US20210087573A1 - Methods and compositions for homology-directed repair of cas endonuclease mediated double strand breaks - Google Patents

Methods and compositions for homology-directed repair of cas endonuclease mediated double strand breaks Download PDF

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US20210087573A1
US20210087573A1 US17/052,320 US201917052320A US2021087573A1 US 20210087573 A1 US20210087573 A1 US 20210087573A1 US 201917052320 A US201917052320 A US 201917052320A US 2021087573 A1 US2021087573 A1 US 2021087573A1
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sequence
double
polynucleotide
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Stephen Lawrence Gasior
Sergi Svitashev
Joshua K Young
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Pioneer Hi Bred International Inc
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

  • sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 7664USPCT_SequenceListing_ST25.txt created on 15 Oct. 2020 and having a size of 20,147 bytes and is filed concurrently with the specification.
  • sequence listing comprised in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
  • the disclosure relates to the field of molecular biology, in particular to compositions of guide polynucleotide/endonuclease systems, and compositions and methods for modifying the genome of a cell.
  • Recombinant DNA technology has made it possible to insert DNA sequences at targeted genomic locations and/or modify specific endogenous chromosomal sequences.
  • Site-specific integration techniques which employ site-specific recombination systems, as well as other types of recombination technologies, have been used to generate targeted insertions of genes of interest in a variety of organism.
  • Genome-editing techniques such as designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or homing meganucleases, are available for producing targeted genome perturbations, but these systems tend to have low specificity and employ designed nucleases that need to be redesigned for each target site, which renders them costly and time-consuming to prepare.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • HDR homology-directed repair
  • HDR of a target site DSB is promoted by recurrent cutting (RC) of the target site.
  • Recurrent cutting may be accomplished by creating a first double-strand break with a DSB agent, such as a Cas endonuclease, at the target site, and allowing it to repair via the NHEJ pathway.
  • the repaired double-strand break is then provided an RNA-guided Cas endonuclease, wherein the guide RNA comprises a sequence that is capable of hybridizing with the repaired DSB, which becomes a new “target sequence”.
  • a second double-strand break is create by the Cas endonuclease.
  • a single recurrent cut is performed.
  • recursive iterations of recurrent cuts are performed, e.g. a subsequent DSB is created at the newly repaired previously DSB, that becomes a new target site. In some aspects, three, four, five, six, or more than six recurrent cuts are performed in such a recursive fashion.
  • the DSB agent is a Cas endonuclease, for example, a Cas9 endonuclease. It is contemplated that any Cas endonuclease that cleaves a target polynucleotide within the recognition site may be used for any of the recurrent or recursive methods described herein. In principle, any DSB agent may be used.
  • HDR of a target site DSB is promoted by the creation of one or more nick sites adjacent to the double-strand break of the target site. In some aspects, this is accomplished by using one or multiple single-strand endonucleases targeted to the flanking regions of the double-strand break in a strand specific manner. In some aspects, the single-strand endonuclease(s) must be heterologous to the double-strand endonuclease when using RNA guides for targeting. In some aspects, a non-RNA guided single-strand endonuclease may be designed to target the flanking sequences. In some aspects, two nicks are created, each one flanking one side of the target site or the DSB.
  • the DSB agent is a Cas endonuclease, for example, a Cas9 endonuclease.
  • the nickase is a Cas endonuclease that has been modified to retain single strand nicking activity but lacks double strand breaking activity.
  • any DSB agent in combination with any nickase agent may be used.
  • the distance between a nick and the DSB is at least 20, between 20 and 30, at least 30, between 30 and 40, at least 40, between 40 and 50, at least 50, between 50 and 60, at least 60, between 60 and 70, at least 70, between 70 and 80, at least 80, between 80 and 90, at least 90, between 90 and 100, at least 100, between 100 and 125, at least 125, between 125 and 150, at least 150, between 150 and 175, at least 175, between 175 and 200, at least 200, or even greater than 200 base pairs.
  • the method further includes providing to the double-strand break a heterologous polynucleotide that comprises a DNA sequence, optionally flanked by polynucleotides sharing homology to sequences flanking the target site.
  • the DNA sequence is further flanked by polynucleotides sharing homology to the target site sequence.
  • the DNA sequences is further flanked by two polynucleotides, wherein each of the two polynucleotides shares homology to a mutation created by a repair of the double-strand break.
  • the mutation may be one of the NHEJ mutations that arise after the initial repair of the DSB, for example the two polynucleotides may each share homology to the first most prevalent, second most prevalent, or any other NHEJ mutation that arises after the initial repair of the DSB.
  • a plurality of DNA sequences that comprise different flanking homology regions maybe provided. Said plurality may be DNA sequences that are identical to each other and to either the original target site or to new target sites created by DSB repair, or said plurality may be a population of different DNA sequences, each sharing homology with a combination of any of the original target site and/or one or more new target sites created by subsequent DSB creation and repair.
  • the heterologous polynucleotide that comprises a DNA sequence is a “donor” DNA molecule that becomes inserted at the site of a DSB. In some aspects, the heterologous polynucleotide that comprises a DNA sequence is a “modification template” that provides the basis for template-directed repair of a DSB.
  • any target polynucleotide may be used in any aspect of the method described herein.
  • the target polynucleotide is in an in vitro setting.
  • the target polynucleotide is in a cell, for example in the genome of a cell.
  • the cell is an animal cell, a fungal cell, a bacterial cell, or a plant cell.
  • a method for obtaining a multicellular organism, tissue, or part from a cell that has had its genome modified by any of the methods or compositions described herein, wherein the multicellular organism, tissue, or part retains the modification.
  • FIG. 1 depicts some examples of DNA expression cassettes and circular double-stranded plasmid DNA molecules, that may be a donor DNA molecule for insertion at a DSB and/or a template used for repair (“Donor/Repair Template”, “DRT”) that may be used for recurrent cleavage of a target polynucleotide to improve the frequency of HDR.
  • FIG. 1A depicts a linear single-stranded DNA flanked by homology regions.
  • FIG. 1B depicts a linear double-stranded DNA flanked by homology regions.
  • FIG. 1C depicts a linear double-stranded DNA flanked by homology regions and further flanked by sequences sharing homology with the target site.
  • FIG. 1D depicts a circular double-stranded DNA flanked by homology regions.
  • FIG. 1E depicts a circular double-stranded DNA flanked by both homology regions and further flanked by sequences sharing homology with the target site.
  • FIG. 2A shows one variation of the recurrent cleavage method for improving HDR of a target polynucleotide DSB.
  • FIG. 2B shows one variation of the recursive cleavage method for improving HDR of a target polynucleotide DSB.
  • FIG. 3 depicts a schematic of the break-nick method for improving HDR of a target polynucleotide DSB, with one double-strand break created and two flanking nicks created on either side of the DSB.
  • FIG. 4A depicts target sites of the nickase SpynCas9 guides near the MS45-BLAT2 target sequence, labeled 1, 2, 3, and 4, respectively.
  • FIGS. 4B and 4C show the frequencies of mutations at the target site when: BLATCas9 and SpynCas9 were delivered but no guides were provided (no guide); when BLAT and its cognate guide were used by itself (BLAT only); and when BLAT and its cognate guide was co-delivered with SpynCas9 and specified Spy guides. Mutant reads for each are shown in FIG. 4B and HDR frequencies for each are shown in FIG. 4C .
  • FIG. 4B shows HDR frequencies for each of FIG. 4C .
  • FIGS. 4E and 4F show the frequencies of mutations at the target site for: BlatCas9 and nSpynCas9 were delivered but no guides were provided (no guide); when Blat and its cognate guide were used by itself (Blat only); when Blat and its cognate guide were co-delivered with nickase and specified paired nick site Spyn guides (5/3 and 5/4 pairs); when SpynCas9 nickase and nick site guides (5/3 pair) and were delivered without Blat or the Blat guide; and when BlatCas9, Blat guide, and SpynGuides were delivered without co-delivering the nickase Spyn Cas9. Mutant reads for each are shown in FIG. 4E and HDR frequencies for each are shown in FIG. 4F .
  • the target site is given as SEQID NO:73.
  • FIG. 5 depicts a “Blat-Spy-Blat” nickase-DSB agent-nickase strategy ( FIG. 5A ) performed at maize target site M16 in one germplasm line, for five replicates.
  • the M16 sgRNA guide sequence is given as SEQID NO:77.
  • Average mutant reads for are shown in FIG. 5B and HDR frequencies shown in FIG. 5C , for: SpyCas9 delivered with no guides; SpyCas9 delivered with its cognate gRNA; SpyCas9, its cognate gRNA, nickase Blat and its guides; and Blat nickase with its guides only, with no SpyCas9 or its cognate gRNA.
  • the Blat-Spy-Blat strategy ( FIG. 5D ) was also performed at a different maize target site (NLB-CR8) in a different germplasm line, for five replicates.
  • the NLB_8 sgRNA guide sequence is given as SEQID NO:78.
  • Average mutant reads for are shown in FIG. 5E and HDR frequencies shown in FIG. 5F , for: SpyCas9 delivered with no guides; SpyCas9 delivered with its cognate gRNA; SpyCas9, its cognate gRNA, nickase Blat and its guides; and Blat nickase with its guides only, with no SpyCas9 or its cognate gRNA.
  • FIG. 6 depicts different strategies with S. pyogenes Cas9 as either a nickase or DSB agent, and S. aureus Cas9 as either a DSB agent or nickase, respectively.
  • a “Spy-Sa-Spy” strategy ( FIG. 6A ) was performed at the M545 maize genomic target site, for four replicates.
  • the MS45 Sa sgRNA guide sequence is given as SEQID NO:75. Average mutant reads for are shown in FIG. 6B and HDR frequencies shown in FIG.
  • SaCas9 delivered with no guides for: SaCas9 delivered with no guides; SaCas9 delivered with its cognate gRNA; SaCas9, its cognate gRNA, nickase SpynCas9 and its guides; and SpynCas9 nickase with its guides only, with no SaCas9 or its cognate gRNA.
  • a “Sa-Spy-Sa” strategy ( FIG. 6D ) was performed at the TS50 maize genomic target site, for three replicates.
  • the TS50 sgRNA guide sequence is given as SEQID NO:76. Average mutant reads for are shown in FIG. 6E and HDR frequencies shown in FIG.
  • SpyCas9 delivered with no guides for: SpyCas9 delivered with no guides; SpyCas9 delivered with its cognate gRNA; SpyCas9, its cognate gRNA, nickase SaCas9 and its guides; and SaCas9 nickase with its guides only, with no SpyCas9 or its cognate gRNA.
  • the “Sa-Spy-Sa” strategy (The “Sa-Spy-Sa” strategy ( FIG. 6G ) was performed at a different maize genomic target site (TS45), for two replicates. Average mutant reads for are shown in FIG. 6H and HDR frequencies shown in FIG.
  • SpyCas9 delivered with no guides for: SpyCas9 delivered with its cognate gRNA; SpyCas9, its cognate gRNA, nickase SaCas9 and its guides; and SaCas9 nickase with its guides only, with no SpyCas9 or its cognate gRNA.
  • FIG. 7 shows the top 5 most prevalent NHEJ mutations for the Zea mays LIG1 target site ( FIG. 7A ), MS26 target site ( FIG. 7B ), MS45 target site ( FIG. 7C ), and TS45 target site ( FIG. 7D ).
  • Streptococcus pyogenes Cas9 targets are underlined with the protospacer adjacent motif (PAM) being underlined with a solid line and the guide RNA target being underlined with a dashed line.
  • PAM protospacer adjacent motif
  • Target sites targeted for cleavage by recurrent Cas9, the initial target, NHEJ mutation 1, NHEJ mutation 2 are boxed.
  • FIG. 8 shows the HDR frequencies with and without recurrent Cas9 cleavage for the Zea mays LIG1 target site.
  • % HDR was increased over single cutting at the initial target (iTarget) when both the iTarget and the first most prevalent NHEJ mutation (NHEJ1) were both targeted for cleavage.
  • % HDR was increased over single cutting at the initial target (iTarget) when both the iTarget and the first and second most prevalent NHEJ mutations (NHEJ1 and NHEJ2) were targeted for cleavage. Relative to experiments targeting only the iTarget (and without flanking DNA targets), the fold increase in HDR was nearly 20-fold.
  • FIG. 9 shows a recurrent cutting (RC) and template-directed repair HDR strategy for a template flanked by both homology regions and sequences sharing homology to the initial target (iTarget) site ( FIG. 9A ), for a template flanked by both homology regions and sequences sharing homology to the first most prevalent NHEJ mutation (NHEJ1) ( FIG. 9B ), and for a template flanked by both homology regions and sequences sharing no homology to either the iTarget or to the TS first most prevalent mutation, but instead to site with no homology to the iTarget ( FIG. 9C ).
  • RC recurrent cutting
  • NHEJ1 first most prevalent NHEJ mutation
  • FIG. 10 shows that flanking the DRT with a target site increased the frequency of HDR and frequencies were further elevated using RC Cas9. Notably, HDR outcomes were the highest when RC Cas9 and a DRT with partially homologous flanking target sites were used.
  • FIG. 11 shows the HDR frequencies with recurrent cutting (RC) and different flanking targets for the Zea mays MS26 target site.
  • FIG. 12 shows the HDR frequencies with recurrent cutting (RC) and different flanking targets for the Zea mays MS45 target site.
