G 7 4 1 R 4 o mo R4 E 4 2 G E 3 5 G E 8 5 2 0 8 8 R7 ( R1 ( R2 8 8 9 1 ( 1 n 3 4. 6 t e 0 . 0 8 n e 3 2 t n e 6 7 4 9 8 t e .. 3 2 n e g a G 0 0 3 0 0 m 0 0 m 0 3 8 6 0 m P I 0 B 0 8 _ 5 0 6 0 e l p 0 0 e l p 0 0 0 3 0 e l 4 _ m _ m _ 0 8 _ p C C8 C C C c N4 Wm N N 4 No c No 9 No c ) 6 ) 0 0 3 4 4 0 9 6 9 7 D 6 I 2 0 5 2 3 2 2 7 2 G 4 0 1 0 e ne 7 2 9 5 2 0 1 7 5 9 T 5 C G1 G 1 1 1 4 2 2 2 O 2 1 1 3 8 A ( 0 1 L ( T A si s m s si p m i u s p n a g o s n e o ms n o d i s o o d i r m e o i s p u o o H e i b r a e b a O Ha s M . p S a s r A . b S u t r A ri a ri e p a ri a e l s e p p l e r 1 e 1 e 2 e 1 / l a s a ae e e s r e r e l a e s e s n I n a g n o e ci i e i s t s r d l c c m i e u l m i a e l m i a e l e v r O t a ) a e l a l n o ) u 3 v n e r p c ) ox m e u 1 r n x p c ) e e u 2 r n x p c ) 2 it e e u n x e c e n e d l Aa r 1 c , D u n 1 p o h 1 n D r o c o D o x 7 9 1 6 E a e r o N h t x r e r o e T ( h t x r e r o e T ( h t x r f e l e T ( Do Ng e P o P l ti P E P D DD ( x e Dh c mD ( t 2 A 3 0 9 2
[0083] In some embodiments, the exonuclease exhibits 3’ to 5’ exonuclease activity at the double stranded break site. In some embodiments, the exonuclease exhibits 5’ to 3’ exonuclease activity at the double stranded break site. [0084] In some embodiments, the exonuclease is selected from the group consisting of Trex1, Trex2, and DPD1. In some embodiments, the exonuclease is Trex2. In some embodiments, the exonuclease is DPD1. [0085] In some embodiments, the exonuclease sequence is a gene selected from Table 1. [0086] In some embodiments, the exonuclease comprises SEQ ID NO: 1 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 1 or a sequence at least 85% identical thereto. [0087] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 1. [0088] In some embodiments, the exonuclease comprises SEQ ID NO: 2 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 2 or a sequence at least 85% identical thereto. [0089] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 2. [0090] In some embodiments, the exonuclease comprises SEQ ID NO: 3 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 3 or a sequence at least 85% identical thereto. Page 19 of 61 290326197 v3
[0091] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 3. [0092] In some embodiments, the exonuclease comprises SEQ ID NO: 4 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 4 or a sequence at least 85% identical thereto. [0093] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 4. [0094] In some embodiments, the exonuclease comprises SEQ ID NO: 5 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 5 or a sequence at least 85% identical thereto. [0095] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 5. [0096] In some embodiments, the exonuclease comprises SEQ ID NO: 6 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 6 or a sequence at least 85% identical thereto. Page 20 of 61 290326197 v3
[0097] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 6. [0098] In some embodiments, the exonuclease comprises SEQ ID NO: 7 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 7 or a sequence at least 85% identical thereto. [0099] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 7. [0100] In some embodiments, the exonuclease comprises SEQ ID NO: 8 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 8 or a sequence at least 85% identical thereto. [0101] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 8. [0102] In some embodiments, the exonuclease comprises SEQ ID NO: 9 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 9 or a sequence at least 85% identical thereto. Page 21 of 61 290326197 v3
[0103] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 9. [0104] In some embodiments, the exonuclease comprises SEQ ID NO: 10 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 10 or a sequence at least 85% identical thereto. [0105] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 10. [0106] In some embodiments, the exonuclease comprises SEQ ID NO: 11 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 11 or a sequence at least 85% identical thereto. [0107] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 11. [0108] In some embodiments, the exonuclease comprises SEQ ID NO: 12 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 12 or a sequence at least 85% identical thereto. Page 22 of 61 290326197 v3
[0109] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 12. [0110] In some embodiments, the exonuclease comprises SEQ ID NO: 13 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 13 or a sequence at least 85% identical thereto. [0111] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 13. [0112] In some embodiments, the exonuclease comprises SEQ ID NO: 14 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 14 or a sequence at least 85% identical thereto. [0113] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 14. [0114] In some embodiments, the exonuclease comprises SEQ ID NO: 15 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 15 or a sequence at least 85% identical thereto. Page 23 of 61 290326197 v3
[0115] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 15. [0116] In some embodiments, the exonuclease comprises SEQ ID NO: 16 or a sequence at least 75% identical thereto. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 16 or a sequence at least 85% identical thereto. [0117] In some embodiments, the exonuclease sequence comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 16. Silencing of the nonhomologous end joining pathway (NHEJ) [0118] In some embodiments, the methods of enhanced targeted knock-in frequency comprise concurrent silencing of a NHEJ pathway gene. Methods of gene silencing are well known in the art (see for example Yan H, et al., New construct approaches for efficient gene silencing in plants. Plant Physiol. 2006 Aug;141(4):1508-18; Wesley SV, et al., Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J.2001 Sep;27(6):581-90; Helliwell CA and Waterhouse PM, Constructs and Methods for Hairpin RNA-Mediated Gene Silencing in Plants. Methods in Enzymology 2005392:24-35; El-Sappah A. et al., Comprehensive Mechanism of Gene Silencing and Its Role in Plant Growth and Development. Front Plant Sci. 2021 Sep 7;12:705249). [0119] Exemplary genes in the NHEJ pathway that may be targeted by a silencing construct used with the methods disclosed herein are shown below in Table 2. Page 24 of 61 290326197 v3
2 6 6 2 D - I 7 7 5 7 1 3 O W 1 0 / 6 8 0-I S R J
m u s s u u u i o s r o s s u s u s r o r o r o o n a e g b e b e e r e r e r u t u b u b u b u b . t. t t t u t O S S . S . S . S . S ]e n ) t s i e a t 4 n e 2 e t 0 n e 2 e 0 o b t A i o Ne h e s d a n s g e a c 8 d s a 7 r i U n e c i U- 1 P e s d n - r e s p D g ( t e 1 s e s e i l p e l d e K p l K h t Da i e e h t i r a a e e s r e n i I a n Hr a e g i l m -P n d A u [ a i C A A - b P An u m b y l y l ci ma I m l y l t n B y l A a N TN TN A D s o p o p h p c E N N D A Du s u e o o T o p D I 1 H J ) 3 C Q v 7 E e Hn 4 0 0 P I L 9 1 g 8 7 R BO 1 g 6 2 Ne g i u u L K K A P E T P ( i L 3 0 9 2
-7 7 5 7 1 3 O W 1 0 / 6
8 n . 0 i o 9 -I e t N0 9 S o 9 0 9 0 9 0 9 0 r n 6 9 1 6 9 9 9 1 6 1 6 6 R J P oi I s 5 B s e 1 5 0 1 5 1 0 1 5 1 0 1 5 0 1 0 Cc c _ P _ P _ P _ P _ P N A X X X X X tp i 1 . 1 1 1 1 c . 4 . . . . r s o 0 5 0 6 0 7 0 8 na N6 3 6 6 6 0 6 r n 1 3 3 3 3 o 3 1 5 3 1 5 3 1 1 5 3 5 3 T i I s B s 1 1 1 1 5 1 e 0 _ 0 _ 0 _ 0 _ 0 _ Cc c MMMMM N A X X X X X c i m. o o n N e n 1 Go 6 i f I s o B s e 6 Cc c 2 N A e g a ) P Dl I o e b ne my G S I e B n Ce NG ( msi n a g r O e ma N 3 J v 7 E e 9 Hn 1 6 Ne 2 g 3 0 9 2
[0120] In some embodiments, the NHEJ gene targeted for silencing is selected from Table 2. [0121] In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 17 or a sequence at least 75% identical thereto. In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 17 or a sequence at least 85% identical thereto. [0122] In some embodiments, the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 17. [0123] In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 18 or a sequence at least 75% identical thereto. In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 18 or a sequence at least 85% identical thereto. [0124] In some embodiments, the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 18. [0125] In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 19 or a sequence at least 75% identical thereto. In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 19 or a sequence at least 85% identical thereto. [0126] In some embodiments, the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 19. [0127] In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 20 or a sequence at least 75% identical thereto. In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 20 or a sequence at least 85% identical thereto. Page 27 of 61 290326197 v3
[0128] In some embodiments, the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 20. [0129] In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 21 or a sequence at least 75% identical thereto. In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 21 or a sequence at least 85% identical thereto. [0130] In some embodiments, the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 21. [0131] In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 22 or a sequence at least 75% identical thereto. In some embodiments, the NHEJ gene targeted for silencing comprises SEQ ID NO: 22 or a sequence at least 85% identical thereto. [0132] In some embodiments, the NHEJ gene targeted for silencing comprises a sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 22. Expression vectors and cassettes [0133] In some embodiments, the expression vector is a plasmid. In such embodiments, the methods described herein may include a first plasmid that, when delivered to a cell, expresses the CRISPR system components, CAS endonuclease and the guide RNA sequence; a second plasmid that comprises a donor DNA template; and a third plasmid that, when delivered to a cell with the first plasmid, co-expresses an exonuclease. In some embodiments, the exonuclease Page 28 of 61 290326197 v3