  • FIG. 13 shows the averages of HDR frequencies with RC Cas9 and different flanking targets across all target sites tested.
  • RC Cas9 with a repair template flanked by a site partially homologous to the iTarget provided a 28-fold increase in HDR frequencies relative to experiments when only the iTarget (without DRT flanking sequences) was cleaved.
  • flanking targets that contained homology with the iTarget enhanced HDR with the highest frequencies being recovered for targets with partial homology (NHEJ1) to the iTarget.
  • NHEJ1 partial homology
  • FIG. 14 shows a PCR-based method for detecting HDR ( FIG. 14A ) and an agarose gel depicting the frequency of transformed callus tissue positive for HDR with results from the iTarget only and no sites flanking the DRT ( FIG. 14B ) and for the recurrent cutting Cas9 experiment with the NHEJ1 flanking the DRT ( FIG. 14C ) being shown.
  • the MS26 HDR edit was detectable in almost all of the transformed tissues as compared to only a few instances in the control.
  • FIG. 15 shows the percentage of regenerated plantlets with HDR from cleavage (iTarget only) or recurrent cleavage (with NHEJ1 flanking the DRT) at two different target sites in maize (LIG1 and MS26).
  • RC significantly improves the HDR outcome not only in the germline, but also increased the frequency of bi-allelic HDR.
  • FIG. 16 shows the strategy for an RC approach for the insertion of larger DNA fragments (e.g. transgenes).
  • FIG. 17 shows HDR frequencies for single cleavage without flanking targets (iTarget only, No Sites Flanking the DRT) and with flanking targets (iTarget only, iTarget Flanking the DRT), as compared to RC with NHEJ1 flanking the DRT.
  • flanking targets iTarget only, No Sites Flanking the DRT
  • flanking targets iTarget only, iTarget Flanking the DRT
  • the proportion of HDR edits for RC Cas9 that were determined to be bi-allelic was significantly enhanced compared to the other treatments (3.3% for RC Cas9, 0.7% for iTarget (with DRTs with iTarget sites), and 0.0% for iTarget (without sites flanking the DRT).
  • the proportion of clean plants, defined as having no other DNA integrated (e.g. Cas9, sgRNA, BBM, Wus) except the NPTII expression cassette at the target site was also elevated for RC Cas9.
  • sequence descriptions and sequence listing attached hereto comply with the rules governing nucleotide and amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. ⁇ 1.821 and 1.825.
  • sequence descriptions comprise the three letter codes for amino acids as defined in 37 C.F.R. ⁇ 1.821 and 1.825, which are incorporated herein by reference.
  • compositions and methods are provided for increasing the probability of homology-directed repair as the preferred outcome of double-strand-break repair of a polynucleotide.
  • nucleic acid means a polynucleotide and includes a single or a double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include fragments and modified nucleotides. Thus, the terms “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence” and “nucleic acid fragment” are used interchangeably to denote a polymer of RNA and/or DNA and/or RNA-DNA that is single- or double-stranded, optionally comprising synthetic, non-natural, or altered nucleotide bases.
  • Nucleotides are referred to by their single letter designation as follows: “A” for adenosine or deoxyadenosine (for RNA or DNA, respectively), “C” for cytosine or deoxycytosine, “G” for guanosine or deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
  • gene as it applies to a prokaryotic and eukaryotic cell or organism cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondria, or plastid) of the cell.
  • ORF Open reading frame
  • sequences include reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids.
  • Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) with each other.
  • stringent conditions or “stringent hybridization conditions” includes reference to conditions under which a probe will selectively hybridize to its target sequence in an in vitro hybridization assay. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salt(s)) at pH 7.0 to 8.3, and at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5 ⁇ to 1 ⁇ SSC at 55 to 60° C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1 ⁇ SSC at 60 to 65° C.
  • homology is meant DNA sequences that are similar.
  • a “region of homology to a genomic region” that is found on the donor DNA is a region of DNA that has a similar sequence to a given “genomic region” in the cell or organism genome.
  • a region of homology can be of any length that is sufficient to promote homologous recombination at the cleaved target site.
  • the region of homology can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900, 5-3000, 5-3100 or more bases in length such that the region of homology has sufficient homology to undergo homologous recombination with the corresponding genomic region.
  • “Sufficient homology” indicates that two polynucleotide sequences have sufficient structural similarity to act as substrates for a homologous recombination reaction.
  • the structural similarity includes overall length of each polynucleotide fragment, as well as the sequence similarity of the polynucleotides. Sequence similarity can be described by the percent sequence identity over the whole length of the sequences, and/or by conserved regions comprising localized similarities such as contiguous nucleotides having 100% sequence identity, and percent sequence identity over a portion of the length of the sequences.
  • genomic region is a segment of a chromosome in the genome of a cell that is present on either side of the target site or, alternatively, also comprises a portion of the target site.
  • the genomic region can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800. 5-2900, 5-3000, 5-3100 or more bases such that the genomic region has sufficient homology to undergo homologous recombination with the corresponding region
  • homologous recombination includes the exchange of DNA fragments between two DNA molecules at the sites of homology.
  • the frequency of homologous recombination is influenced by a number of factors. Different organisms vary with respect to the amount of homologous recombination and the relative proportion of homologous to non-homologous recombination.
  • the length of the region of homology affects the frequency of homologous recombination events: the longer the region of homology, the greater the frequency.
  • the length of the homology region needed to observe homologous recombination is also species-variable.
  • Sequence identity or “identity” in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity.
  • percent sequence identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any percentage from 50% to 100%. These identities can be determined using any of the programs described herein.
  • Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
  • sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified.
  • default values will mean any set of values or parameters that originally load with the software when first initialized.
  • Clustal V method of alignment corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci 8:189-191) and found in the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
  • Clustal W method of alignment corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci 8:189-191) and found in the MegAlignTM v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
  • sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) using the following parameters: % identity and % similarity for a nucleotide sequence using a gap creation penalty weight of 50 and a gap length extension penalty weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using a GAP creation penalty weight of 8 and a gap length extension penalty of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).
  • GAP uses the algorithm of Needleman and Wunsch, (1970) J Mol Biol 48:443-53, to find an alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps, using a gap creation penalty and a gap extension penalty in units of matched bases.
  • BLAST is a searching algorithm provided by the National Center for Biotechnology Information (NCBI) used to find regions of similarity between biological sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches to identify sequences having sufficient similarity to a query sequence such that the similarity would not be predicted to have occurred randomly.
  • BLAST reports the identified sequences and their local alignment to the query sequence. It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides from other species or modified naturally or synthetically wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any percentage from 50% to 100%.
  • any amino acid identity from 50% to 100% may be useful in describing the present disclosure, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • Polynucleotide and polypeptide sequences, variants thereof, and the structural relationships of these sequences can be described by the terms “homology”, “homologous”, “substantially identical”, “substantially similar” and “corresponding substantially” which are used interchangeably herein. These refer to polypeptide or nucleic acid sequences wherein changes in one or more amino acids or nucleotide bases do not affect the function of the molecule, such as the ability to mediate gene expression or to produce a certain phenotype. These terms also refer to modification(s) of nucleic acid sequences that do not substantially alter the functional properties of the resulting nucleic acid relative to the initial, unmodified nucleic acid.
  • nucleic acid fragments include deletion, substitution, and/or insertion of one or more nucleotides in the nucleic acid fragment.
  • Substantially similar nucleic acid sequences encompassed may be defined by their ability to hybridize (under moderately stringent conditions, e.g., 0.5 ⁇ SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein.
  • Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.
  • centimorgan or “map unit” is the distance between two polynucleotide sequences, linked genes, markers, target sites, loci, or any pair thereof, wherein 1% of the products of meiosis are recombinant.
  • a centimorgan is equivalent to a distance equal to a 1% average recombination frequency between the two linked genes, markers, target sites, loci, or any pair thereof.
  • an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, or protein, or biologically active portion thereof is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment.
  • an isolated or purified polynucleotide or polypeptide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an “isolated” polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.
  • the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived.
  • Isolated polynucleotides may be purified from a cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
  • fragment refers to a contiguous set of nucleotides or amino acids. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater than 20 contiguous nucleotides. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater than 20 contiguous amino acids. A fragment may or may not exhibit the function of a sequence sharing some percent identity over the length of said fragment.
  • fragment that is functionally equivalent and “functionally equivalent fragment” are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment or polypeptide that displays the same activity or function as the longer sequence from which it derives. In one example, the fragment retains the ability to alter gene expression or produce a certain phenotype whether or not the fragment encodes an active protein. For example, the fragment can be used in the design of genes to produce the desired phenotype in a modified plant. Genes can be designed for use in suppression by linking a nucleic acid fragment, whether or not it encodes an active enzyme, in the sense or antisense orientation relative to a plant promoter sequence.
  • Gene includes a nucleic acid fragment that expresses a functional molecule such as, but not limited to, a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence.
  • “Native gene” refers to a gene as found in its natural endogenous location with its own regulatory sequences.
  • endogenous it is meant a sequence or other molecule that naturally occurs in a cell or organism.
  • an endogenous polynucleotide is normally found in the genome of a cell; that is, not heterologous.
  • an “allele” is one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same, that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ, that plant is heterozygous at that locus.
  • Coding sequence refers to a polynucleotide sequence which codes for a specific amino acid sequence.
  • Regulatory sequences refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, promoters, translation leader sequences, 5′ untranslated sequences, 3′ untranslated sequences, introns, polyadenylation target sequences, RNA processing sites, effector binding sites, and stem-loop structures.
  • a “mutated gene” is a gene that has been altered through human intervention. Such a “mutated gene” has a sequence that differs from the sequence of the corresponding non-mutated gene by at least one nucleotide addition, deletion, or substitution. In certain embodiments of the disclosure, the mutated gene comprises an alteration that results from a guide polynucleotide/Cas endonuclease system as disclosed herein.
  • a mutated plant is a plant comprising a mutated gene.
  • a “targeted mutation” is a mutation in a gene (referred to as the target gene), including a native gene, that was made by altering a target sequence within the target gene using any method known to one skilled in the art, including a method involving a guided Cas endonuclease system as disclosed herein.
  • knock-out represents a DNA sequence of a cell that has been rendered partially or completely inoperative by targeting with a Cas protein; for example, a DNA sequence prior to knock-out could have encoded an amino acid sequence, or could have had a regulatory function (e.g., promoter).
  • knock-in represents the replacement or insertion of a DNA sequence at a specific DNA sequence in cell by targeting with a Cas protein (for example by homologous recombination (HR), wherein a suitable donor DNA polynucleotide is also used).
  • a Cas protein for example by homologous recombination (HR), wherein a suitable donor DNA polynucleotide is also used.
  • knock-ins are a specific insertion of a heterologous amino acid coding sequence in a coding region of a gene, or a specific insertion of a transcriptional regulatory element in a genetic locus.
  • domain it is meant a contiguous stretch of nucleotides (that can be RNA, DNA, and/or RNA-DNA-combination sequence) or amino acids.
  • conserved domain or “motif” means a set of polynucleotides or amino acids conserved at specific positions along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential to the structure, the stability, or the activity of a protein. Because they are identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers, or “signatures”, to determine if a protein with a newly determined sequence belongs to a previously identified protein family.
  • a “codon-modified gene” or “codon-preferred gene” or “codon-optimized gene” is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.
  • An “optimized” polynucleotide is a sequence that has been optimized for improved expression in a particular heterologous host cell.
  • a “plant-optimized nucleotide sequence” is a nucleotide sequence that has been optimized for expression in plants, particularly for increased expression in plants.
  • a plant-optimized nucleotide sequence includes a codon-optimized gene.
  • a plant-optimized nucleotide sequence can be synthesized by modifying a nucleotide sequence encoding a protein such as, for example, a Cas endonuclease as disclosed herein, using one or more plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage.
  • a “promoter” is a region of DNA involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • An “enhancer” is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, and/or comprise synthetic DNA segments.
  • promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity.
  • inducible promoter refers to a promoter that selectively express a coding sequence or functional RNA in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals.
  • inducible or regulated promoters include, for example, promoters induced or regulated by light, heat, stress, flooding or drought, salt stress, osmotic stress, phytohormones, wounding, or chemicals such as ethanol, abscisic acid (ABA), jasmonate, salicylic acid, or safeners.
  • Translation leader sequence refers to a polynucleotide sequence located between the promoter sequence of a gene and the coding sequence.
  • the translation leader sequence is present in the mRNA upstream of the translation start sequence.
  • the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (e.g., Turner and Foster, (1995) Mol Biotechnol 3:225-236).
  • 3′ non-coding sequences refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor.
  • the use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al., (1989) Plant Cell 1:671-680.
  • RNA transcript refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complimentary copy of the DNA sequence, it is referred to as the primary transcript or pre-mRNA. An RNA transcript is referred to as the mature RNA or mRNA when it is an RNA sequence derived from post-transcriptional processing of the primary transcript pre-mRNA. “Messenger RNA” or “mRNA” refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a DNA that is complementary to, and synthesized from, an mRNA template using the enzyme reverse transcriptase.
  • RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro.
  • Antisense RNA refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA, and that blocks the expression of a target gene (see, e.g., U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence.
  • RNA refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.
  • complement and “reverse complement” are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.
  • genomic refers to the entire complement of genetic material (genes and non-coding sequences) that is present in each cell of an organism, or virus or organelle; and/or a complete set of chromosomes inherited as a (haploid) unit from one parent.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other.
  • a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.
  • the complementary RNA regions can be operably linked, either directly or indirectly, 5′ to the target mRNA, or 3′ to the target mRNA, or within the target mRNA, or a first complementary region is 5′ and its complement is 3′ to the target mRNA.
  • a “host” refers to an organism or cell into which a heterologous component (polynucleotide, polypeptide, other molecule, cell) has been introduced.
  • a “host cell” refers to an in vivo or in vitro eukaryotic cell, prokaryotic cell (e.g., bacterial or archaeal cell), or cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, into which a heterologous polynucleotide or polypeptide has been introduced.
  • the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, an insect cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell.
  • the cell is in vitro. In some cases, the cell is in vivo.
  • recombinant refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis, or manipulation of isolated segments of nucleic acids by genetic engineering techniques.
  • Plasmid refers to a linear or circular extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of double-stranded DNA.
  • Such elements may be autonomously replicating sequences, genome integrating sequences, phage, or nucleotide sequences, in linear or circular form, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a polynucleotide of interest into a cell.
  • Transformation cassette refers to a specific vector comprising a gene and having elements in addition to the gene that facilitates transformation of a particular host cell.
  • Expression cassette refers to a specific vector comprising a gene and having elements in addition to the gene that allow for expression of that gene in a host.
  • a recombinant DNA construct comprises an artificial combination of nucleic acid sequences, e.g., regulatory and coding sequences that are not all found together in nature.
  • a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • Such a construct may be used by itself or may be used in conjunction with a vector.
  • a vector is used, then the choice of vector is dependent upon the method that will be used to introduce the vector into the host cells as is well known to those skilled in the art.
  • a plasmid vector can be used.
  • the skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells.
  • the skilled artisan will also recognize that different independent transformation events may result in different levels and patterns of expression (Jones et al., (1985) EMBO J 4:2411-2418; De Almeida et al., (1989) Mol Gen Genetics 218:78-86), and thus that multiple events are typically screened in order to obtain lines displaying the desired expression level and pattern.
  • Such screening may be accomplished standard molecular biological, biochemical, and other assays including Southern analysis of DNA, Northern analysis of mRNA expression, PCR, real time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblotting analysis of protein expression, enzyme or activity assays, and/or phenotypic analysis.
  • Southern analysis of DNA Northern analysis of mRNA expression, PCR, real time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblotting analysis of protein expression, enzyme or activity assays, and/or phenotypic analysis.
  • heterologous refers to the difference between the original environment, location, or composition of a particular polynucleotide or polypeptide sequence and its current environment, location, or composition.
  • Non-limiting examples include differences in taxonomic derivation (e.g., a polynucleotide sequence obtained from Zea mays would be heterologous if inserted into the genome of an Oryza sativa plant, or of a different variety or cultivar of Zea mays ; or a polynucleotide obtained from a bacterium was introduced into a cell of a plant), or sequence (e.g., a polynucleotide sequence obtained from Zea mays , isolated, modified, and re-introduced into a maize plant).
  • heterologous in reference to a sequence can refer to a sequence that originates from a different species, variety, foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.
  • one or more regulatory region(s) and/or a polynucleotide provided herein may be entirely synthetic.
  • expression refers to the production of a functional end-product (e.g., an mRNA, guide RNA, or a protein) in either precursor or mature form.
  • a functional end-product e.g., an mRNA, guide RNA, or a protein
  • a “mature” protein refers to a post-translationally processed polypeptide (i.e., one from which any pre- or propeptides present in the primary translation product have been removed).
  • Precursor protein refers to the primary product of translation of mRNA (i.e., with pre- and propeptides still present). Pre- and propeptides may be but are not limited to intracellular localization signals.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • a CRISPR locus can consist of a CRISPR array, comprising short direct repeats (CRISPR repeats) separated by short variable DNA sequences (called spacers), which can be flanked by diverse Cas (CRISPR-associated) genes.
  • an “effector” or “effector protein” is a protein that encompasses an activity including recognizing, binding to, and/or cleaving or nicking a polynucleotide target.
  • An effector, or effector protein may also be an endonuclease.
  • the “effector complex” of a CRISPR system includes Cas proteins involved in crRNA and target recognition and binding. Some of the component Cas proteins may additionally comprise domains involved in target polynucleotide cleavage.
  • Cas protein refers to a polypeptide encoded by a Cas (CRISPR-associated) gene.
  • a Cas protein includes but is not limited to: a Cas9 protein, a Cpf1 (Cas12) protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, Cas10, or combinations or complexes of these.
  • a Cas protein may be a “Cas endonuclease” or “Cas effector protein”, that when in complex with a suitable polynucleotide component, is capable of recognizing, binding to, and optionally nicking or cleaving all or part of a specific polynucleotide target sequence.
  • a Cas endonuclease described herein comprises one or more nuclease domains.
  • the endonucleases of the disclosure may include those having one or more RuvC nuclease domains.
  • a Cas protein is further defined as a functional fragment or functional variant of a native Cas protein, or a protein that shares at least 50%, between 50% and 55%, at least 55%, between 55% and 60%, at least 60%, between 60% and 65%, at least 65%, between 65% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 85%, at least 85%, between 85% and 90%, at least 90%, between 90% and 95%, at least 95%, between 95% and 96%, at least 96%, between 96% and 97%, at least 97%, between 97% and 98%, at least 98%, between 98% and 99%, at least 99%, between 99% and 100%, or 100% sequence identity with at least 50, between 50 and 100, at least 100, between 100 and 150, at least 150, between 150 and 200, at least 200, between 200 and 250, at least 250, between 250 and 300, at least 300, between 300 and 350, at least 350, between 350 and 400, at
  • a “Cas endonuclease” may comprise domains that enable it to function as a double-strand-break-inducing agent.
  • a “Cas endonuclease” may also comprise one or more modifications or mutations that abolish or reduce its ability to cleave a double-strand polynucleotide (dCas).
  • the Cas endonuclease molecule may retain the ability to nick a single-strand polynucleotide (for example, a D10A mutation in a Cas9 endonuclease molecule) (nCas9).
  • a “functional fragment”, “fragment that is functionally equivalent” and “functionally equivalent fragment” of a Cas endonuclease are used interchangeably herein, and refer to a portion or subsequence of the Cas endonuclease of the present disclosure in which the ability to recognize, bind to, and optionally unwind, nick or cleave (introduce a single or double-strand break in) the target site is retained.
  • the portion or subsequence of the Cas endonuclease can comprise a complete or partial (functional) peptide of any one of its domains such as for example, but not limiting to a complete of functional part of a Cas3 HD domain, a complete of functional part of a Cas3 Helicase domain, complete of functional part of a Cascade protein (such as but not limiting to a Cas5, Cas5d, Cas7 and Cas8b1).
  • a Cas endonuclease may also include a multifunctional Cas endonuclease.
  • multifunctional Cas endonuclease and “multifunctional Cas endonuclease polypeptide” are used interchangeably herein and includes reference to a single polypeptide that has Cas endonuclease functionality (comprising at least one protein domain that can act as a Cas endonuclease) and at least one other functionality, such as but not limited to, the functionality to form a cascade (comprises at least a second protein domain that can form a cascade with other proteins).
  • the multifunctional Cas endonuclease comprises at least one additional protein domain relative (either internally, upstream (5′), downstream (3′), or both internally 5′ and 3′, or any combination thereof) to those domains typical of a Cas endonuclease.
  • cascade and “cascade complex” are used interchangeably herein and include reference to a multi-subunit protein complex that can assemble with a polynucleotide forming a polynucleotide-protein complex (PNP).
  • PNP polynucleotide-protein complex
  • Cascade is a PNP that relies on the polynucleotide for complex assembly and stability, and for the identification of target nucleic acid sequences.
  • Cascade functions as a surveillance complex that finds and optionally binds target nucleic acids that are complementary to a variable targeting domain of the guide polynucleotide.
  • cleavage-ready Cascade is used interchangeably herein and include reference to a multi-subunit protein complex that can assemble with a polynucleotide forming a polynucleotide-protein complex (PNP), wherein one of the cascade proteins is a Cas endonuclease capable of recognizing, binding to, and optionally unwinding, nicking, or cleaving all or part of a target sequence.
  • PNP polynucleotide-protein complex
  • RNA polymerase II RNA polymerase II
  • RNA capping occurs generally as follows: The most terminal 5′ phosphate group of the mRNA transcript is removed by RNA terminal phosphatase, leaving two terminal phosphates. A guanosine monophosphate (GMP) is added to the terminal phosphate of the transcript by a guanylyl transferase, leaving a 5′-5′ triphosphate-linked guanine at the transcript terminus. Finally, the 7-nitrogen of this terminal guanine is methylated by a methyl transferase.
  • GMP guanosine monophosphate
  • RNA having, for example, a 5′-hydroxyl group instead of a 5′-cap Such RNA can be referred to as “uncapped RNA”, for example. Uncapped RNA can better accumulate in the nucleus following transcription, since 5′-capped RNA is subject to nuclear export. One or more RNA components herein are uncapped.
  • guide polynucleotide relates to a polynucleotide sequence that can form a complex with a Cas endonuclease, including the Cas endonuclease described herein, and enables the Cas endonuclease to recognize, optionally bind to, and optionally cleave a DNA target site.
  • the guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (an RNA-DNA combination sequence).
  • RNA, crRNA or tracrRNA are used interchangeably herein, and refer to a portion or subsequence of the guide RNA, crRNA or tracrRNA, respectively, of the present disclosure in which the ability to function as a guide RNA, crRNA or tracrRNA, respectively, is retained.
  • RNA, crRNA or tracrRNA are used interchangeably herein, and refer to a variant of the guide RNA, crRNA or tracrRNA, respectively, of the present disclosure in which the ability to function as a guide RNA, crRNA or tracrRNA, respectively, is retained.
  • single guide RNA and “sgRNA” are used interchangeably herein and relate to a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variable targeting domain (linked to a tracr mate sequence that hybridizes to a tracrRNA), fused to a tracrRNA (trans-activating CRISPR RNA).
  • CRISPR RNA crRNA
  • variable targeting domain linked to a tracr mate sequence that hybridizes to a tracrRNA
  • trans-activating CRISPR RNA trans-activating CRISPR RNA
  • the single guide RNA can comprise a crRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of the type II CRISPR/Cas system that can form a complex with a type II Cas endonuclease, wherein said guide RNA/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, optionally bind to, and optionally nick or cleave (introduce a single or double-strand break) the DNA target site.
  • variable targeting domain or “VT domain” is used interchangeably herein and includes a nucleotide sequence that can hybridize (is complementary) to one strand (nucleotide sequence) of a double strand DNA target site.
  • the percent complementation between the first nucleotide sequence domain (VT domain) and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • variable targeting domain can be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the variable targeting domain comprises a contiguous stretch of 12 to 30 nucleotides.
  • the variable targeting domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.
  • CER domain of a guide polynucleotide
  • CER domain includes a nucleotide sequence that interacts with a Cas endonuclease polypeptide.
  • a CER domain comprises a (trans-acting) tracrNucleotide mate sequence followed by a tracrNucleotide sequence.
  • the CER domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see for example US20150059010A1, published 26 Feb. 2015), or any combination thereof.
  • guide polynucleotide/Cas endonuclease complex As used herein, the terms “guide polynucleotide/Cas endonuclease complex”, “guide polynucleotide/Cas endonuclease system”, “guide polynucleotide/Cas complex”, “guide polynucleotide/Cas system” and “guided Cas system” “Polynucleotide-guided endonuclease”, “PGEN” are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease, that are capable of forming a complex, wherein said guide polynucleotide/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a
  • a guide polynucleotide/Cas endonuclease complex herein can comprise Cas protein(s) and suitable polynucleotide component(s) of any of the known CRISPR systems (Horvath and Barrangou, 2010 , Science 327:167-170; Makarova et al. 2015 , Nature Reviews Microbiology Vol. 13:1-15; Zetsche et al., 2015 , Cell 163, 1-13; Shmakov et al., 2015 , Molecular Cell 60, 1-13).
  • guide RNA/Cas endonuclease complex refers to at least one RNA component and at least one Cas endonuclease that are capable of forming a complex, wherein said guide RNA/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double-strand break) the DNA target site.
  • target site refers to a polynucleotide sequence such as, but not limited to, a nucleotide sequence on a chromosome, episome, a locus, or any other DNA molecule in the genome (including chromosomal, chloroplastic, mitochondrial DNA, plasmid DNA) of a cell, at which a guide polynucleotide/Cas endonuclease complex can recognize, bind to, and optionally nick or cleave.
  • the target site can be an endogenous site in the genome of a cell, or alternatively, the target site can be heterologous to the cell and thereby not be naturally occurring in the genome of the cell, or the target site can be found in a heterologous genomic location compared to where it occurs in nature.
  • endogenous target sequence and “native target sequence” are used interchangeable herein to refer to a target sequence that is endogenous or native to the genome of a cell and is at the endogenous or native position of that target sequence in the genome of the cell.
  • An “artificial target site” or “artificial target sequence” are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell. Such an artificial target sequence can be identical in sequence to an endogenous or native target sequence in the genome of a cell but be located in a different position (i.e., a non-endogenous or non-native position) in the genome of a cell.