is Trex2. In some embodiments, the methods disclosed herein include a fourth plasmid comprising a NHEJ pathway gene silencing construct. Alternatively, the methods disclosed herein may include a plasmid that comprises all three or all four expression cassettes and donor DNA template, or may include three plasmids, the first of which expresses the CAS endonuclease, the second of which expresses the guide RNA sequence, and the third of which expresses an endonuclease, such as Trex2. [0134] According to some embodiments, the methods described herein may be used to knock- in donor DNA to a target genomic nucleotide sequence in a cell. Such methods may include transfecting a cell with one or more expression cassettes that, when inserted into an expression vector, express an endonuclease (or endonuclease system) and an exonuclease. In certain embodiments, the endonuclease system is a CRISPR system and the exonuclease is Trex2. [0135] In some embodiments, the method includes providing to a plant cell: (i) a first expression cassette that includes a nucleotide sequence that encodes a CAS9 endonuclease, (ii) a second expression cassette that that includes a nucleotide sequence that encodes a guide RNA sequence designed to be complementary to a target genomic nucleotide sequence in a cell, (iii) a third expression cassette that includes a nucleotide sequence that encodes a Trex2 exonuclease, and (iv) a donor DNA template. [0136] In some embodiments, the method includes providing (i) a first expression cassette that includes a nucleotide sequence that encodes a CAS9 endonuclease, (ii) a second expression cassette that that includes a nucleotide sequence that encodes a guide RNA sequence designed to be complementary to a target genomic nucleotide sequence in a cell, (iii) a third expression cassette that includes a nucleotide sequence that encodes a Trex2 exonuclease, (iv) a fourth expression cassette that includes a gene silencing construct, and (v) a donor DNA template. [0137] In some embodiments, the method provides one expression cassette to a host cell. In some embodiments, the method provides two expression cassettes to a host cell. In some embodiments, the method provides three expression cassettes to a host cell. In some embodiments, the method provides four expression cassettes to a host cell. [0138] In some embodiments, the method provides one expression vector to a host cell. In some embodiments, the method provides two expression vectors to a host cell. In some embodiments, the method provides three expression vectors to a host cell. In some embodiments, the method provides four expression vectors to a host cell. [0139] In some embodiments, the expression vector(s) comprises Trex2 and a silencing construct for Lig4. In some embodiments, the expression vector(s) comprises Trex2 and a silencing construct for Ku80. Page 29 of 61 290326197 v3
[0140] In some embodiments, the expression vector(s) comprises DPD1 and a silencing construct for Lig4. In some embodiments, the expression vector(s) comprises DPD1 and a silencing construct for Ku80. [0141] In some embodiments, the expression vector(s) comprises POLQ and a silencing construct for Lig4. In some embodiments, the expression vector(s) comprises POLQ and a silencing construct for Ku80. [0142] In some embodiments, the methods described herein insert a DNA sequence of interest into a host cell’s genome with at least a 5% frequency, at least a 10% frequency, at least a 20% frequency, at least a 30% frequency, at least a 40% frequency; at least a 50% frequency, at least a 60% frequency, at least a 70% frequency, at least an 80% frequency, at least a 90% frequency, or a 100% frequency. [0143] In some embodiments, the methods described herein insert a DNA sequence of interest into a host cell’s genome with about 5% frequency to about 10% frequency, about a 10% frequency to about 20% frequency, about a 20% frequency to about 30% frequency, about a 30% frequency to about 40% frequency, about a 40% frequency to about 50% frequency; about a 50% frequency to about 60% frequency, about a 60% frequency to about 70% frequency, about a 70% frequency to about 80% frequency, about an 80% frequency to about 90% frequency, or about a 90% frequency to about 100% frequency. [0144] In some embodiments, the methods described herein insert a DNA sequence into a host cell’s genome with at least a 5% frequency, at least a 10% frequency, at least a 20% frequency, at least a 30% frequency, at least a 40% frequency; at least a 50% frequency, at least a 60% frequency, at least a 70% frequency, at least an 80% frequency, at least a 90% frequency, or a 100% frequency. [0145] In some embodiments, the methods described herein insert a DNA sequence of interest into a host cell’s genome with about 5% frequency to about 10% frequency, about a 10% frequency to about 20% frequency, about a 20% frequency to about 30% frequency, about a 30% frequency to about 40% frequency, about a 40% frequency to about 50% frequency; about a 50% frequency to about 60% frequency, about a 60% frequency to about 70% frequency, about a 70% frequency to about 80% frequency, about an 80% frequency to about 90% frequency, or about a 90% frequency to about 100% frequency. Expression Vectors for Transformation: Marker Genes [0146] Expression vectors usually include at least one genetic marker, operably linked to a regulatory element (a promoter, for example) that allows transformed cells containing the marker to be either recovered by negative selection, i.e., inhibiting growth of cells that do not Page 30 of 61 290326197 v3
contain the selectable marker gene, or by positive selection, i.e., screening for the product encoded by the genetic marker. Many commonly used selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or an herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art. [0147] Some commonly used selectable marker genes for plant transformation include, but are not limited to, neomycin phosphotransferase II (nptII) (Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983)), aminoglycoside phosphotransferases APH(3')II and APH(3')I (Davies and Smith, 1978; Jimenez and Davies, 1980), kanamycin resistance (KmR) gene ,(Gray and Fitch, 1983), hygromycin phosphotransferase gene (Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985)), gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3'-adenyl transferase, and the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab et al., Plant Mol. Biol. 14:197 (1990) Hille et al., Plant Mol. Biol. 7:171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or bromoxynil. Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) and Stalker et al., Science 242:419-423 (1988). [0148] Selectable marker genes for plant transformation not of bacterial origin include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet.13:67 (1987), Shah et al., Science 233:478 (1986), Charest et al., Plant Cell Rep.8:643 (1990). [0149] Another class of marker genes for plant transformation requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. Commonly used genes for screening presumptively transformed cells include beta-glucuronidase (GUS), beta-galactosidase, luciferase and chloramphenicol acetyltransferase. Jefferson, R. A., Plant Mol. Biol. Rep.5:387 (1987), Teeri et al., EMBO J.8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci. USA 84:131 (1987), DeBlock et al., EMBO J.3:1681 (1984). In vivo methods for visualizing GUS activity that do not require destruction of plant tissue are available. Molecular Probes publication 2908, IMAGENE GREEN, p.1-4 (1993) and Naleway et al., J. Cell Biol.115:151a (1991). Expression Vectors for Transformation: Promoters [0150] Genes included in expression vectors are typically driven by a nucleotide sequence comprising a regulatory element, for example, a promoter. Several types of promoters are well Page 31 of 61 290326197 v3
known in the transformation arts as are other regulatory elements that can be used alone or in combination with promoters. [0151] A “plant promoter” is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissue-preferred”. Promoters that initiate transcription only in a certain tissue are referred to as “tissue-specific”. A “cell- type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter that is active under most environmental conditions, and cell types. Expression Vectors for Transformation: Terminators [0152] As used herein, the term “terminator” or “termination sequence” generally refers to a 3′ flanking region of a gene that contains nucleotide sequences which regulate transcription termination and typically confer RNA stability. [0153] Terminator sequences that find use in proper transcriptional processing of recombinant nucleic acids in the vectors taught herein are well known in the art. Although terminator sequences do not by themselves initiate gene transcription, their presence can increase accurate processing and termination of the RNA transcript, and result in message stability. The use of recombinant terminator sequences is established in the art. It is appreciated that an understanding of the molecular mechanisms underlying terminator sequence activity are not required to make or use the present disclosure. Plants for use with the disclosed methods [0154] The disclosure has use over a broad range of plants, monocots and dicots, including species from the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and Zea. Examples include tobacco and Arabidopsis, cereal crops such as maize, wheat, rice, soybean barley, rye, oats, sorghum, alfalfa, clover and the like, oil-producing plants such as canola, Page 32 of 61 290326197 v3