  • a “protospacer adjacent motif” herein refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by a guide polynucleotide/Cas endonuclease system described herein.
  • the Cas endonuclease may not successfully recognize a target DNA sequence if the target DNA sequence is not followed by a PAM sequence.
  • the sequence and length of a PAM herein can differ depending on the Cas protein or Cas protein complex used.
  • the PAM sequence can be of any length but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long.
  • altered target site refers to a target sequence as disclosed herein that comprises at least one alteration when compared to non-altered target sequence.
  • alterations include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).
  • a “modified nucleotide” or “edited nucleotide” refers to a nucleotide sequence of interest that comprises at least one alteration when compared to its non-modified nucleotide sequence. Such “alterations” include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).
  • Methods for “modifying a target site” and “altering a target site” are used interchangeably herein and refer to methods for producing an altered target site.
  • donor DNA is a DNA construct that comprises a polynucleotide of interest to be inserted into the target site of a Cas endonuclease.
  • polynucleotide modification template includes a polynucleotide that comprises at least one nucleotide modification when compared to the nucleotide sequence to be edited.
  • a nucleotide modification can be at least one nucleotide substitution, addition or deletion.
  • the polynucleotide modification template can further comprise homologous nucleotide sequences flanking the at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.
  • plant-optimized Cas endonuclease refers to a Cas protein, including a multifunctional Cas protein, encoded by a nucleotide sequence that has been optimized for expression in a plant cell or plant.
  • a “plant-optimized nucleotide sequence encoding a Cas endonuclease”, “plant-optimized construct encoding a Cas endonuclease” and a “plant-optimized polynucleotide encoding a Cas endonuclease” are used interchangeably herein and refer to a nucleotide sequence encoding a Cas protein, or a variant or functional fragment thereof, that has been optimized for expression in a plant cell or plant.
  • a plant comprising a plant-optimized Cas endonuclease includes a plant comprising the nucleotide sequence encoding for the Cas sequence and/or a plant comprising the Cas endonuclease protein.
  • the plant-optimized Cas endonuclease nucleotide sequence is a maize-optimized, rice-optimized, wheat-optimized, soybean-optimized, cotton-optimized, or canola-optimized Cas endonuclease.
  • plant generically includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds and progeny of the same.
  • Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.
  • a “plant element” is intended to reference either a whole plant or a plant component, which may comprise differentiated and/or undifferentiated tissues, for example but not limited to plant tissues, parts, and cell types.
  • a plant element is one of the following: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, keiki, shoot, bud, tumor tissue, and various forms of cells and culture (e.g., single cells, protoplasts, embryos, callus tissue).
  • plant organ refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant.
  • a “plant element” is synonymous to a “portion” of a plant, and refers to any part of the plant, and can include distinct tissues and/or organs, and may be used interchangeably with the term “tissue” throughout.
  • a “plant reproductive element” is intended to generically reference any part of a plant that is able to initiate other plants via either sexual or asexual reproduction of that plant, for example but not limited to: seed, seedling, root, shoot, cutting, scion, graft, stolon, bulb, tuber, corm, keiki, or bud.
  • the plant element may be in plant or in a plant organ, tissue culture, or cell culture.
  • “Progeny” comprises any subsequent generation of a plant.
  • plant part refers to plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like, as well as the parts themselves.
  • Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species.
  • Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides.
  • the term “monocotyledonous” or “monocot” refers to the subclass of angiosperm plants also known as “monocotyledoneae”, whose seeds typically comprise only one embryonic leaf, or cotyledon.
  • the term includes references to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of the same.
  • dicotyledonous or “dicot” refers to the subclass of angiosperm plants also knows as “dicotyledoneae”, whose seeds typically comprise two embryonic leaves, or cotyledons.
  • the term includes references to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of the same.
  • a “male sterile plant” is a plant that does not produce male gametes that are viable or otherwise capable of fertilization.
  • a “female sterile plant” is a plant that does not produce female gametes that are viable or otherwise capable of fertilization. It is recognized that male-sterile and female-sterile plants can be female-fertile and male-fertile, respectively. It is further recognized that a male fertile (but female sterile) plant can produce viable progeny when crossed with a female fertile plant and that a female fertile (but male sterile) plant can produce viable progeny when crossed with a male fertile plant.
  • non-conventional yeast refers to any yeast that is not a Saccharomyces (e.g., S. cerevisiae ) or Schizosaccharomyces yeast species. (see “Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology: Practical Protocols”, K. Wolf, K. D. Breunig, G. Barth, Eds., Springer-Verlag, Berlin, Germany, 2003).
  • crossing means the fusion of gametes via pollination to produce progeny (i.e., cells, seeds, or plants).
  • progeny i.e., cells, seeds, or plants.
  • the term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, i.e., when the pollen and ovule (or microspores and megaspores) are from the same plant or genetically identical plants).
  • introgression refers to the transmission of a desired allele of a genetic locus from one genetic background to another.
  • introgression of a desired allele at a specified locus can be transmitted to at least one progeny plant via a sexual cross between two parent plants, where at least one of the parent plants has the desired allele within its genome.
  • transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome.
  • the desired allele can be, e.g., a transgene, a modified (mutated or edited) native allele, or a selected allele of a marker or QTL.
  • isoline is a comparative term, and references organisms that are genetically identical, but differ in treatment.
  • two genetically identical maize plant embryos may be separated into two different groups, one receiving a treatment (such as the introduction of a CRISPR-Cas effector endonuclease) and one control that does not receive such treatment. Any phenotypic differences between the two groups may thus be attributed solely to the treatment and not to any inherency of the plant's endogenous genetic makeup.
  • “Introducing” is intended to mean presenting to a target, such as a cell or organism, a polynucleotide or polypeptide or polynucleotide-protein complex, in such a manner that the component(s) gains access to the interior of a cell of the organism or to the cell itself.
  • a “polynucleotide of interest” includes any nucleotide sequence encoding a protein or polypeptide that improves desirability of crops, i.e. a trait of agronomic interest.
  • Polynucleotides of interest include, but are not limited to, polynucleotides encoding important traits for agronomics, herbicide-resistance, insecticidal resistance, disease resistance, nematode resistance, herbicide resistance, microbial resistance, fungal resistance, viral resistance, fertility or sterility, grain characteristics, commercial products, phenotypic marker, or any other trait of agronomic or commercial importance.
  • a polynucleotide of interest may additionally be utilized in either the sense or anti-sense orientation. Further, more than one polynucleotide of interest may be utilized together, or “stacked”, to provide additional benefit.
  • a “complex trait locus” includes a genomic locus that has multiple transgenes genetically linked to each other.
  • compositions and methods herein may provide for an improved “agronomic trait” or “trait of agronomic importance” or “trait of agronomic interest” to a plant, which may include, but not be limited to, the following: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, yield improvement, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, modulation of a metabolite, modulation of the proteome, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition, as compared to an isoline plant not comprising a modification derived from the methods or compositions herein.
  • Agronomic trait potential is intended to mean a capability of a plant element for exhibiting a phenotype, preferably an improved agronomic trait, at some point during its life cycle, or conveying said phenotype to another plant element with which it is associated in the same plant.
  • a decrease in a characteristic may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, between 5% and 10%, at least 10%, between 10% and 20%, at least 15%, at least 20%, between 20% and 30%, at least 25%, at least 30%, between 30% and 40%, at least 35%, at least 40%, between 40% and 50%, at least 45%, at least 50%, between 50% and 60%, at least about 60%, between 60% and 70%, between 70% and 80%, at least 75%, at least about 80%, between 80% and 90%, at least about 90%, between 90% and 100%, at least 100%, between 100% and 200%, at least 200%, at least about 300%, at least about 400%) or more lower than the untreated control and an increase may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, between 5% and 10%, at least 10%, between 10% and 20%, at least 15%, at least 20%, between 20% and 30%, at least 25%, at least 30%, between 30% and 40%, at least 3
  • the term “before”, in reference to a sequence position, refers to an occurrence of one sequence upstream, or 5′, to another sequence.
  • Double-Strand-Break (DSB) Inducing Agents (DSB Agents)
  • Double-strand breaks induced by double-strand-break-inducing agents can result in the induction of DNA repair mechanisms, including the non-homologous end-joining pathway, and homologous recombination.
  • Endonucleases include a range of different enzymes, including restriction endonucleases (see e.g. Roberts et al., (2003) Nucleic Acids Res 1:418-20), Roberts et al., (2003) Nucleic Acids Res 31:1805-12, and Belfort et al., (2002) in Mobile DNA II, pp. 761-783, Eds.
  • site-specific base conversions can also be achieved to engineer one or more nucleotide changes to create one or more EMEs described herein into the genome.
  • site-specific base edit mediated by an C ⁇ G to T ⁇ A or an A ⁇ T to G ⁇ C base editing deaminase enzymes (Gaudelli et al., Programmable base editing of A ⁇ T to G ⁇ C in genomic DNA without DNA cleavage.” Nature (2017); Nishida et al. “Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.” Science 353 (6305) (2016); Komor et al. “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature 533 (7603) (2016):420-4.
  • Any double-strand-break or -nick or -modification inducing agent may be used for the methods described herein, including for example but not limited to: Cas endonucleases, recombinases, TALENs, zinc finger nucleases, restriction endonucleases, meganucleases, and deaminases.
  • Class I Cas endonucleases comprise multisubunit effector complexes (Types I, III, and IV), while Class 2 systems comprise single protein effectors (Types II, V, and VI) (Makarova et al. 2015 , Nature Reviews Microbiology Vol. 13:1-15; Zetsche et al., 2015 , Cell 163, 1-13; Shmakov et al., 2015 , Molecular Cell 60, 1-13; Haft et al., 2005 , Computational Biology, PLoS Comput Biol 1(6): e60; and Koonin et al.
  • the Cas endonuclease acts in complex with a guide RNA (gRNA) that directs the Cas endonuclease to cleave the DNA target to enable target recognition, binding, and cleavage by the Cas endonuclease.
  • gRNA guide RNA
  • the gRNA comprises a Cas endonuclease recognition (CER) domain that interacts with the Cas endonuclease, and a Variable Targeting (VT) domain that hybridizes to a nucleotide sequence in a target DNA.
  • CER Cas endonuclease recognition
  • VT Variable Targeting
  • the gRNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) to guide the Cas endonuclease to its DNA target.
  • the crRNA comprises a spacer region complementary to one strand of the double strand DNA target and a region that base pairs with the tracrRNA, forming an RNA duplex.
  • the gRNA is a “single guide RNA” (sgRNA) that comprises a synthetic fusion of crRNA and tracrRNA.
  • sgRNA single guide RNA
  • the Cas endonuclease-guide polynucleotide complex recognizes a short nucleotide sequence adjacent to the target sequence (protospacer), called a “protospacer adjacent motif” (PAM).
  • Cas endonuclease examples include but are not limited to Cas9 and Cpf1.
  • Cas9 (formerly referred to as Cas5, Csn1, or Csx12) is a Class 2 Type II Cas endonuclease (Makarova et al. 2015 , Nature Reviews Microbiology Vol. 13:1-15).
  • a Cas9-gRNA complex recognizes a 3′ PAM sequence (NGG for the S. pyogenes Cas9) at the target site, permitting the spacer of the guide RNA to invade the double-stranded DNA target, and, if sufficient homology between the spacer and protospacer exists, generate a double-strand break cleavage.
  • Cas9 endonucleases comprise RuvC and HNH domains that together produce double strand breaks, and separately can produce single strand breaks.
  • the double-strand break leaves a blunt end.
  • Cpf1 is a Class 2 Type V Cas endonuclease, and comprises nuclease RuvC domain but lacks an HNH domain (Yamane et al., 2016, Cell 165:949-962). Cpf1 endonucleases create “sticky” overhang ends.
  • Cas9-gRNA systems at a genomic target site include but are not limited to insertions, deletions, substitutions, or modifications of one or more nucleotides at the target site; modifying or replacing nucleotide sequences of interest (such as a regulatory elements); insertion of polynucleotides of interest; gene knock-out; gene-knock in; modification of splicing sites and/or introducing alternate splicing sites; modifications of nucleotide sequences encoding a protein of interest; amino acid and/or protein fusions; and gene silencing by expressing an inverted repeat into a gene of interest.
  • a “polynucleotide modification template” comprises at least one nucleotide modification when compared to the nucleotide sequence to be edited.
  • a nucleotide modification can be at least one nucleotide substitution, addition, deletion, or chemical alteration.
  • the polynucleotide modification template can further comprise homologous nucleotide sequences flanking the at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.
  • a polynucleotide of interest is inserted at a target site and provided as part of a “donor DNA” molecule.
  • donor DNA is a DNA construct that comprises a polynucleotide of interest to be inserted into the target site of a Cas endonuclease.
  • the donor DNA construct further comprises a first and a second region of homology that flank the polynucleotide of interest.
  • the first and second regions of homology of the donor DNA share homology to a first and a second genomic region, respectively, present in or flanking the target site of the cell or organism genome.
  • the donor DNA can be tethered to the guide polynucleotide.
  • Tethered donor DNAs can allow for co-localizing target and donor DNA, useful in genome editing, gene insertion, and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous HR machinery is expected to be highly diminished (Mali et al., 2013 , Nature Methods Vol. 10: 957-963).
  • the amount of homology or sequence identity shared by a target and a donor polynucleotide can vary and includes total lengths and/or regions.
  • the process for editing a genomic sequence at a Cas9-gRNA double-strand-break site with a modification template generally comprises: providing a host cell with a Cas9-gRNA complex that recognizes a target sequence in the genome of the host cell and is able to induce a single- or double-strand-break in the genomic sequence, and optionally at least one polynucleotide modification template comprising at least one nucleotide alteration when compared to the nucleotide sequence to be edited.
  • the polynucleotide modification template can further comprise nucleotide sequences flanking the at least one nucleotide alteration, in which the flanking sequences are substantially homologous to the chromosomal region flanking the double-strand break.
  • Genome editing using double-strand-break-inducing agents has been described, for example in US20150082478 published on 19 Mar. 2015, WO2015026886 published on 26 Feb. 2015, WO2016007347 published 14 Jan. 2016, and WO2016025131 published on 18 Feb. 2016.
  • the gene comprising the Cas endonuclease may be optimized as described in WO2016186953 published 24 Nov. 2016, and then delivered into cells as DNA expression cassettes by methods known in the art.
  • the Cas endonuclease is provided as a polypeptide.
  • the Cas endonuclease is provided as a polynucleotide encoding a polypeptide.
  • the guide RNA is provided as a DNA molecule encoding one or more RNA molecules.
  • the guide RNA is provide as RNA or chemically-modified RNA.
  • the Cas endonuclease protein and guide RNA are provided as a ribonucleoprotein complex (RNP).
  • a double-strand-break-inducing agent such a guided Cas endonuclease can recognize, bind to a DNA target sequence and introduce a single strand (nick) or double-strand break.
  • a single or double-strand break is induced in the DNA, the cell's DNA repair mechanism is activated to repair the break, for example via nonhomologous end-joining (NHEJ) or Homology-Directed Repair (HDR) processes which can lead to modifications at the target site.
  • NHEJ nonhomologous end-joining
  • HDR Homology-Directed Repair
  • the most common repair mechanism to bring the broken ends together is the nonhomologous end-joining (NHEJ) pathway (Bleuyard et al., (2006) DNA Repair 5:1-12).
  • chromosomes The structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements (such as chromosomal translocations) are possible (Siebert and Puchta, 2002 , Plant Cell 14:1121-31; Pacher et al., 2007 , Genetics 175:21-9).
  • NHEJ is often error-prone and can introduce small mutations in the target site. In plants, NHEJ is often the major pathway by which DSBs are remediated; therefore, methods and compositions to improve the probability of HDR or HR in plants are desirable.
  • SDN1 covers the application of a SDN without an additional donor DNA or repair template.
  • the reaction outcome clearly depends on the DSB repair pathway of the plant genome.
  • SDN1a small insertions or deletions can occur (SDN1a).
  • SDN1b larger deletions can be obtained (SDN1b).
  • SDN1c inversions
  • SDN1d translocations
  • SDN2 describes the use of a SDN with an additional DNA “polynucleotide modification template” to introduce small mutations in a controlled manner.
  • a template mainly homologous to the target sequence is provided to be the substrate for HR-mediated DSB repair following the induction of one or two adjacent DSBs.
  • This approach allows the introduction of small mutations that could also occur naturally, per se. Taking the size of plant genomes into account, small modifications up to 20 nucleotides can statistically be regarded as GE that resembles naturally occurring genome changes. Therefore, targeted genome modifications using ODM are also regarded comparable to SDN2.
  • SDN3 describes the use of a SDN with an additional “donor polynucleotide” or “donor DNA” to introduce large stretches of exogenous DNA at a pre-determined locus, adding or replacing genetic information.
  • this process relies on HR-mediated DSB repair like SDN2, and the discrimination is arbitrary as the size of the sequence inserted can vary significantly.
  • Both SDN2 and SDN3 are types of homology-directed repair (HDR) of a double-strand break in a polynucleotide, and involve methods of introducing a heterologous polynucleotide as either a template for repair of the double strand break (SDN2), or insertion of a new double-stranded polynucleotide at the double strand break site (SDN3).
  • SDN2 repairs may be detected by the presence of one or a few nucleotide changes (mutations).
  • SDN3 repairs may be detected by the presence of a novel contiguous heterologous polynucleotide.
  • Modification of a target polynucleotide includes any one or more of the following: insertion of at least one nucleotide, deletion of at least one nucleotide, chemical alteration of at least one nucleotide, replacement of at least one nucleotide, or mutation of at least one nucleotide.
  • the DNA repair mechanism creates an imperfect repair of the double-strand break, resulting in a change of a nucleotide at the break site.
  • a polynucleotide template may be provided to the break site, wherein the repair results in a template-directed repair of the break.
  • a donor polynucleotide may be provided to the break site, wherein the repair results in the incorporation of the donor polynucleotide into the break site.
  • the methods and compositions described herein improve the probability of a non-NHEJ repair mechanism outcome at a DSB. In one aspect, an increase of the HDR to NHEJ repair ratio is effected.
  • Homology-directed repair is a mechanism in cells to repair double-stranded and single stranded DNA breaks.
  • Homology-directed repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem. 79:181-211).
  • HR homologous recombination
  • SSA single-strand annealing
  • Other forms of HDR include single-stranded annealing (SSA) and breakage-induced replication, and these require shorter sequence homology relative to HR.
  • homology is meant DNA sequences that are similar.
  • a “region of homology to a genomic region” that is found on the donor DNA is a region of DNA that has a similar sequence to a given “genomic region” in the cell or organism genome.
  • a region of homology can be of any length that is sufficient to promote homologous recombination at the cleaved target site.
  • the region of homology can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900, 5-3000, 5-3100 or more bases in length such that the region of homology has sufficient homology to undergo homologous recombination with the corresponding genomic region.
  • “Sufficient homology” indicates that two polynucleotide sequences have sufficient structural similarity to act as substrates for a homologous recombination reaction.
  • the structural similarity includes overall length of each polynucleotide fragment, as well as the sequence similarity of the polynucleotides. Sequence similarity can be described by the percent sequence identity over the whole length of the sequences, and/or by conserved regions comprising localized similarities such as contiguous nucleotides having 100% sequence identity, and percent sequence identity over a portion of the length of the sequences.
  • the amount of homology or sequence identity shared by a target and a donor polynucleotide can vary and includes total lengths and/or regions having unit integral values in the ranges of about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total length of the target site.
  • ranges include every integer within the range, for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bps.
  • the amount of homology can also be described by percent sequence identity over the full aligned length of the two polynucleotides which includes percent sequence identity of about at least 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • Sufficient homology includes any combination of polynucleotide length, global percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the target locus. Sufficient homology can also be described by the predicted ability of two polynucleotides to specifically hybridize under high stringency conditions, see, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology , Ausubel et al., Eds (1994) Current Protocols , (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes , (Elsevier, New York).
  • DNA double-strand breaks can be an effective factor to stimulate homologous recombination pathways (Puchta et al., (1995) Plant Mol Biol 28:281-92; Tzfira and White, (2005) Trends Biotechnol 23:567-9; Puchta, (2005) J Exp Bot 56:1-14).
  • DNA-breaking agents a two- to nine-fold increase of homologous recombination was observed between artificially constructed homologous DNA repeats in plants (Puchta et al., (1995) Plant Mol Biol 28:281-92).
  • experiments with linear DNA molecules demonstrated enhanced homologous recombination between plasmids (Lyznik et al., (1991) Mol Gen Genet 230:209-18).
  • ES embryonic stem cell lines
  • the inventors have also conceived that because recombinogenic intermediates involve 3′ overhangs, additional single strand breaks flanking the double-strand break site will produce destabilized duplexes, leading to a recombinogenic intermediate.
  • different endonucleases e.g., from different source organisms or CRISPR loci, or engineered enzymes, or nickases.
  • the fraction or percent of HR reads is greater than of a comparator, such as a control sample, sample with NHEJ repair, or as compared to the total mutant reads. In some aspects, the fraction or percent of HR reads is greater than of the control sample (no DSB agent). In some aspects, the fraction or percent of HR reads is greater than the fraction or percent of NHEJ reads. In some aspects, the fraction or percent of HR reads is greater than the fraction or percent of total mutant reads (NHEJ+HR).
  • the fraction of HR reads relative to a comparator is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, between 10 and 15, 15, between 15 and 20, 20, between 20 and 25, 25, between 25 and 30, 30, between 30 and 40, 40, between 40 and 50, 50, between 50 and 60, 60, between 60 and 70, 70, between 70 and 80, 80, between 80 and 90, 90, between 90 and 100, 100, between 100 and 125, 125, between 125 and 150, greater than 150, or infinitely greater.
  • the percent of HR reads relative a comparator is at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 20%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
  • the percent of HR reads is greater than zero.
  • a double-strand-break is created, repaired, and recurrently cleaved by any method or composition, for example but not limited to a Cas endonuclease and guide RNA.
  • a DSB inducing agent e.g., Cas endonuclease and first guide RNA
  • a first double-strand-break is created, and repaired.
  • the repair results in a change of the target site polynucleotide sequence (for example, but not limited to, an insertion of a nucleotide, a deletion of a nucleotide, or a replacement of a nucleotide).
  • a repair template is provided for a specific target polynucleotide repair composition outcome.
  • the repair template is flanked with inverted target site (PAM on the inside).
  • a second guide RNA is introduced that is complementary to the mutation that was created by the repair by the first double-strand break.
  • the DSB repair composition outcome is determined by the introduction of a donor polynucleotide template or insertion, and the second guide RNA designed to be complementary to that determined target sequence outcome.
  • the second guide RNA is designed to be complementary to the most commonly created repair mutation.
  • the second guide RNA is designed to be complementary to a desired DNA repair outcome.
  • a library of second guide RNAs is designed that are complementary to all possible mutations of the target site. The mutation(s) created by the first double-strand-break repair may be either known or predicted bioinformatically.
  • the second guide RNA acts in concert with the Cas endonuclease (either provided de novo or the same Cas endonuclease that was present for the first DSB) to create a second double-strand-break at the same site (within the on-target recognition sequence of the Cas endonuclease/first guide RNA complex).
  • another DSB inducing reagent may be introduced instead of a second guide RNA and a Cas endonuclease creating the second DSB.
  • the second DSB has a higher probability of repair by HDR than NHEJ, as compared to the repair of the first DSB (i.e., the probability of HDR is increased, or the frequency of HDR is increased, or the ratio of HDR to NHEJ is increased).
  • the probability of HDR is increased, or the frequency of HDR is increased, or the ratio of HDR to NHEJ is increased.
  • there is a subsequent cut at a previous cut site which in some aspects can be accomplished by the introduction of another Cas endonuclease/gRNA complex. Continued cleavage in a sequential manner increases the frequency of HDR as a DSB repair mechanism.
  • a double-strand-break is created, repaired, and recursively cleaved by any method or composition, for example but not limited to a Cas endonuclease and guide RNA.
  • a DSB inducing agent e.g., Cas endonuclease and first guide RNA
  • the first guide RNA is provided as a DNA sequence on a plasmid that further comprises a spacer sequence.
  • the DNA encoding the gRNA is operably linked to a regulatory expression element.
  • a first double-strand-break is created, and repaired.
  • the composition of the repaired target polynucleotide is used as the basis of a mutation generated by Cas editing of the spacer on the plasmid comprising the gRNA DNA and spacer.
  • the mutated spacer composition directs the generation of a second gRNA that is complementary to the sequence of the repaired targeted polynucleotide of the first DSB, and a second double-strand break is induced at the target site by the Cas endonuclease and the second gRNA.
  • the cycle may then repeat, with sequence of the newly repaired second DSB then being used as a template for the composition of a third gRNA that is complementary to the sequence of the repaired second DSB polynucleotide, and so forth.
  • a loop of DSB generation and repair occurs, with each subsequent repair after the first having a higher probability of repair via HDR than NHEJ, as compared to the mechanism of the first repair.
  • the process may stop by any of a number of methods, including but not limited to: titrated reagent availability, mutation induced in the region of the gRNA DNA expression construct that renders the expression cassette or transcribed gRNA to be non-functional, an external factor that may optionally be inducible or repressible, or via the introduction of another molecule.
  • a nick (cleavage of double-stranded DNA on only one of the two phosphate backbones) is created adjacent to a double-strand-break on a target polynucleotide.
  • a single nick is created.
  • two nicks are created.
  • two nicks are created, one each flanking the two sides of the DSB.
  • the double-strand-break is created by one Cas endonuclease, and the nick(s) is(are) created by a different molecule (e.g., a molecule derived from a different organism, or a Cas endonuclease that lacks double strand break creation functionality but possesses nickase activity (for example, nCas9)). Due to the presence of adjacent nick(s), double-strand-break repair of the DSB at the target site has a higher probability of being repaired by HDR than by NHEJ, or has a higher frequency of HDR as compared to a DSB at the same locus that does not have one or more nicks adjacent to the DSB.