safflower, sunflower, peanut and the like, vegetable crops such as tomato tomatillo, potato, pepper, eggplant, sugar beet, carrot, cucumber, lettuce, pea and the like, horticultural plants such as aster, begonia, chrysanthemum, delphinium, zinnia, lawn and turfgrasses and the like. [0155] The Solanaceae family contains several well-known cultivated crops such as tomato (Solanum lycopersicum also referred to as Lycopersicon esculentum), eggplant (Solanum melogena), tobacco (Nicotiana tabacum), pepper (Capsicum annuum) and potato (Solanum tuberosum). Within the genus Solanum, over a thousand species have been recognized. Potatoes will not hybridize with non-tuber bearing Solanum (tomato, eggplant, etc.) species including weeds commonly found in and around commercial potato fields (Love 1994). [0156] The genus Solanum is divided into several subsections, of which the subsection potato contains all tuber-bearing potatoes. The subsection potato is divided into series, of which tuberosa is relevant to this document. Within the series tuberosa approximately 54 species of wild and cultivated potatoes are found. One of these is S. tuberosum. [0157] S. tuberosum is divided into two subspecies: tuberosum and andigena. The subspecies tuberosum is the cultivated potato widely in use as a crop plant in, for example, North America and Europe. The subspecies andigena is also a cultivated species, but cultivation is restricted to Central and South America (Hanneman 1994). Z. Huamán and D. M. Spooner reclassified all cultivated potatoes as a single species, S. tuberosum, with various groups, including a Tuberosum Group (S. tuberosum subsp. Tuberosum) for the modern cultivars (Huamán et al., Am. J. of Botany 89(6): 947-965.2002). [0158] With respect to potato plants, Solanum tuberosum subsp. tuberosum is an example of one of the most widely cultivated potato varieties, although there are thousands of potato varieties worldwide. Examples of well-known cultivated varieties that may be used with the methods and sequences disclosed herein include, but are not limited to, russets, reds, whites, yellows (also called Yukons) purples, Adirondack Blue, Adirondack Red, Agata, Almond, Amandine, Anya, Arran Victory, Atlantic, Bamberg, Belle de Fontenay, BF-15, Bildtstar, Bintje, Blue Congo, Bonnotte, Cabritas, Camota, Chelina, Chiloé, Cielo, Clavela Blanca, Désirée, Fianna, Fingerling, Flava, Golden Wonder, Innovator, Jersey Royal, Kerr's Pink, Kestrel, King Edward, Kipfler, Lady Balfour, Linda, Marfona, Maris Piper, Marquis, Nicola, Pachacoñ a, Pink Eye, Pink Fir Apple, Primura, Ratte, Red Norland, Red Pontiac, Rooster, Russet Burbank, Russet Norkotah, Selma, Shepody, Sieglinde, Sirco, Spunta, Stobrawa, Vivaldi, Vitelotte, Yellow Finn, and Yukon Gold. Any of these cultivated varieties may be used with the methods disclosed herein. Page 33 of 61 290326197 v3
[0159] In some embodiments, the host cell is from the Solanum genus. In some embodiments, the host cell is a Solanum tuberosum cell. In some embodiments, the host cell is a potato cell. In some embodiments, the host cell is a Solanum lycopersicum cell. In some embodiments, the host cell is a tomato cell. EXAMPLES Example 1. Trex2 Exonuclease Increases Knock-In Frequency of Donor DNA [0160] The purpose of this study was to determine a method to increase the targeted knock-in efficiency of donor DNA in plant genomes. [0161] CRISPR/Cas9 can be used to induce targeted DSBs (double-strand breaks) in the genome. There are several applications for CRISPR/Cas9-based gene targeting (FIG. 1), however, targeted KI efficiency has been very low in plants. [0162] Since expression across different loci varies greatly, several loci were screened to identify the loci with highest expression to use in subsequent screening analyses. The RFP coding sequences without both promoter and terminator are used to target the endogenous locus (Bam9 or SSR2) (FIG.2). If NLS-RFP KI is successful, the potato protoplasts will express NLS-RFP signals. NLS stands for nuclear localization signal, which can be used to concentrate RFP protein to nucleus. The Bam9 loci had the highest expression in either CW protoplasts or DE protoplasts, and thus was used in most RFP KI experiments (FIG.3). [0163] To determine the effect of exonuclease on induced DSBs at PPO2 locus, three different exonucleases were co-expressed: NLS-DPD1, DPD1, and Trex2. DPD1 is an exonuclease from Arabidopsis. As shown in FIG. 4, smaller PCR products flanking PPO2 cleavage site suggest that DNA resection occurs at DSB cleavage site when NLS-DPD1, DPD1 or Trex2 is co-expressed with CRISPR/Cas9. [0164] The next experiment was to test if co-expression of Trex2 nuclease could enhance targeted NLS-RFP KI efficiency in potato protoplasts. As shown in FIGs.5A and 5B, many protoplasts with NLS-RFP signals occur only when Trex2 is co-delivered with RFP donor template. This experiment was repeated with several other RFP constructs, FIG. 6. As a control to ensure the RFP signal was not due to integration at a random locus, a stop codon was introduced in the upstream Bam9 homology arm (p3984, FS RFP). The stop codon resulted in no RFP signal (FIG.7). [0165] As shown in FIGs. 8 and 9, the presence of Trex2 consistently results in a high percentage of RFP(+) FDA cells. FIG.8 shows that NLS-RFP KI efficiency can reach 40% among transfected protoplasts (GFP positive) with Trex2 co-expression. As a control when Page 34 of 61 290326197 v3
co-delivered with a GFP plasmid DNA, NLS-RFP signals are observed among 1% of GFP- positive protoplasts. These results indicate the RFP signal is from the precision of the RFP integration at the Bam9 locus. [0166] To further determine the precision of Trex2 exonuclease in targeted donor DNA integration, a PCR amplification of the 5’ and 3’ ends was performed using the strategy described in FIG.10A. As shown in FIG.10B, expression of Trex2 improves the precision of donor DNA integration at the 5’ and 3’ KI junction by yielding only one major PCR amplification product. Example 2. The effect of different homology arm lengths on targeted KI in potato protoplasts [0167] To study the effect of homology arm lengths on targeted KI, multiple RFP donor constructs were designed with the homology arm length ranges from 50-bp to 400-bp (Table 3). Homology arm was only present at one side for p6081 or p6082. There was no homology arm for p6083. Two additional plasmid constructs (p6084 and p6085) served as additional negative controls. Table 3: RFP donor constructs with various homology arm lengths
[0168] When the homology arm length reduces from 400-bp to 50-bp, observed NLS-RFP KI efficiency also decreases (FIG. 11). The presence of a left homology arm resulted in more targeted RFP KI than a right homology arm (data from p6081/p3899, p6082/p3899 and p6083/p3899, FIG. 11). Very few protoplasts with NLS-RFP signals were observed for the two negative controls (p6084/p3899 and p6085/p3899). Page 35 of 61 290326197 v3
Example 3. The effect of suppressing NHEJ pathway on Trex2-mediated RFP knock-in in potato protoplasts [0169] Lig4 and Ku80 are two important genes involved in the canonical nonhomologous end joining (NHEJ) pathway. To study if suppressing NHEJ pathway can increase Trex2-mediated targeted KI, two silencing constructs (p6091 and p6093) (FIG.12A and 12B) comprising the inverted sequence of Lig4 (FIG.12A) and Ku80 (FIG.12B) were co-delivered to protoplasts together with RFP donor template (p6076) and Trex2 (p3899). As shown in FIG.13, the RFP KI efficiency can reach 40% with Trex2, however when Lig4 or Ku80 are also suppressed, the RFP KI efficiency increased to between 50% and 60%. Example 4. The effect of an exonuclease from Arabidopsis on targeted RFP knock-in [0170] Since Trex2 exonuclease is from a mammalian system, exonucleases from Arabidopsis were tested to determine if they too could be used for enhancing targeted RFP knock-in in potato protoplasts. FIG. 14 shows that the co-expression of p5762 (AtExonuclease, AT1G02270) can enhance targeted RFP knock-in at Bam9 to 30% compared to only 2% for the control. The result demonstrates that other exonucleases can be used to enhance targeted KI in potato protoplasts. [0171] To determine the which exonucleases are most effective at increasing targeting knock- in frequency, multiple exonucleases were tested (FIG.15). The 3’-5’ exonucleases yielded the highest percent positive RFP cells: Trex2 ~43% RFP(+) cells and AtDPD1 ~23% RFP(+) cells. Both the 5’-3’ exonuclease and GFP control yielded less than 5% RFP(+) cells. [0172] The study demonstrates that inclusion of exonucleases can significantly increase the knock-in frequency of donor DNA. Page 36 of 61 290326197 v3
NUMBERED EMBODIMENTS [0173] Notwithstanding the appended claims, the disclosure sets forth the following numbered embodiments: [0174] Embodiment 1. A method of targeted DNA sequence insertion into a host cell genome, comprising: a) providing a host cell; b) selecting a target site nucleic acid sequence in the host cell genome for DNA sequence insertion; c) introducing into the host cell a guide RNA (gRNA) with a guide sequence having complementarity to the target site nucleic acid sequence in the host genome, and a Cas endonuclease that interacts with the gRNA and is capable of creating a double stranded break in the host genome; d) introducing into the host cell an exonuclease; and e) introducing into the host cell a donor DNA sequence template comprising: a DNA sequence of interest and left and right homology arm sequences, wherein the gRNA guide sequence binds to a complementary target site nucleic acid sequence in the host genome and the Cas endonuclease creates a double stranded break in the host genome; and wherein the DNA sequence of interest is inserted into the host genome via a homology dependent repair mechanism. [0175] Embodiment 2. The method of embodiment 1, wherein the target site nucleic acid sequence is within a coding sequence. [0176] Embodiment 3. The method of any one of embodiments 1 to 2, wherein the target site nucleic acid sequence is within a gene. [0177] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the target site nucleic acid sequence is within a non-coding sequence. [0178] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the target site nucleic acid sequence is within a non-coding regulatory sequence. [0179] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the target site nucleic acid sequence is within a promoter sequence region. [0180] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the host cell is a eukaryotic cell. [0181] Embodiment 8. The method of any one of embodiments 1 to 6, wherein the host cell is a prokaryotic cell. Page 37 of 61 290326197 v3