  • a different molecule e.g., a molecule derived from a different organism, or a Cas endonuclease that lacks double strand break creation functionality but possesses nickase activity (for example, nCas9). Due to the presence of adjacent nick
  • the distance between the nick and the DSB site is 10 basepairs, between 10 and 20 basepairs, 20 basepairs, between 20 and 30 basepairs, 30 basepairs, between 30 and 40 basepairs, 40 basepairs, between 40 and 50 basepairs, 50 basepairs, between 50 and 60 basepairs, 60 basepairs, between 60 and 70 basepairs, 70 basepairs, between 70 and 80 basepairs, 80 basepairs, between 80 and 90 basepairs, 90 basepairs, between 90 and 100 basepairs, 100 basepairs, between 100 and 110 basepairs, 110 basepairs, between 110 and 120 basepairs, or greater than 120 basepairs in length.
  • DNA repair outcomes that are contemplated to be improved with the methods described herein include gene targeting, gene editing, gene drop-out, gene swap (deletion plus insertion), and promoter swap (deletion plus insertion).
  • compositions and methods described herein can be used for gene targeting.
  • DNA targeting can be performed by cleaving one or both strands at a specific polynucleotide sequence in a cell with a Cas endonuclease associated with a suitable guide polynucleotide component.
  • a Cas endonuclease associated with a suitable guide polynucleotide component.
  • the cell's DNA repair mechanism is activated to repair the break via nonhomologous end-joining (NHEJ) or Homology-Directed Repair (HDR) processes which can lead to modifications at the target site.
  • NHEJ nonhomologous end-joining
  • HDR Homology-Directed Repair
  • the length of the DNA sequence at the target site can vary, and includes, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length. It is further possible that the target site can be palindromic, that is, the sequence on one strand reads the same in the opposite direction on the complementary strand.
  • the nick/cleavage site can be within the target sequence or the nick/cleavage site could be outside of the target sequence.
  • the cleavage could occur at nucleotide positions immediately opposite each other to produce a blunt end cut or, in other cases, the incisions could be staggered to produce single-stranded overhangs, also called “sticky ends”, which can be either 5′ overhangs, or 3′ overhangs.
  • Active variants of genomic target sites can also be used. Such active variants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the given target site, wherein the active variants retain biological activity and hence are capable of being recognized and cleaved by an Cas endonuclease.
  • Assays to measure the single or double-strand break of a target site by an endonuclease are known in the art and generally measure the overall activity and specificity of the agent on DNA substrates comprising recognition sites.
  • a targeting method herein can be performed in such a way that two or more DNA target sites are targeted in the method, for example. Such a method can optionally be characterized as a multiplex method. Two, three, four, five, six, seven, eight, nine, ten, or more target sites can be targeted at the same time in certain embodiments.
  • a multiplex method is typically performed by a targeting method herein in which multiple different RNA components are provided, each designed to guide a guide polynucleotide/Cas endonuclease complex to a unique DNA target site.
  • the process for editing a genomic sequence combining DSB and modification templates generally comprises: introducing into a host cell a DSB-inducing agent, or a nucleic acid encoding a DSB-inducing agent, that recognizes a target sequence in the chromosomal sequence and is able to induce a DSB in the genomic sequence, and at least one polynucleotide modification template comprising at least one nucleotide alteration when compared to the nucleotide sequence to be edited.
  • the polynucleotide modification template can further comprise nucleotide sequences flanking the at least one nucleotide alteration, in which the flanking sequences are substantially homologous to the chromosomal region flanking the DSB.
  • Genome editing using DSB-inducing agents such as Cas-gRNA complexes, has been described, for example in US20150082478 published on 19 Mar. 2015, WO2015026886 published on 26 Feb. 2015, WO2016007347 published 14 Jan. 2016, and WO/2017/025131 published on 18 Feb. 2016.
  • RNA/Cas endonuclease systems include but are not limited to modifying or replacing nucleotide sequences of interest (such as a regulatory elements), insertion of polynucleotides of interest, gene drop-out, gene knock-out, gene-knock in, modification of splicing sites and/or introducing alternate splicing sites, modifications of nucleotide sequences encoding a protein of interest, amino acid and/or protein fusions, and gene silencing by expressing an inverted repeat into a gene of interest.
  • nucleotide sequences of interest such as a regulatory elements
  • Proteins may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known. For example, amino acid sequence variants of the protein(s) can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations include, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel et al., (1987) Meth Enzymol 154:367-82; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.
  • amino acid substitutions not likely to affect biological activity of the protein are found, for example, in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, D.C.). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferable. Conservative deletions, insertions, and amino acid substitutions are not expected to produce radical changes in the characteristics of the protein, and the effect of any substitution, deletion, insertion, or combination thereof can be evaluated by routine screening assays. Assays for double-strand-break-inducing activity are known and generally measure the overall activity and specificity of the agent on DNA substrates comprising target sites.
  • crCascade Cleavage Ready Cascade
  • components of the cleavage ready Cascade (crCascade) complex and associated CRISPR RNA (crRNA) may be utilized to modify chromosomal DNA in other organisms including plants.
  • crCascade cleavage ready Cascade
  • crRNA CRISPR RNA
  • the genes comprising the crCascade may be optimized as described in WO2016186953 published 24 Nov. 2016, and then delivered into cells as DNA expression cassettes by methods known in the art.
  • the components necessary to comprise an active crCascade complex may also be delivered as RNA with or without modifications that protect the RNA from degradation or as mRNA capped or uncapped (Zhang, Y. et al., 2016 , Nat. Commun. 7:12617) or Cas protein guide polynucleotide complexes (WO2017070032 published 27 Apr. 2017), or any combination thereof. Additionally, a part or part(s) of the crCascade complex and crRNA may be expressed from a DNA construct while other components are delivered as RNA with or without modifications that protect the RNA from degradation or as mRNA capped or uncapped (Zhang et al. 2016 Nat. Commun.
  • tRNA derived elements may also be used to recruit endogenous RNAses to cleave crRNA transcripts into mature forms capable of guiding the crCascade complex to its DNA target site, as described, for example, in WO2017105991 published 22 Jun. 2017. crCascade nickase complexes may be utilized separately or concertedly to generate a single or multiple DNA nicks on one or both DNA strands.
  • cleavage activity of the Cas endonuclease may be deactivated by altering key catalytic residues in its cleavage domain (Sinkunas, T. et al., 2013 , EMBO J. 32:385-394) resulting in an RNA guided helicase that may be used to enhance homology-directed repair, induce transcriptional activation, or remodel local DNA structures.
  • the activity of the Cas cleavage and helicase domains may both be knocked-out and used in combination with other DNA cutting, DNA nicking, DNA binding, transcriptional activation, transcriptional repression, DNA remodeling, DNA deamination, DNA unwinding, DNA recombination enhancing, DNA integration, DNA inversion, and DNA repair agents.
  • the transcriptional direction of the tracrRNA for the CRISPR-Cas system (if present) and other components of the CRISPR-Cas system (such as variable targeting domain, crRNA repeat, loop, anti-repeat) can be deduced as described in WO2016186946 published 24 Nov. 2016, and WO2016186953 published 24 Nov. 2016.
  • the PAM preferences for each new system disclosed herein may be examined. If the cleavage ready Cascade (crCascade) complex results in degradation of the randomized PAM library, the crCascade complex can be converted into a nickase by disabling the ATPase dependent helicase activity either through mutagenesis of critical residues or by assembling the reaction in the absence of ATP as described previously (Sinkunas, T. et al., 2013 , EMBO J. 32:385-394). Two regions of PAM randomization separated by two protospacer targets may be utilized to generate a double-stranded DNA break which may be captured and sequenced to examine the PAM sequences that support cleavage by the respective crCascade complex.
  • the invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing into a cell at least one Cas endonuclease and guide RNA, and identifying at least one cell that has a modification at the target site.
  • the nucleotide to be edited can be located within or outside a target site recognized and cleaved by a Cas endonuclease.
  • the at least one nucleotide modification is not a modification at a target site recognized and cleaved by a Cas endonuclease.
  • a knock-out may be produced by an indel (insertion or deletion of nucleotide bases in a target DNA sequence through NHEJ), or by specific removal of sequence that reduces or completely destroys the function of sequence at or near the targeting site.
  • a guide polynucleotide/Cas endonuclease induced targeted mutation can occur in a nucleotide sequence that is located within or outside a genomic target site that is recognized and cleaved by the Cas endonuclease.
  • the method for editing a nucleotide sequence in the genome of a cell can be a method without the use of an exogenous selectable marker by restoring function to a non-functional gene product.
  • the invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing into a cell at least one PGEN described herein and at least one donor DNA, wherein said donor DNA comprises a polynucleotide of interest, and optionally, further comprising identifying at least one cell that said polynucleotide of interest integrated in or near said target site.
  • the methods disclosed herein may employ homologous recombination (HR) to provide integration of the polynucleotide of interest at the target site.
  • HR homologous recombination
  • a polynucleotide of interest is introduced into the organism cell via a donor DNA construct.
  • donor DNA is a DNA construct that comprises a polynucleotide of interest to be inserted into the target site of a Cas endonuclease.
  • the donor DNA construct further comprises a first and a second region of homology that flank the polynucleotide of interest.
  • the first and second regions of homology of the donor DNA share homology to a first and a second genomic region, respectively, present in or flanking the target site of the cell or organism genome.
  • the donor DNA can be tethered to the guide polynucleotide. Tethered donor DNAs can allow for co-localizing target and donor DNA, useful in genome editing, gene insertion, and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous HR machinery is expected to be highly diminished (Mali et al., 2013 , Nature Methods Vol. 10: 957-963).
  • the amount of homology or sequence identity shared by a target and a donor polynucleotide can vary and includes total lengths and/or regions having unit integral values in the ranges of about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total length of the target site.
  • ranges include every integer within the range, for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bps.
  • the amount of homology can also be described by percent sequence identity over the full aligned length of the two polynucleotides which includes percent sequence identity of about at least 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • Sufficient homology includes any combination of polynucleotide length, global percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the target locus. Sufficient homology can also be described by the predicted ability of two polynucleotides to specifically hybridize under high stringency conditions, see, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology , Ausubel et al., Eds (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes , (Elsevier, New York).
  • Episomal DNA molecules can also be ligated into the double-strand break, for example, integration of T-DNAs into chromosomal double-strand breaks (Chilton and Que, (2003) Plant Physiol 133:956-65; Salomon and Puchta, (1998) EMBO 1 17:6086-95).
  • gene conversion pathways can restore the original structure if a homologous sequence is available, such as a homologous chromosome in non-dividing somatic cells, or a sister chromatid after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52).
  • Ectopic and/or epigenic DNA sequences may also serve as a DNA repair template for homologous recombination (Puchta, (1999) Genetics 152:1173-81).
  • the disclosure comprises a method for editing a nucleotide sequence in the genome of a cell, the method comprising introducing into at least one PGEN described herein, and a polynucleotide modification template, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence, and optionally further comprising selecting at least one cell that comprises the edited nucleotide sequence.
  • the guide polynucleotide/Cas endonuclease system can be used in combination with at least one polynucleotide modification template to allow for editing (modification) of a genomic nucleotide sequence of interest.
  • Polynucleotides of interest and/or traits can be stacked together in a complex trait locus as described in WO2012129373 published 27 Sep. 2012, and in WO2013112686, published 1 Aug. 2013.
  • the guide polynucleotide/Cas9 endonuclease system described herein provides for an efficient system to generate double-strand breaks and allows for traits to be stacked in a complex trait locus.
  • a guide polynucleotide/Cas system as described herein, mediating gene targeting can be used in methods for directing heterologous gene insertion and/or for producing complex trait loci comprising multiple heterologous genes in a fashion similar as disclosed in WO2012129373 published 27 Sep. 2012, where instead of using a double-strand break inducing agent to introduce a gene of interest, a guide polynucleotide/Cas system as disclosed herein is used.
  • the transgenes can be bred as a single genetic locus (see, for example, US20130263324 published 3 Oct. 2013 or WO2012129373 published 14 Mar. 2013).
  • plants comprising (at least) one transgenes can be crossed to form an F1 that comprises both transgenes.
  • progeny from these F1 F2 or BC1
  • progeny from these F1 F2 or BC1
  • progeny from these F1 F2 or BC1
  • progeny from these F1 F2 or BC1 1/500 progeny would have the two different transgenes recombined onto the same chromosome.
  • the complex locus can then be bred as single genetic locus with both transgene traits. This process can be repeated to stack as many traits as desired.
  • nucleotide sequences of interest such as a regulatory elements
  • insertion of polynucleotides of interest gene knock-out, gene-knock in, modification of splicing sites and/or introducing alternate splicing sites, modifications of nucleotide sequences encoding a protein of interest, amino acid and/or protein fusions, and gene silencing by expressing an inverted repeat into a gene of interest.
  • Chromosomal intervals that correlate with a phenotype or trait of interest can be identified.
  • a variety of methods well known in the art are available for identifying chromosomal intervals.
  • the boundaries of such chromosomal intervals are drawn to encompass markers that will be linked to the gene controlling the trait of interest.
  • the chromosomal interval is drawn such that any marker that lies within that interval (including the terminal markers that define the boundaries of the interval) can be used as a marker for a particular trait.
  • the chromosomal interval comprises at least one QTL, and furthermore, may indeed comprise more than one QTL.
  • QTL quantitative trait locus
  • An “allele of a QTL” can comprise multiple genes or other genetic factors within a contiguous genomic region or linkage group, such as a haplotype.
  • An allele of a QTL can denote a haplotype within a specified window wherein said window is a contiguous genomic region that can be defined, and tracked, with a set of one or more polymorphic markers.
  • a haplotype can be defined by the unique fingerprint of alleles at each marker within the specified window.
  • compositions disclosed herein may further be used as compositions for use in in vitro methods, in some aspects with isolated polynucleotide sequence(s).
  • Said isolated polynucleotide sequence(s) may comprise one or more target sequence(s) for modification.
  • said isolated polynucleotide sequence(s) may be genomic DNA, a PCR product, or a synthesized oligonucleotide.
  • Modification of a target sequence may be in the form of a nucleotide insertion, a nucleotide deletion, a nucleotide substitution, the addition of an atom molecule to an existing nucleotide, a nucleotide modification, or the binding of a heterologous polynucleotide or polypeptide to said target sequence.
  • the insertion of one or more nucleotides may be accomplished by the inclusion of a donor polynucleotide in the reaction mixture: said donor polynucleotide is inserted into a double-strand break created by said Cas endonuclease.
  • the insertion may be via non-homologous end joining or via homologous recombination.
  • sequence of the target polynucleotide is known prior to modification, and compared to the sequence(s) of polynucleotide(s) that result from treatment with the compositions described herein. In one aspect, the sequence of the target polynucleotide is not known prior to modification.
  • any or all of the possible polynucleotide components of the reaction may be provided as part of a vector, a construct, a linearized or circularized plasmid, or as part of a chimeric molecule. Each component may be provided to the reaction mixture separately or together.
  • one or more of the polynucleotide components are operably linked to a heterologous noncoding regulatory element that regulates its expression.
  • the method for modification of a target polynucleotide comprises combining the minimal elements into a reaction mixture comprising: a Cas endonuclease (or variant, fragment, or other related molecule as described above), a guide polynucleotide comprising a sequence that is substantially complementary to, or selectively hybridizes to, the target polynucleotide sequence of the target polynucleotide, and a target polynucleotide for modification.
  • the Cas endonuclease is provided as a polypeptide.
  • the Cas endonuclease is provided as a cas polynucleotide.
  • the guide polynucleotide is provided as an RNA molecule, a DNA molecule, an RNA:DNA hybrid, or a polynucleotide molecule comprising a chemically-modified nucleotide.
  • the storage buffer of any one of the components, or the reaction mixture may be optimized for stability, efficacy, or other parameters. Additional components of the storage buffer or the reaction mixture may include a buffer composition, Tris, EDTA, dithiothreitol (DTT), phosphate-buffered saline (PBS), sodium chloride, magnesium chloride, HEPES, glycerol, BSA, a salt, an emulsifier, a detergent, a chelating agent, a redox reagent, an antibody, nuclease-free water, a proteinase, and/or a viscosity agent.
  • Tris Tris
  • EDTA dithiothreitol
  • PBS phosphate-buffered saline
  • sodium chloride magnesium chloride
  • HEPES glycerol
  • BSA a salt
  • an emulsifier a detergent
  • a chelating agent a chelating agent
  • the storage buffer or reaction mixture further comprises a buffer solution with at least one of the following components: HEPES, MgCl2, NaCl, EDTA, a proteinase, Proteinase K, glycerol, nuclease-free water.
  • the temperature is preferably at least 10 degrees Celsius, between 10 and 15, at least 15, between 15 and 17, at least 17, between 17 and 20, at least 20, between 20 and 22, at least 22, between 22 and 25, at least 25, between 25 and 27, at least 27, between 27 and 30, at least 30, between 30 and 32, at least 32, between 32 and 35, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, or even greater than 40 degrees Celsius.
  • the time of incubation is at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, or even greater than 10 minutes.
  • sequence(s) of the polynucleotide(s) in the reaction mixture prior to, during, or after incubation may be determined by any method known in the art.
  • modification of a target polynucleotide may be ascertained by comparing the sequence(s) of the polynucleotide(s) purified from the reaction mixture to the sequence of the target polynucleotide prior to combining with the Cas enodnuclease.
  • kits comprises a Cas endonuclease or a polynucleotide cas encoding such, optionally further comprising buffer components to enable efficient storage, and one or more additional compositions that enable the introduction of said Cas endonuclease or cas to a heterologous polynucleotide, wherein said Cas endonuclease or cas is capable of effecting a modification, addition, deletion, or substitution of at least one nucleotide of said heterologous polynucleotide.
  • a Cas endonuclease disclosed herein may be used for the enrichment of one or more polynucleotide target sequences from a mixed pool.
  • a Cas enodnuclease disclosed herein may be immobilized on a matrix for use in in vitro target polynucleotide detection, binding, and/or modification
  • the disclosed guide polynucleotides, Cas endonucleases, polynucleotide modification templates, donor DNAs, guide polynucleotide/Cas endonuclease systems disclosed herein, and any one combination thereof, optionally further comprising one or more polynucleotide(s) of interest, can be introduced into a cell.
  • Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cells as well as plants and seeds produced by the methods described herein.
  • Vectors and constructs include circular plasmids, and linear polynucleotides, comprising a polynucleotide of interest and optionally other components including linkers, adapters, regulatory or analysis.
  • a recognition site and/or target site can be comprised within an intron, coding sequence, 5′ UTRs, 3′ UTRs, and/or regulatory regions.
  • the invention further provides expression constructs for expressing in a prokaryotic or eukaryotic cell/organism a guide RNA/Cas system that is capable of recognizing, binding to, and optionally nicking, unwinding, or cleaving all or part of a target sequence.
  • the expression constructs of the disclosure comprise a promoter operably linked to a nucleotide sequence encoding a Cas gene (or plant optimized, including a Cas endonuclease gene described herein) and a promoter operably linked to a guide RNA of the present disclosure.
  • the promoter is capable of driving expression of an operably linked nucleotide sequence in a prokaryotic or eukaryotic cell/organism.
  • Nucleotide sequence modification of the guide polynucleotide, VT domain and/or CER domain can be selected from, but not limited to, the group consisting of a 5′ cap, a 3′ polyadenylated tail, a riboswitch sequence, a stability control sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide poly nucleotide to a subcellular location, a modification or sequence that provides for tracking, a modification or sequence that provides a binding site for proteins, a Locked Nucleic Acid (LNA), a 5-methyl dC nucleotide, a 2,6-Diaminopurine nucleotide, a 2′-Fluoro A nucleotide, a 2′-Fluoro U nucleotide; a 2′-O-Methyl RNA nucleotide, a phosphorothioate bond, linkage to a cholesterol molecule, linkage to
  • the additional beneficial feature is selected from the group of a modified or regulated stability, a subcellular targeting, tracking, a fluorescent label, a binding site for a protein or protein complex, modified binding affinity to complementary target sequence, modified resistance to cellular degradation, and increased cellular permeability.
  • RNA polymerase III RNA polymerase III promoters
  • This strategy has been successfully applied in cells of several different species including maize and soybean (US20150082478 published 19 Mar. 2015). Methods for expressing RNA components that do not have a 5′ cap have been described (WO2016/025131 published 18 Feb. 2016).
  • compositions can be employed to obtain a cell or organism having a polynucleotide of interest inserted in a target site for a Cas endonuclease.
  • Such methods can employ homologous recombination (HR) to provide integration of the polynucleotide of interest at the target site.
  • HR homologous recombination
  • a polynucleotide of interest is introduced into the organism cell via a donor DNA construct.
  • the donor DNA construct further comprises a first and a second region of homology that flank the polynucleotide of interest.
  • the first and second regions of homology of the donor DNA share homology to a first and a second genomic region, respectively, present in or flanking the target site of the cell or organism genome.
  • the donor DNA can be tethered to the guide polynucleotide. Tethered donor DNAs can allow for co-localizing target and donor DNA, useful in genome editing, gene insertion, and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous HR machinery is expected to be highly diminished (Mali et al., 2013 , Nature Methods Vol. 10: 957-963).
  • the amount of homology or sequence identity shared by a target and a donor polynucleotide can vary and includes total lengths and/or regions having unit integral values in the ranges of about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total length of the target site.
  • ranges include every integer within the range, for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bps.
  • the amount of homology can also be described by percent sequence identity over the full aligned length of the two polynucleotides which includes percent sequence identity at least of about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, between 98% and 99%, 99%, between 99% and 100%, or 100%.
  • Sufficient homology includes any combination of polynucleotide length, global percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the target locus. Sufficient homology can also be described by the predicted ability of two polynucleotides to specifically hybridize under high stringency conditions, see, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology , Ausubel et al., Eds (1994) Current Protocols , (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, (Elsevier, New York).
  • the structural similarity between a given genomic region and the corresponding region of homology found on the donor DNA can be any degree of sequence identity that allows for homologous recombination to occur.
  • the amount of homology or sequence identity shared by the “region of homology” of the donor DNA and the “genomic region” of the organism genome can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that the sequences undergo homologous recombination
  • the region of homology on the donor DNA can have homology to any sequence flanking the target site. While in some instances the regions of homology share significant sequence homology to the genomic sequence immediately flanking the target site, it is recognized that the regions of homology can be designed to have sufficient homology to regions that may be further 5′ or 3′ to the target site. The regions of homology can also have homology with a fragment of the target site along with downstream genomic regions
  • the first region of homology further comprises a first fragment of the target site and the second region of homology comprises a second fragment of the target site, wherein the first and second fragments are dissimilar.
  • Polynucleotides of interest are further described herein and include polynucleotides reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest change, and as developing nations open up world markets, new crops and technologies will emerge also. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increase, the choice of genes for genetic engineering will change accordingly.
  • polynucleotides of interest include, for example, genes of interest involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific polynucleotides of interest include, but are not limited to, genes involved in traits of agronomic interest such as but not limited to: crop yield, grain quality, crop nutrient content, starch and carbohydrate quality and quantity as well as those affecting kernel size, sucrose loading, protein quality and quantity, nitrogen fixation and/or utilization, fatty acid and oil composition, genes encoding proteins conferring resistance to abiotic stress (such as drought, nitrogen, temperature, salinity, toxic metals or trace elements, or those conferring resistance to toxins such as pesticides and herbicides), genes encoding proteins conferring resistance to biotic stress (such as attacks by fungi, viruses, bacteria, insects, and nematodes, and development of diseases associated with these organisms).
  • genes of interest involved in information such as zinc fingers
  • Agronomically important traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.
  • Polynucleotide sequences of interest may encode proteins involved in providing disease or pest resistance.
  • Disease resistance or “pest resistance” is intended that the plants avoid the harmful symptoms that are the outcome of the plant-pathogen interactions.
  • Pest resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like.
  • Disease resistance and insect resistance genes such as lysozymes or cecropins for antibacterial protection, or proteins such as defensins, glucanases or chitinases for antifungal protection, or Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, lectins, or glycosidases for controlling nematodes or insects are all examples of useful gene products.
  • Genes encoding disease resistance traits include detoxification genes, such as against fumonisin (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al.
  • Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like.
  • Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109); and the like.
  • an “herbicide resistance protein” or a protein resulting from expression of an “herbicide resistance-encoding nucleic acid molecule” includes proteins that confer upon a cell the ability to tolerate a higher concentration of an herbicide than cells that do not express the protein, or to tolerate a certain concentration of an herbicide for a longer period of time than cells that do not express the protein.
  • Herbicide resistance traits may be introduced into plants by genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS, also referred to as acetohydroxyacid synthase, AHAS), in particular the sulfonylurea (UK: sulphonylurea) type herbicides, genes coding for resistance to herbicides that act to inhibit the action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), glyphosate (e.g., the EPSP synthase gene and the GAT gene), HPPD inhibitors (e.g, the HPPD gene) or other such genes known in the art. See, for example, U.S.
  • the polynucleotide of interest may also comprise antisense sequences complementary to at least a portion of the messenger RNA (mRNA) for a targeted gene sequence of interest.
  • Antisense nucleotides are constructed to hybridize with the corresponding mRNA. Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding mRNA. In this manner, antisense constructions having 70%, 80%, or 85% sequence identity to the corresponding antisense sequences may be used. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater may be used.
  • the polynucleotide of interest may also be used in the sense orientation to suppress the expression of endogenous genes in plants.
  • Methods for suppressing gene expression in plants using polynucleotides in the sense orientation are known in the art.
  • the methods generally involve transforming plants with a DNA construct comprising a promoter that drives expression in a plant operably linked to at least a portion of a nucleotide sequence that corresponds to the transcript of the endogenous gene.
  • a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, generally greater than about 65% sequence identity, about 85% sequence identity, or greater than about 95% sequence identity. See U.S. Pat. Nos. 5,283,184 and 5,034,323.
  • the polynucleotide of interest can also be a phenotypic marker.
  • a phenotypic marker is screenable or a selectable marker that includes visual markers and selectable markers whether it is a positive or negative selectable marker. Any phenotypic marker can be used.
  • a selectable or screenable marker comprises a DNA segment that allows one to identify, or select for or against a molecule or a cell that comprises it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like.