[0182] Embodiment 9. The method of any one of embodiments 1 to 6, wherein the host cell is a mammalian cell. [0183] Embodiment 9.1 The method of any one of embodiments 1 to 6, wherein the host cell is a plant cell. [0184] Embodiment 10. The method of embodiment 9.1, wherein the host cell is a plant protoplast cell. [0185] Embodiment 11. The method of any one of embodiments 1-6, wherein the host cell is an Angiosperm cell. [0186] Embodiment 12. The method of any one of embodiments 1-6, wherein the host cell is a dicot cell. [0187] Embodiment 12.1 The method of any one of embodiments 1-6, wherein the host cell is a monocot cell. [0188] Embodiment 13. The method of any one of embodiments 1-6, wherein the host cell is from the Solanum genus. [0189] Embodiment 14. The method of any one of embodiments 1-6, wherein the host cell is a Solanum tuberosum cell. [0190] Embodiment 15. The method of any one of embodiments 1-6, wherein the host cell is a potato cell. [0191] Embodiment 16. The method of any one of embodiments 1-6, wherein the host cell is a Solanum lycopersicum cell. [0192] Embodiment 17. The method of any one of embodiments 1-6, wherein the host cell is a tomato cell. [0193] Embodiment 18. The method of any one of embodiments 1-17, wherein the gRNA is a single gRNA. [0194] Embodiment 19. The method of any one of embodiments 1-18, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex). [0195] Embodiment 20. The method of any one of embodiments 1-19, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro. [0196] Embodiment 21. The method of any one of embodiments 1-20, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro and the exonuclease is expressed on a transient vector. Page 38 of 61 290326197 v3
[0197] Embodiment 22. The method of any one of embodiments 1-21, wherein the gRNA, Cas, and/or exonuclease are expressed on the same vector. [0198] Embodiment 23. The method of any one of embodiments 1-22, wherein the gRNA, Cas, and/or exonuclease are expressed on a different vector. [0199] Embodiment 24. The method of any one of embodiments 1-23, wherein the gRNA, Cas, and/or exonuclease are expressed from a nucleotide sequence integrated into the host cell’s genome. [0200] Embodiment 25. The method of any one of embodiments 1-24, wherein at least one of the gRNA, Cas, and/or exonuclease are expressed on a vector, and wherein at least one of the gRNA, Cas, and/or exonuclease are expressed from a nucleotide sequence integrated into the host cell’s genome. [0201] Embodiment 26. The method of any one of embodiments 1-25, wherein the gRNA and exonuclease are expressed on a vector and the Cas is expressed from a nucleotide sequence integrated into the host cell’s genome. [0202] Embodiment 27. The method of any one of embodiments 1-26, wherein the Cas is a Class II endonuclease. [0203] Embodiment 28. The method of any one of embodiments 1-27, wherein the Cas is a Type II endonuclease. [0204] Embodiment 29. The method of any one of embodiments 1-28, wherein the Cas is a Cas9. [0205] Embodiment 30. The method of any one of embodiments 1-29, wherein the Cas is a Type V endonuclease. [0206] Embodiment 31. The method of any one of embodiments 1-30, wherein the Cas is a Cas12a. [0207] Embodiment 32. The method of any one of embodiments 1-31, wherein the exonuclease exhibits 3’ to 5’ exonuclease activity at the double stranded break site. [0208] Embodiment 32.1 The method of any one of embodiments 1-31, wherein the exonuclease exhibits 5’ to 3’ exonuclease activity at the double stranded break site. [0209] Embodiment 32.2. The method of any one of embodiments 1-31, wherein the exonuclease is selected from the group consisting of: Trex1, Trex2, and DPD1. [0210] Embodiment 33. The method of any one of embodiments 1-32.2, wherein the exonuclease is Trex2. [0211] Embodiment 34. The method of any one of embodiments 1-32.2, wherein the exonuclease is DPD1. Page 39 of 61 290326197 v3
[0212] Embodiment 34.1. The method of any one of embodiments 1-32.2, wherein the exonuclease is POLQ. [0213] Embodiment 34.2. The method of any one of embodiments 1-31, wherein the exonuclease is a gene selected from Table 1. [0214] Embodiment 34.3. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 1 or a sequence at least 75% identical thereto. [0215] Embodiment 34.4. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 2 or a sequence at least 75% identical thereto. [0216] Embodiment 34.5. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 3 or a sequence at least 75% identical thereto. [0217] Embodiment 34.6. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 4 or a sequence at least 75% identical thereto. [0218] Embodiment 34.7. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 5 or a sequence at least 75% identical thereto. [0219] Embodiment 34.8. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 6 or a sequence at least 75% identical thereto. [0220] Embodiment 34.9. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 7 or a sequence at least 75% identical thereto. [0221] Embodiment 34.10. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 8 or a sequence at least 75% identical thereto. [0222] Embodiment 34.11. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 9 or a sequence at least 75% identical thereto. [0223] Embodiment 34.12. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 10 or a sequence at least 75% identical thereto. [0224] Embodiment 34.13. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 11 or a sequence at least 75% identical thereto. [0225] Embodiment 34.14. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 12 or a sequence at least 75% identical thereto. [0226] Embodiment 34.15. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 13 or a sequence at least 75% identical thereto. [0227] Embodiment 34.16. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 14 or a sequence at least 75% identical thereto. [0228] Embodiment 34.17. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 15 or a sequence at least 75% identical thereto. Page 40 of 61 290326197 v3
[0229] Embodiment 34.18. The method of any one of embodiments 1-31, wherein the exonuclease comprises SEQ ID NO: 16 or a sequence at least 75% identical thereto. [0230] Embodiment 35. The method of any one of embodiments 1-34, wherein each of the left and right homology arms are at least 100-bp long. [0231] Embodiment 36. The method of any one of embodiments 1-34, wherein each of the left and right homology arms are at least 200-bp long. [0232] Embodiment 37. The method of any one of embodiments 1-34, wherein each of the left and right homology arms are at least 300-bp long. [0233] Embodiment 38. The method of any one of embodiments 1-34, wherein each of the left and right homology arms are at least 400-bp long. [0234] Embodiment 39. The method of any one of embodiments 1-34, wherein each of the left and right homology arms are at least between 100-bp and 800-bp long. [0235] Embodiment 40. The method of any one of embodiments 1-34, wherein each of the left and right homology arms are at least between 100-bp and 1000-bp long. [0236] Embodiment 40.1. The method of any one of embodiments 1-34, wherein the left homology arm is at least 100-bp long. [0237] Embodiment 40.2. The method of any one of embodiments 1-34, wherein the left homology arm is at least 200-bp long. [0238] Embodiment 40.3. The method of any one of embodiments 1-34, wherein the left homology arm is at least 300-bp long. [0239] Embodiment 40.4. The method of any one of embodiments 1-34, wherein the left homology arm is at least 400-bp long. [0240] Embodiment 40.5. The method of any one of embodiments 1-34, wherein the left homology arm is at least between 100-bp and 800-bp long. [0241] Embodiment 40.6. The method of any one of embodiments 1-34, wherein the left homology arm is at least between 100-bp and 1000-bp long. [0242] Embodiment 41. The method of any one of embodiments 1-40.6, wherein the DNA sequence of interest is a coding sequence. [0243] Embodiment 42. The method of any one of embodiments 1-41, wherein the DNA sequence of interest is a gene. [0244] Embodiment 43. The method of any one of embodiments 1-42, wherein the DNA sequence of interest is a heterologous gene. [0245] Embodiment 44. The method of any one of embodiments 1-43, wherein the DNA sequence of interest is a non-heterologous gene. Page 41 of 61 290326197 v3
[0246] Embodiment 45. The method of any one of embodiments 1-44, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome. [0247] Embodiment 46. The method of any one of embodiments 1-45, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome, which upon insertion causes a single base pair edit. [0248] Embodiment 47. The method of any one of embodiments 1-46, wherein the DNA sequence of interest causes a point mutation upon insertion into the host cell’s genome. [0249] Embodiment 48. The method of any one of embodiments 1-47, wherein the DNA sequence of interest is a non-coding sequence. [0250] Embodiment 49. The method of any one of embodiments 1-48, wherein the DNA sequence of interest is a non-coding regulatory sequence. [0251] Embodiment 50. The method of any one of embodiments 1-49, wherein the DNA sequence of interest is a promoter. [0252] Embodiment 51. The method of any one of embodiments 1-50, wherein the DNA sequence of interest is inserted into the host cell’s genome via single strand annealing. [0253] Embodiment 52. The method of any one of embodiments 1-51, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 5% frequency. [0254] Embodiment 53. The method of any one of embodiments 1-52, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 10% frequency. [0255] Embodiment 54. The method of any one of embodiments 1-53, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 20% frequency. [0256] Embodiment 55. The method of any one of embodiments 1-54, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 30% frequency. [0257] Embodiment 56. The method of any one of embodiments 1-55, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 40% frequency. [0258] Embodiment 57. The method of any one of embodiments 1-56, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 50% frequency. [0259] Embodiment 58. The method of any one of embodiments 1-57, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 60% frequency. [0260] Embodiment 59. A method of targeted DNA sequence insertion into a host cell genome, comprising: Page 42 of 61 290326197 v3
a) determining a target site nucleic acid sequence in the host cell genome for DNA sequence insertion; b) providing a host cell; c) introducing into the host cell a guide RNA (gRNA) with a guide sequence having complementarity to the target site nucleic acid sequence in the host genome and a Cas endonuclease that interacts with the gRNA and is capable of creating a double stranded break in the host genome; d) introducing into the host cell an exonuclease; e) introducing into the host cell a donor DNA sequence template comprising: a DNA sequence of interest and left and right homology arm sequences, and f) introducing into the host cell a silencing construct, wherein the silencing construct knocks down expression of a nonhomologous end joining pathway gene; wherein the gRNA guide sequence binds to a complementary target site nucleic acid sequence in the host genome and the Cas endonuclease creates a double stranded break in the host genome; and wherein the DNA sequence of interest is inserted into the host genome via a homology dependent repair mechanism. [0261] Embodiment 60. The method of embodiment 59, wherein the target site nucleic acid sequence is within a coding sequence. [0262] Embodiment 61. The method of any one of embodiments 59-60, wherein the target site nucleic acid sequence is within a gene. [0263] Embodiment 62. The method of any one of embodiments 59-61, wherein the target site nucleic acid sequence is within a non-coding sequence. [0264] Embodiment 63. The method of any one of embodiments 59-62, wherein the target site nucleic acid sequence is within a non-coding regulatory sequence. [0265] Embodiment 64. The method of any one of embodiments 59-63, wherein the target site nucleic acid sequence is within a promoter sequence region. [0266] Embodiment 65. The method of any one of embodiments 59-64, wherein the host cell is a eukaryotic cell. [0267] Embodiment 66. The method of any one of embodiments 59-64, wherein the host cell is a prokaryotic cell. [0268] Embodiment 67. The method of any one of embodiments 59-64, wherein the host cell is a mammalian cell. Page 43 of 61 290326197 v3
[0269] Embodiment 68 The method of any one of embodiments 59-64, wherein the host cell is a plant cell. [0270] Embodiment 69. The method of embodiment 68, wherein the host cell is a plant protoplast cell. [0271] Embodiment 70. The method of any one of embodiments 59-64, wherein the host cell is an Angiosperm cell. [0272] Embodiment 71. The method of any one of embodiments 59-64, wherein the host cell is a dicot cell. [0273] Embodiment 72. The method of any one of embodiments 59-64, wherein the host cell is a monocot cell. [0274] Embodiment 73. The method of any one of embodiments 59-64, wherein the host cell is from the Solanum genus. [0275] Embodiment 74. The method of any one of embodiments 59-64, wherein the host cell is a Solanum tuberosum cell. [0276] Embodiment 75. The method of any one of embodiments 59-64, wherein the host cell is a potato cell. [0277] Embodiment 76. The method of any one of embodiments 59-64, wherein the host cell is a Solanum lycopersicum cell. [0278] Embodiment 77. The method of any one of embodiments 59-64, wherein the host cell is a tomato cell. [0279] Embodiment 78. The method of any one of embodiments 59-77, wherein the gRNA is a single gRNA. [0280] Embodiment 79. The method of any one of embodiments 59-78, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex). [0281] Embodiment 80. The method of any one of embodiments 59-79, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro. [0282] Embodiment 81. The method of any one of embodiments 59-80, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro and the exonuclease is expressed on a transient vector. [0283] Embodiment 82. The method of any one of embodiments 59-81, wherein the gRNA, Cas, and/or exonuclease are expressed on the same vector. Page 44 of 61 290326197 v3
[0284] Embodiment 83. The method of any one of embodiments 59-82, wherein the gRNA, Cas, and/or exonuclease are expressed on a different vector. [0285] Embodiment 84. The method of any one of embodiments 59-83, wherein the gRNA, Cas, and/or exonuclease are expressed from a nucleotide sequence integrated into the host cell’s genome. [0286] Embodiment 85. The method of any one of embodiments 59-84, wherein at least one of the gRNA, Cas, and/or exonuclease are expressed on a vector, and wherein at least one of the gRNA, Cas, and/or exonuclease are expressed from a nucleotide sequence integrated into the host cell’s genome. [0287] Embodiment 86. The method of any one of embodiments 59-85, wherein the gRNA and exonuclease are expressed on a vector and the Cas is expressed from a nucleotide sequence integrated into the host cell’s genome. [0288] Embodiment 87. The method of any one of embodiments 59-86, wherein the Cas is a Class II endonuclease. [0289] Embodiment 88. The method of any one of embodiments 59-87, wherein the Cas is a Type II endonuclease. [0290] Embodiment 89. The method of any one of embodiments 59-88, wherein the Cas is a Cas9. [0291] Embodiment 90. The method of any one of embodiments 59-89, wherein the Cas is a Type V endonuclease. [0292] Embodiment 91. The method of any one of embodiments 59-90, wherein the Cas is a Cas12a. [0293] Embodiment 92. The method of any one of embodiments 59-91, wherein the exonuclease exhibits 3’ to 5’ exonuclease activity at the double stranded break site. [0294] Embodiment 93. The method of any one of embodiments 59-91, wherein the exonuclease exhibits 5’ to 3’ exonuclease activity at the double stranded break site. [0295] Embodiment 94. The method of any one of embodiments 59-91, wherein the exonuclease is selected from the group consisting of: Trex1, Trex2, and DPD1. [0296] Embodiment 95. The method of any one of embodiments 59-94, wherein the exonuclease is Trex2. [0297] Embodiment 96. The method of any one of embodiments 59-94, wherein the exonuclease is DPD1. [0298] Embodiment 96.1. The method of any one of embodiments 59-94, wherein the exonuclease is POLQ. Page 45 of 61 290326197 v3
[0299] Embodiment 96.2. The method of any one of embodiments 59-93, wherein the exonuclease is a gene selected from Table 1. [0300] Embodiment 96.3. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 1 or a sequence at least 75% identical thereto. [0301] Embodiment 96.4. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 2 or a sequence at least 75% identical thereto. [0302] Embodiment 96.5. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 3 or a sequence at least 75% identical thereto. [0303] Embodiment 96.6. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 4 or a sequence at least 75% identical thereto. [0304] Embodiment 96.7. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 5 or a sequence at least 75% identical thereto. [0305] Embodiment 96.8. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 6 or a sequence at least 75% identical thereto. [0306] Embodiment 96.9. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 7 or a sequence at least 75% identical thereto. [0307] Embodiment 96.10. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 8 or a sequence at least 75% identical thereto. [0308] Embodiment 96.11. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 9 or a sequence at least 75% identical thereto. [0309] Embodiment 96.12. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 10 or a sequence at least 75% identical thereto. [0310] Embodiment 96.13. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 11 or a sequence at least 75% identical thereto. [0311] Embodiment 96.14. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 12 or a sequence at least 75% identical thereto. [0312] Embodiment 96.15. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 13 or a sequence at least 75% identical thereto. [0313] Embodiment 96.16. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 14 or a sequence at least 75% identical thereto. [0314] Embodiment 96.17. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 15 or a sequence at least 75% identical thereto. [0315] Embodiment 96.18. The method of any one of embodiments 59-93, wherein the exonuclease comprises SEQ ID NO: 16 or a sequence at least 75% identical thereto. Page 46 of 61 290326197 v3
[0316] Embodiment 97. The method of any one of embodiments 59-96, wherein each of the left and right homology arms are at least 100-bp long. [0317] Embodiment 98. The method of any one of embodiments 59-96, wherein each of the left and right homology arms are at least 200-bp long. [0318] Embodiment 99. The method of any one of embodiments 59-96, wherein each of the left and right homology arms are at least 300-bp long. [0319] Embodiment 100. The method of any one of embodiments 59-96, wherein each of the left and right homology arms are at least 400-bp long. [0320] Embodiment 101. The method of any one of embodiments 59-96, wherein each of the left and right homology arms are at least between 100-bp and 800-bp long. [0321] Embodiment 102. The method of any one of embodiments 59-96, wherein each of the left and right homology arms are at least between 100-bp and 1000-bp long. [0322] Embodiment 103. The method of any one of embodiments 59-96, wherein the left homology arm is at least 100-bp long. [0323] Embodiment 104. The method of any one of embodiments 59-96, wherein the left homology arm is at least 200-bp long. [0324] Embodiment 105. The method of any one of embodiments 59-96, wherein the left homology arm is at least 300-bp long. [0325] Embodiment 106. The method of any one of embodiments 59-96, wherein the left homology arm is at least 400-bp long. [0326] Embodiment 107. The method of any one of embodiments 59-96, wherein the left homology arm is at least between 100-bp and 800-bp long. [0327] Embodiment 108. The method of any one of embodiments 59-96, wherein the left homology arm is at least between 100-bp and 1000-bp long. [0328] Embodiment 109. The method of any one of embodiments 59-108, wherein the DNA sequence of interest is a coding sequence. [0329] Embodiment 110. The method of any one of embodiments 59-109, wherein the DNA sequence of interest is a gene. [0330] Embodiment 111. The method of any one of embodiments 59-110, wherein the DNA sequence of interest is a heterologous gene. [0331] Embodiment 112. The method of any one of embodiments 59-111, wherein the DNA sequence of interest is a non-heterologous gene. Page 47 of 61 290326197 v3
[0332] Embodiment 113. The method of any one of embodiments 59-112, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome. [0333] Embodiment 114. The method of any one of embodiments 59-113, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome, which upon insertion causes a single base pair edit. [0334] Embodiment 115. The method of any one of embodiments 59-114, wherein the DNA sequence of interest causes a point mutation upon insertion into the host cell’s genome. [0335] Embodiment 116. The method of any one of embodiments 59-115, wherein the DNA sequence of interest is a non-coding sequence. [0336] Embodiment 117. The method of any one of embodiments 59-116, wherein the DNA sequence of interest is a non-coding regulatory sequence. [0337] Embodiment 118. The method of any one of embodiments 59-117, wherein the DNA sequence of interest is a promoter. [0338] Embodiment 119. The method of any one of embodiments 59-118, wherein the DNA sequence of interest is inserted into the host cell’s genome via single strand annealing. [0339] Embodiment 119.1. The method of any one of embodiments 59-119, wherein the nonhomologous end joining pathway gene is a gene selected from Table 2. [0340] Embodiment 119.2. The method of any one of embodiments 59-119, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 17 or a sequence at least 75% identical thereto. [0341] Embodiment 119.3. The method of any one of embodiments 59-119, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 18 or a sequence at least 75% identical thereto. [0342] Embodiment 119.4. The method of any one of embodiments 59-119, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 19 or a sequence at least 75% identical thereto. [0343] Embodiment 119.5. The method of any one of embodiments 59-119, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 20 or a sequence at least 75% identical thereto. [0344] Embodiment 119.6. The method of any one of embodiments 59-119, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 21 or a sequence at least 75% identical thereto. Page 48 of 61 290326197 v3
[0345] Embodiment 119.7. The method of any one of embodiments 59-119, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 22 or a sequence at least 75% identical thereto. [0346] Embodiment 120. The method of any one of embodiments 59-119, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 5% frequency. [0347] Embodiment 121. The method of any one of embodiments 59-120, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 10% frequency. [0348] Embodiment 122. The method of any one of embodiments 59-121, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 20% frequency. [0349] Embodiment 123. The method of any one of embodiments 59-122, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 30% frequency. [0350] Embodiment 124. The method of any one of embodiments 59-123, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 40% frequency. [0351] Embodiment 125. The method of any one of embodiments 59-124, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 50% frequency. [0352] Embodiment 126. The method of any one of embodiments 59-125, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 60% frequency. INCORPORATION BY REFERENCE [0353] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not, be taken as an acknowledgement or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. Patent Citations 1) US 2019/0093104 2) US 10,745,690 3) EP2633040B1 Non-Patent Citations 1) Rogowsky P. CRISPR-Cas Technology in Plant Science. Potato Research.2017.60:353-360. 2) Cejka P. DNA End Resection: Nucleases Team Up with the Right Partners to Initiate Homologous Recombination. J Biol Chem.2015 Sep 18;290(38):22931-8. Page 49 of 61 290326197 v3
3) Weiss T, Wang C, Kang X, Zhao H, Elena Gamo M, Starker CG, Crisp PA, Zhou P, Springer NM, Voytas DF, Zhang F. Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis. Plant J.2020 Nov;104(3):828-838. 4) Mazur DJ, Perrino FW. Identification and expression of the TREX1 and TREX2 cDNA sequences encoding mammalian 3'-->5' exonucleases. J Biol Chem.1999 Jul 9;274(28):19655- 60. 5) Ceccaldi R, Rondinelli B, D'Andrea AD. Repair Pathway Choices and Consequences at the Double-Strand Break. Trends Cell Biol.2016 Jan;26(1):52-64. 6) Certo MT, Gwiazda KS, Kuhar R, Sather B, Curinga G, Mandt T, Brault M, Lambert AR, Baxter SK, Jacoby K, Ryu BY, Kiem HP, Gouble A, Paques F, Rawlings DJ, Scharenberg AM. Coupling endonucleases with DNA end-processing enzymes to drive gene disruption. Nat Methods.2012 Oct;9(10):973-5. Page 50 of 61 290326197 v3
Claims
CLAIMS What is claimed is: 1. A method of targeted DNA sequence insertion into a host cell genome, comprising: a) providing a host cell; b) selecting a target site nucleic acid sequence in the host cell genome for DNA sequence insertion; c) introducing into the host cell a guide RNA (gRNA) with a guide sequence having complementarity to the target site nucleic acid sequence in the host genome, and a Cas endonuclease that interacts with the gRNA and is capable of creating a double stranded break in the host genome; d) introducing into the host cell an exonuclease; and e) introducing into the host cell a donor DNA sequence template comprising: a DNA sequence of interest and left and right homology arm sequences, wherein the gRNA guide sequence binds to a complementary target site nucleic acid sequence in the host genome and the Cas endonuclease creates a double stranded break in the host genome; and wherein the DNA sequence of interest is inserted into the host genome via a homology dependent repair mechanism.
2. The method of claim 1, wherein the target site nucleic acid sequence is within a coding sequence.
3. The method of claim 1, wherein the target site nucleic acid sequence is within a gene.
4. The method of claim 1, wherein the target site nucleic acid sequence is within a non- coding sequence.
5. The method of claim 1, wherein the target site nucleic acid sequence is within a non- coding regulatory sequence.
6. The method of claim 1, wherein the target site nucleic acid sequence is within a promoter sequence region.
7. The method of claim 1, wherein the host cell is a eukaryotic cell.
8. The method of claim 1, wherein the host cell is a prokaryotic cell.
9. The method of claim 1, wherein the host cell is a mammalian cell.
10. The method of claim 1, wherein the host cell is a plant cell.
11. The method of claim 10, wherein the host cell is a plant protoplast cell.
12. The method of claim 1, wherein the host cell is an Angiosperm cell. Page 51 of 61 290326197 v3
13. The method of claim 1, wherein the host cell is a dicot cell.
14. The method of claim 1, wherein the host cell is a monocot cell.
15. The method of claim 1, wherein the host cell is from the Solanum genus.
16. The method of claim 1, wherein the host cell is a Solanum tuberosum cell.
17. The method of claim 1, wherein the host cell is a potato cell.
18. The method of claim 1, wherein the host cell is a Solanum lycopersicum cell.
19. The method of claim 1, wherein the host cell is a tomato cell.
20. The method of claim 1, wherein the gRNA is a single gRNA.
21. The method of claim 1, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex).
22. The method of claim 1, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro.
23. The method of claim 1, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro and the exonuclease is expressed on a transient vector.
24. The method of claim 1, wherein the gRNA, Cas, and/or exonuclease are expressed on the same vector.
25. The method of claim 1, wherein the gRNA, Cas, and/or exonuclease are expressed on a different vector.
26. The method of claim 1, wherein the gRNA, Cas, and/or exonuclease are expressed from a nucleotide sequence integrated into the host cell’s genome.
27. The method of claim 1, wherein at least one of the gRNA, Cas, and/or exonuclease are expressed on a vector, and wherein at least one of the gRNA, Cas, and/or exonuclease are expressed from a nucleotide sequence integrated into the host cell’s genome.
28. The method of claim 1, wherein the gRNA and exonuclease are expressed on a vector and the Cas is expressed from a nucleotide sequence integrated into the host cell’s genome.
29. The method of claim 1, wherein the Cas is a Class II endonuclease.
30. The method of claim 1, wherein the Cas is a Type II endonuclease.
31. The method of claim 1, wherein the Cas is a Cas9.
32. The method of claim 1, wherein the Cas is a Type V endonuclease.
33. The method of claim 1, wherein the Cas is a Cas12a.
34. The method of claim 1, wherein the exonuclease exhibits 3’ to 5’ exonuclease activity at the double stranded break site. Page 52 of 61 290326197 v3
35. The method of claim 1, wherein the exonuclease exhibits 5’ to 3’ exonuclease activity at the double stranded break site.
36. The method of claim 1, wherein the exonuclease is selected from the group consisting of: Trex1, Trex2, and DPD1.
37. The method of claim 1, wherein the exonuclease is Trex2.
38. The method of claim 1, wherein the exonuclease is DPD1.
39. The method of claim 1, wherein the exonuclease is POLQ.
40. The method of claim 1, wherein the exonuclease is a gene selected from Table 1.
41. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 1 or a sequence at least 75% identical thereto.
42. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 2 or a sequence at least 75% identical thereto.
44. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 4 or a sequence at least 75% identical thereto.
45. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 5 or a sequence at least 75% identical thereto.
46. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 6 or a sequence at least 75% identical thereto.
47. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 7 or a sequence at least 75% identical thereto.
48. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 8 or a sequence at least 75% identical thereto.
49. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 9 or a sequence at least 75% identical thereto.
51. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 11 or a sequence at least 75% identical thereto.
52. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 12 or a sequence at least 75% identical thereto.
53. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: or a sequence at least 75% identical thereto. Page 53 of 61 290326197 v3
54. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 14 or a sequence at least 75% identical thereto.
55. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 15 or a sequence at least 75% identical thereto.
56. The method of claim 1, wherein the exonuclease comprises SEQ ID NO: 16 or a sequence at least 75% identical thereto.
57. The method of claim 1, wherein each of the left and right homology arms are at least 100-bp long.
58. The method of claim 1, wherein each of the left and right homology arms are at least 200-bp long.
59. The method of claim 1, wherein each of the left and right homology arms are at least 300-bp long.
60. The method of claim 1, wherein each of the left and right homology arms are at least 400-bp long.
61. The method of claim 1, wherein each of the left and right homology arms are at least between 100-bp and 800-bp long.
62. The method of claim 1, wherein each of the left and right homology arms are at least between 100-bp and 1000-bp long.
63. The method of claim 1, wherein the left homology arm is at least 100-bp long.
64. The method of claim 1, wherein the left homology arm is at least 200-bp long.
65. The method of claim 1, wherein the left homology arm is at least 300-bp long.
66. The method of claim 1, wherein the left homology arm is at least 400-bp long.
67. The method of claim 1, wherein the left homology arm is at least between 100-bp and 800-bp long.
68. The method of claim 1, wherein the left homology arm is at least between 100-bp and 1000-bp long.
69. The method of claim 1, wherein the DNA sequence of interest is a coding sequence.
70. The method of claim 1, wherein the DNA sequence of interest is a gene.
71. The method of claim 1, wherein the DNA sequence of interest is a heterologous gene.
72. The method of claim 1, wherein the DNA sequence of interest is a non-heterologous gene.
73. The method of claim 1, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome. Page 54 of 61 290326197 v3
74. The method of claim 1, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome, which upon insertion causes a single base pair edit.
75. The method of claim 1, wherein the DNA sequence of interest causes a point mutation upon insertion into the host cell’s genome.
76. The method of claim 1, wherein the DNA sequence of interest is a non-coding sequence.
77. The method of claim 1, wherein the DNA sequence of interest is a non-coding regulatory sequence.
78. The method of claim 1, wherein the DNA sequence of interest is a promoter.
79. The method of claim 1, wherein the DNA sequence of interest is inserted into the host cell’s genome via single strand annealing.
80. The method of claim 1, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 5% frequency.
81. The method of claim 1, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 10% frequency.
82. The method of claim 1, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 20% frequency.
83. The method of claim 1, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 30% frequency.
84. The method of claim 1, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 40% frequency.
85. The method of claim 1, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 50% frequency.
86. The method of claim 1, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 60% frequency.
87. A method of targeted DNA sequence insertion into a host cell genome, comprising: a) determining a target site nucleic acid sequence in the host cell genome for DNA sequence insertion; b) providing a host cell; c) introducing into the host cell a guide RNA (gRNA) with a guide sequence having complementarity to the target site nucleic acid sequence in the host genome and a Cas endonuclease that interacts with the gRNA and is capable of creating a double stranded break in the host genome; Page 55 of 61 290326197 v3
d) introducing into the host cell an exonuclease; e) introducing into the host cell a donor DNA sequence template comprising: a DNA sequence of interest and left and right homology arm sequences, and f) introducing into the host cell a silencing construct, wherein the silencing construct knocks down expression of a nonhomologous end joining pathway gene; wherein the gRNA guide sequence binds to a complementary target site nucleic acid sequence in the host genome and the Cas endonuclease creates a double stranded break in the host genome; and wherein the DNA sequence of interest is inserted into the host genome via a homology dependent repair mechanism.
88. The method of claim 87, wherein the target site nucleic acid sequence is within a coding sequence.
89. The method of claim 87, wherein the target site nucleic acid sequence is within a gene.
90. The method of claim 87, wherein the target site nucleic acid sequence is within a non- coding sequence.
91. The method of claim 87, wherein the target site nucleic acid sequence is within a non- coding regulatory sequence.
92. The method of claim 87, wherein the target site nucleic acid sequence is within a promoter sequence region.
93. The method of claim 87, wherein the host cell is a eukaryotic cell.
94. The method of claim 87, wherein the host cell is a prokaryotic cell.
95. The method of claim 87, wherein the host cell is a mammalian cell.
96. The method of claim 87, wherein the host cell is a plant cell.
97. The method of claim 96, wherein the host cell is a plant protoplast cell.
98. The method of claim 87, wherein the host cell is an Angiosperm cell.
99. The method of claim 87, wherein the host cell is a dicot cell.
100. The method of claim 87, wherein the host cell is a monocot cell.
101. The method of claim 87, wherein the host cell is from the Solanum genus.
102. The method of claim 87, wherein the host cell is a Solanum tuberosum cell.
103. The method of claim 87, wherein the host cell is a potato cell.
104. The method of claim 87, wherein the host cell is a Solanum lycopersicum cell.
105. The method of claim 87, wherein the host cell is a tomato cell.
106. The method of claim 87, wherein the gRNA is a single gRNA. Page 56 of 61 290326197 v3
107. The method of claim 87, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex).
108. The method of claim 87, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro.
109. The method of claim 87, wherein the gRNA is complexed with the Cas endonuclease to form a CRISPR Cas ribonucleoprotein complex (RNP-complex) and the RNP-complex is provided to the host cell in vitro and the exonuclease is expressed on a transient vector.
110. The method of claim 87, wherein the gRNA, Cas, and/or exonuclease are expressed on the same vector.
111. The method of claim 87, wherein the gRNA, Cas, and/or exonuclease are expressed on a different vector.
112. The method of claim 87, wherein the gRNA, Cas, and/or exonuclease are expressed from a nucleotide sequence integrated into the host cell’s genome.
113. The method of claim 87, wherein at least one of the gRNA, Cas, and/or exonuclease are expressed on a vector, and wherein at least one of the gRNA, Cas, and/or exonuclease are expressed from a nucleotide sequence integrated into the host cell’s genome.
114. The method of claim 87, wherein the gRNA and exonuclease are expressed on a vector and the Cas is expressed from a nucleotide sequence integrated into the host cell’s genome.
115. The method of claim 87, wherein the Cas is a Class II endonuclease.
116. The method of claim 87, wherein the Cas is a Type II endonuclease.
117. The method of claim 87, wherein the Cas is a Cas9.
118. The method of claim 87, wherein the Cas is a Type V endonuclease.
119. The method of claim 87, wherein the Cas is a Cas12a.
120. The method of claim 87, wherein the exonuclease exhibits 3’ to 5’ exonuclease activity at the double stranded break site.
121. The method of claim 87, wherein the exonuclease exhibits 5’ to 3’ exonuclease activity at the double stranded break site.
122. The method of claim 87, wherein the exonuclease is selected from the group consisting of: Trex1, Trex2, and DPD1.
123. The method of claim 87, wherein the exonuclease is Trex2.
124. The method of claim 87, wherein the exonuclease is DPD1.
125. The method of claim 87, wherein the exonuclease is POLQ.
126. The method of claim 87, wherein the exonuclease is a gene selected from Table 1. Page 57 of 61 290326197 v3
127. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 1 or a sequence at least 75% identical thereto.
128. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 2 or a sequence at least 75% identical thereto.
129. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 3 or a sequence at least 75% identical thereto.
130. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 4 or a sequence at least 75% identical thereto.
131. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 5 or a sequence at least 75% identical thereto.
132. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 6 or a sequence at least 75% identical thereto.
133. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 7 or a sequence at least 75% identical thereto.
134. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 8 or a sequence at least 75% identical thereto.
135. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 9 or a sequence at least 75% identical thereto.
136. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 10 or a sequence at least 75% identical thereto.
137. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 11 or a sequence at least 75% identical thereto.
138. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 12 or a sequence at least 75% identical thereto.
139. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 13 or a sequence at least 75% identical thereto.
140. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 14 or a sequence at least 75% identical thereto.
141. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 15 or a sequence at least 75% identical thereto.
142. The method of claim 87, wherein the exonuclease comprises SEQ ID NO: 16 or a sequence at least 75% identical thereto.
143. The method of claim 87, wherein each of the left and right homology arms are at least 100-bp long. Page 58 of 61 290326197 v3
144. The method of claim 87, wherein each of the left and right homology arms are at least 200-bp long.
145. The method of claim 87, wherein each of the left and right homology arms are at least 300-bp long.
146. The method of claim 87, wherein each of the left and right homology arms are at least 400-bp long.
147. The method of claim 87, wherein each of the left and right homology arms are at least between 100-bp and 800-bp long.
148. The method of claim 87, wherein each of the left and right homology arms are at least between 100-bp and 1000-bp long.
149. The method of claim 87, wherein the left homology arm is at least 100-bp long.
150. The method of claim 87, wherein the left homology arm is at least 200-bp long.
151. The method of claim 87, wherein the left homology arm is at least 300-bp long.
152. The method of claim 87, wherein the left homology arm is at least 400-bp long.
153. The method of claim 87, wherein the left homology arm is at least between 100-bp and 800-bp long.
154. The method of claim 87, wherein the left homology arm is at least between 100-bp and 1000-bp long.
155. The method of claim 87, wherein the DNA sequence of interest is a coding sequence.
156. The method of claim 87, wherein the DNA sequence of interest is a gene.
157. The method of claim 87, wherein the DNA sequence of interest is a heterologous gene.
158. The method of claim 87, wherein the DNA sequence of interest is a non-heterologous gene.
159. The method of claim 87, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome.
160. The method of claim 87, wherein the DNA sequence of interest comprises a single nucleotide difference, as compared to a target site nucleic acid sequence in the host cell’s genome, which upon insertion causes a single base pair edit.
161. The method of claim 87, wherein the DNA sequence of interest causes a point mutation upon insertion into the host cell’s genome.
162. The method of claim 87, wherein the DNA sequence of interest is a non-coding sequence. Page 59 of 61 290326197 v3
163. The method of claim 87, wherein the DNA sequence of interest is a non-coding regulatory sequence.
164. The method of claim 87, wherein the DNA sequence of interest is a promoter.
165. The method of claim 87, wherein the DNA sequence of interest is inserted into the host cell’s genome via single strand annealing.
166. The method of claim 87, wherein the nonhomologous end joining pathway gene is a gene selected from Table 2.
167. The method of claim 87, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 17 or a sequence at least 75% identical thereto.
168. The method of claim 87, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 18 or a sequence at least 75% identical thereto.
169. The method of claim 87, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 19 or a sequence at least 75% identical thereto.
170. The method of claim 87, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 20 or a sequence at least 75% identical thereto.
171. The method of claim 87, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 21 or a sequence at least 75% identical thereto.
172. The method of claim 87, wherein the nonhomologous end joining pathway gene comprises SEQ ID NO: 22 or a sequence at least 75% identical thereto.
173. The method of claim 87, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 5% frequency.
174. The method of claim 87, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 10% frequency.
175. The method of claim 87, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 20% frequency.
176. The method of claim 87, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 30% frequency.
177. The method of claim 87, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 40% frequency.
178. The method of claim 87, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 50% frequency.
179. The method of claim 87, wherein the DNA sequence of interest is inserted into the host cell’s genome with at least a 60% frequency. Page 60 of 61 290326197 v3
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8697359B1 (en) | 2012-12-12 | 2014-04-15 | The Broad Institute, Inc. | CRISPR-Cas systems and methods for altering expression of gene products |
US8871445B2 (en) | 2012-12-12 | 2014-10-28 | The Broad Institute Inc. | CRISPR-Cas component systems, methods and compositions for sequence manipulation |
US8889418B2 (en) | 2012-12-12 | 2014-11-18 | The Broad Institute Inc. | Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation |
US20190093104A1 (en) | 2015-04-14 | 2019-03-28 | City Of Hope | Coexpression of cas9 and trex2 for targeted mutagenesis |
EP2633040B1 (en) | 2010-10-27 | 2019-07-10 | Cellectis | Method for increasing the efficiency of double-strand break-induced mutagenesis |
US10745690B2 (en) | 2011-02-28 | 2020-08-18 | Seattle Children's Hospital | Coupling endonucleases with end-processing enzymes drives high efficiency gene disruption |
-
2023
- 2023-09-01 WO PCT/US2023/073361 patent/WO2024050544A2/en unknown
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2633040B1 (en) | 2010-10-27 | 2019-07-10 | Cellectis | Method for increasing the efficiency of double-strand break-induced mutagenesis |
US10745690B2 (en) | 2011-02-28 | 2020-08-18 | Seattle Children's Hospital | Coupling endonucleases with end-processing enzymes drives high efficiency gene disruption |
US8697359B1 (en) | 2012-12-12 | 2014-04-15 | The Broad Institute, Inc. | CRISPR-Cas systems and methods for altering expression of gene products |
US8771945B1 (en) | 2012-12-12 | 2014-07-08 | The Broad Institute, Inc. | CRISPR-Cas systems and methods for altering expression of gene products |
US8871445B2 (en) | 2012-12-12 | 2014-10-28 | The Broad Institute Inc. | CRISPR-Cas component systems, methods and compositions for sequence manipulation |
US8889418B2 (en) | 2012-12-12 | 2014-11-18 | The Broad Institute Inc. | Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation |
US20190093104A1 (en) | 2015-04-14 | 2019-03-28 | City Of Hope | Coexpression of cas9 and trex2 for targeted mutagenesis |
Non-Patent Citations (35)
Title |
---|
"Coupling endonucleases with DNA end-processing enzymes to drive gene disruption", NAT METHODS, vol. 9, no. 10, October 2012 (2012-10-01), pages 973 - 5 |
"Molecular Probes publication 2908", IMAGENE GREEN, 1993, pages 1 - 4 |
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 10 |
CECCALDI RRONDINELLI BD'ANDREA AD: "Repair Pathway Choices and Consequences at the Double-Strand Break", TRENDS CELL BIOL, vol. 26, no. 1, January 2016 (2016-01-01), pages 52 - 64, XP029373553, DOI: 10.1016/j.tcb.2015.07.009 |
CEJKA P.: "DNA End Resection: Nucleases Team Up with the Right Partners to Initiate Homologous Recombination", J BIOL CHEM., vol. 290, no. 38, 18 September 2015 (2015-09-18), pages 22931 - 8 |
CHAREST ET AL., PLANT CELL REP, vol. 8, 1990, pages 643 |
COMAI ET AL., NATURE, vol. 317, 1985, pages 741 - 744 |
DEBLOCK ET AL., EMBO J., vol. 3, 1984, pages 1681 |
EICHHOLTZ ET AL., SOMATIC CELL MOL. GENET., vol. 13, 1987, pages 67 |
EL-SAPPAH A. ET AL.: "Comprehensive Mechanism of Gene Silencing and Its Role in Plant Growth and Development", FRONT PLANT SCI, vol. 12, 7 September 2021 (2021-09-07), pages 705249 |
FRALEY ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 80, 1983, pages 4803 |
GORDON-KAMM ET AL., PLANT CELL, vol. 2, 1990, pages 603 - 618 |
HAYFORD ET AL., PLANT PHYSIOL., vol. 86, 1988, pages 1216 |
HELLIWELL CAWATERHOUSE PM: "Constructs and Methods for Hairpin RNA-Mediated Gene Silencing in Plants", METHODS IN ENZYMOLOGY, vol. 392, 2005, pages 24 - 35 |
HILLE ET AL., PLANT MOL. BIOL., vol. 7, 1986, pages 171 |
HUAMAN ET AL., AM. J. OF BOTANY, vol. 89, no. 6, 2002, pages 947 - 965 |
JEFFERSON, R. A., PLANT MOL. BIOL. REP., vol. 5, pages 387 |
JONES ET AL., MOL. GEN. GENET., vol. 210, 1987, pages 86 |
KONCZ ET AL., PROC. NATL. ACAD. SCI. USA, vol. 84, 1987, pages 131 |
LARKIN ET AL., BIOINFORMATICS, vol. 23, 2007, pages 2947 - 294 |
MAKAROVA, K. S., NAT REV MICROBIOL, vol. 9, no. 6, 9 May 2011 (2011-05-09), pages 467 - 477 |
MAZUR DJPERRINO FW: "Identification and expression of the TREX1 and TREX2 cDNA sequences encoding mammalian 3'-->5' exonucleases", J BIOL CHEM., vol. 274, no. 28, 9 July 1999 (1999-07-09), pages 19655 - 60 |
MEYERSMILLER, COMPUTER APPLIC. BIOL. SCI., vol. 4, 1988, pages 11 - 17 |
NALEWAY ET AL., J. CELL BIOL., vol. 115, 1991, pages 151a |
ROGOWSKY P.: "CRISPR-Cas Technology in Plant Science", POTATO RESEARCH, vol. 60, 2017, pages 353 - 360, XP036581428, DOI: 10.1007/s11540-018-9387-y |
SHAH ET AL., SCIENCE, vol. 233, 1986, pages 478 |
STALKER ET AL., SCIENCE, vol. 242, 1988, pages 419 - 423 |
SVAB ET AL., PLANT MOL. BIOL., vol. 14, 1990, pages 197 |
TEERI ET AL., EMBO J., vol. 8, 1989, pages 343 |
TINLAND ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 7442 - 6 |
VANDEN ELZEN ET AL., PLANT MOL. BIOL., vol. 5, 1985, pages 299 |
WEISS TWANG CKANG XZHAO HELENA GAMO MSTARKER CGCRISP PAZHOU PSPRINGER NMVOYTAS DF: "Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis", PLANT J, vol. 104, no. 3, November 2020 (2020-11-01), pages 828 - 838 |
WESLEY SV ET AL.: "Construct design for efficient, effective and high-throughput gene silencing in plants", PLANT J, vol. 27, no. 6, September 2001 (2001-09-01), pages 581 - 90, XP002187670, DOI: 10.1046/j.1365-313X.2001.01105.x |
WIEDENHEFT ET AL., NATURE, vol. 482, 2012, pages 331 - 8 |
YAN H ET AL.: "New construct approaches for efficient gene silencing in plants", PLANT PHYSIOL., vol. 141, no. 4, August 2006 (2006-08-01), pages 1508 - 18, XP002458540, DOI: 10.1104/pp.106.082271 |
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