  • selectable markers include, but are not limited to, DNA segments that comprise restriction enzyme sites; DNA segments that encode products which provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT)); DNA segments that encode products which are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA segments that encode products which can be readily identified (e.g., phenotypic markers such as ⁇ -galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), and cell surface proteins); the generation of new primer sites for PCR (e.g., the juxtaposition of two DNA sequence not previously juxtaposed), the inclusion of DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA
  • Additional selectable markers include genes that confer resistance to herbicidal compounds, such as sulphonylureas, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • herbicidal compounds such as sulphonylureas, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • ALS Acetolactase synthase
  • imidazolinones imidazolinones
  • triazolopyrimidine sulfonamides pyrimidinylsalicylates
  • sulphonylaminocarbonyl-triazolinones Shaner and Singh, 1997 , Herbicide Activity: Toxicol Biochem Mol Biol 69-110
  • EPSPS glyphosate resistant 5-enolpyruvylshikimate-3-phosphate
  • Polynucleotides of interest includes genes that can be stacked or used in combination with other traits, such as but not limited to herbicide resistance or any other trait described herein. Polynucleotides of interest and/or traits can be stacked together in a complex trait locus as described in US20130263324 published 3 Oct. 2013 and in WO/2013/112686, published 1 Aug. 2013.
  • a polypeptide of interest includes any protein or polypeptide that is encoded by a polynucleotide of interest described herein.
  • identifying at least one plant cell comprising in its genome, a polynucleotide of interest integrated at the target site.
  • a variety of methods are available for identifying those plant cells with insertion into the genome at or near to the target site. Such methods can be viewed as directly analyzing a target sequence to detect any change in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blots, and any combination thereof. See, for example, US20090133152 published 21 May 2009.
  • the method also comprises recovering a plant from the plant cell comprising a polynucleotide of interest integrated into its genome.
  • the plant may be sterile or fertile. It is recognized that any polynucleotide of interest can be provided, integrated into the plant genome at the target site, and expressed in a plant.
  • a plant-optimized nucleotide sequence of the present disclosure comprises one or more of such sequence modifications.
  • Any polynucleotide encoding a Cas protein, other CRISPR system component, or other polynucleotide disclosed herein may be functionally linked to a heterologous expression element, to facilitate transcription or regulation in a host cell.
  • expression elements include but are not limited to: promoter, leader, intron, and terminator.
  • Expression elements may be “minimal”—meaning a shorter sequence derived from a native source, that still functions as an expression regulator or modifier.
  • an expression element may be “optimized”—meaning that its polynucleotide sequence has been altered from its native state in order to function with a more desirable characteristic in a particular host cell (for example, but not limited to, a bacterial promoter may be “maize-optimized” to improve its expression in corn plants).
  • an expression element may be “synthetic”—meaning that it is designed in silico and synthesized for use in a host cell. Synthetic expression elements may be entirely synthetic, or partially synthetic (comprising a fragment of a naturally-occurring polynucleotide sequence).
  • tissue specific promoters or tissue-preferred promoters if the promoters direct RNA synthesis preferably in certain tissues but also in other tissues at reduced levels.
  • a plant promoter includes a promoter capable of initiating transcription in a plant cell.
  • Constitutive promoters include, for example, the core CaMV 35S promoter (Odell et al., (1985) Nature 313:810-2); rice actin (McElroy et al., (1990) Plant Cell 2:163-71); ubiquitin (Christensen et al., (1989) Plant Mol Biol 12:619-32; ALS promoter (U.S. Pat. No. 5,659,026) and the like.
  • Tissue-preferred promoters can be utilized to target enhanced expression within a particular plant tissue.
  • Tissue-preferred promoters include, for example, WO2013103367 published 11 Jul. 2013, Kawamata et al., (1997) Plant Cell Physiol 38:792-803; Hansen et al., (1997) Mol Gen Genet 254:337-43; Russell et al., (1997) Transgenic Res 6:157-68; Rinehart et al., (1996) Plant Physiol 112:1331-41; Van Camp et al., (1996) Plant Physiol 112:525-35; Canevascini et al., (1996) Plant Physiol 112:513-524; Lam, (1994) Results Probl Cell Differ 20:181-96; and Guevara-Garcia et al., (1993) Plant J 4:495-505.
  • Leaf-preferred promoters include, for example, Yamamoto et al., (1997) Plant J 12:255-65; Kwon et al., (1994) Plant Physiol 105:357-67; Yamamoto et al., (1994) Plant Cell Physiol 35:773-8; Gotor et al., (1993) Plant J 3:509-18; Orozco et al., (1993) Plant Mol Biol 23:1129-38; Matsuoka et al., (1993) Proc. Natl. Acad. Sci. USA 90:9586-90; Simpson et al., (1958) EMBO J 4:2723-9; Timko et al., (1988) Nature 318:57-8.
  • Root-preferred promoters include, for example, Hire et al., (1992) Plant Mol Biol 20:207-18 (soybean root-specific glutamine synthase gene); Miao et al., (1991) Plant Cell 3:11-22 (cytosolic glutamine synthase (GS)); Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al., (1990) Plant Mol Biol 14:433-43 (root-specific promoter of A.
  • MAS tumefaciens mannopine synthase
  • Bogusz et al. (1990) Plant Cell 2:633-41 (root-specific promoters isolated from Parasponia andersonii and Trema tomentosa ); Leach and Aoyagi, (1991) Plant Sci 79:69-76 ( A.
  • Seed-preferred promoters include both seed-specific promoters active during seed development, as well as seed-germinating promoters active during seed germination. See, Thompson et al., (1989) BioEssays 10:108. Seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milps (myo-inositol-1-phosphate synthase); and for example those disclosed in WO2000011177 published 2 Mar. 2000 and U.S. Pat. No. 6,225,529.
  • seed-preferred promoters include, but are not limited to, bean ⁇ -phaseolin, napin, ⁇ -conglycinin, soybean lectin, cruciferin, and the like.
  • seed-preferred promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa gamma zein, waxy, shrunken 1, shrunken 2, globulin 1, oleosin, and nuc1. See also, WO2000012733 published 9 Mar. 2000, where seed-preferred promoters from END1 and END2 genes are disclosed.
  • Chemical inducible (regulated) promoters can be used to modulate the expression of a gene in a prokaryotic and eukaryotic cell or organism through the application of an exogenous chemical regulator.
  • the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters include, but are not limited to, the maize In2-2 promoter, activated by benzene sulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-II-27, WO1993001294 published 21 Jan.
  • Pathogen inducible promoters induced following infection by a pathogen include, but are not limited to those regulating expression of PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc.
  • a stress-inducible promoter includes the RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17:287-91).
  • One of ordinary skill in the art is familiar with protocols for simulating stress conditions such as drought, osmotic stress, salt stress and temperature stress and for evaluating stress tolerance of plants that have been subjected to simulated or naturally-occurring stress conditions.
  • ZmCAS1 promoter Another example of an inducible promoter useful in plant cells, is the ZmCAS1 promoter, described in US20130312137 published 21 Nov. 2013.
  • compositions described herein do not depend on a particular method for introducing a sequence into an organism or cell, only that the polynucleotide or polypeptide gains access to the interior of at least one cell of the organism.
  • Introducing includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient (direct) provision of a nucleic acid, protein or polynucleotide-protein complex (PGEN, RGEN) to the cell.
  • Methods for introducing polynucleotides or polypeptides or a polynucleotide-protein complex into cells or organisms are known in the art including, but not limited to, microinjection, electroporation, stable transformation methods, transient transformation methods, ballistic particle acceleration (particle bombardment), whiskers mediated transformation, Agrobacterium -mediated transformation, direct gene transfer, viral-mediated introduction, transfection, transduction, cell-penetrating peptides, mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, topical applications, sexual crossing, sexual breeding, and any combination thereof.
  • microinjection electroporation
  • stable transformation methods including, but not limited to, transient transformation methods, ballistic particle acceleration (particle bombardment), whiskers mediated transformation, Agrobacterium -mediated transformation, direct gene transfer, viral-mediated introduction, transfection, transduction, cell-penetrating peptides, mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, topical
  • the guide polynucleotide (guide RNA, crNucleotide+tracrNucleotide, guide DNA and/or guide RNA-DNA molecule) can be introduced into a cell directly (transiently) as a single stranded or double stranded polynucleotide molecule.
  • the guide RNA (or crRNA+tracrRNA) can also be introduced into a cell indirectly by introducing a recombinant DNA molecule comprising a heterologous nucleic acid fragment encoding the guide RNA (or crRNA+tracrRNA), operably linked to a specific promoter that is capable of transcribing the guide RNA (crRNA+tracrRNA molecules) in said cell.
  • the specific promoter can be, but is not limited to, an RNA polymerase III promoter, which allow for transcription of RNA with precisely defined, unmodified, 5′- and 3′-ends (Ma et al., 2014 , Mol. Ther. Nucleic Acids 3:e161; DiCarlo et al., 2013 , Nucleic Acids Res. 41: 4336-4343; WO2015026887, published 26 Feb. 2015).
  • Any promoter capable of transcribing the guide RNA in a cell can be used and includes a heat shock/heat inducible promoter operably linked to a nucleotide sequence encoding the guide RNA.
  • the Cas endonuclease such as the Cas endonuclease described herein, can be introduced into a cell by directly introducing the Cas polypeptide itself (referred to as direct delivery of Cas endonuclease), the mRNA encoding the Cas protein, and/or the guide polynucleotide/Cas endonuclease complex itself, using any method known in the art.
  • the Cas endonuclease can also be introduced into a cell indirectly by introducing a recombinant DNA molecule that encodes the Cas endonuclease.
  • the endonuclease can be introduced into a cell transiently or can be incorporated into the genome of the host cell using any method known in the art.
  • a Cell Penetrating Peptide as described in WO2016073433 published 12 May 2016.
  • Any promoter capable of expressing the Cas endonuclease in a cell can be used and includes a heat shock/heat inducible promoter operably linked to a nucleotide sequence encoding the Cas endonuclease.
  • Direct delivery of a polynucleotide modification template into plant cells can be achieved through particle mediated delivery, and any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery can be successfully used for delivering a polynucleotide modification template in eukaryotic cells, such as plant cells.
  • PEG polyethylene glycol
  • MSN mesoporous silica nanoparticle
  • the donor DNA can be introduced by any means known in the art.
  • the donor DNA may be provided by any transformation method known in the art including, for example, Agrobacterium -mediated transformation or biolistic particle bombardment.
  • the donor DNA may be present transiently in the cell or it could be introduced via a viral replicon. In the presence of the Cas endonuclease and the target site, the donor DNA is inserted into the transformed plant's genome.
  • Direct delivery of any one of the guided Cas system components can be accompanied by direct delivery (co-delivery) of other mRNAs that can promote the enrichment and/or visualization of cells receiving the guide polynucleotide/Cas endonuclease complex components.
  • direct co-delivery of the guide polynucleotide/Cas endonuclease components (and/or guide polynucleotide/Cas endonuclease complex itself) together with mRNA encoding phenotypic markers (such as but not limiting to transcriptional activators such as CRC (Bruce et al. 2000 The Plant Cell 12:65-79) can enable the selection and enrichment of cells without the use of an exogenous selectable marker by restoring function to a non-functional gene product as described in WO2017070032 published 27 Apr. 2017.
  • Introducing a guide RNA/Cas endonuclease complex described herein, (representing the cleavage ready cascade described herein) into a cell includes introducing the individual components of said complex either separately or combined into the cell, and either directly (direct delivery as RNA for the guide and protein for the Cas endonuclease and protein subunits, or functional fragments thereof) or via recombination constructs expressing the components (guide RNA, Cas endonuclease, protein subunits, or functional fragments thereof).
  • Introducing a guide RNA/Cas endonuclease complex (RGEN) into a cell includes introducing the guide RNA/Cas endonuclease complex as a ribonucleotide-protein into the cell.
  • the ribonucleotide-protein can be assembled prior to being introduced into the cell as described herein.
  • the components comprising the guide RNA/Cas endonuclease ribonucleotide protein (at least one Cas endonuclease, at least one guide RNA, at least one protein subunit) can be assembled in vitro or assembled by any means known in the art prior to being introduced into a cell (targeted for genome modification as described herein).
  • Plant cells differ from human and animal cells in that plant cells comprise a plant cell wall which may act as a barrier to the direct delivery of the ribonucleoproteins and/or of the direct delivery of the components.
  • Direct delivery of a ribonucleoprotein comprising a Cas endonuclease protein and a guide RNA into plant cells may be achieved through particle mediated delivery (particle bombardment.
  • particle mediated delivery particle bombardment.
  • any other direct method of delivery such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, electroporation, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, can be successfully used for delivering RGEN ribonucleoproteins into plant cells.
  • PEG polyethylene glycol
  • MSN mesoporous silica nanoparticle
  • Direct delivery of the ribonucleoprotein allows for genome editing at a target site in the genome of a cell which can be followed by rapid degradation of the complex, and only a transient presence of the complex in the cell. This transient presence of the complex may lead to reduced off-target effects.
  • delivery of components via RNA, Cas9 endonuclease
  • plasmid DNA sequences can result in constant expression from these plasmids which in some cases may promote off target cleavage (Cradick, T. J. et al. (2013) Nucleic Acids Res 41:9584-9592; Fu, Y et al. (2014) Nat. Biotechnol. 31:822-826).
  • Direct delivery can be achieved by combining any one component of the guide RNA/Cas endonuclease complex, representing the cleavage ready cascade described herein, (such as at least one guide RNA, at least one Cas protein, and optionally one additional protein), with a particle delivery matrix comprising a microparticle (such as but not limited to of a gold particle, tungsten particle, and silicon carbide whisker particle) (see also WO2017070032 published 27 Apr. 2017).
  • a particle delivery matrix comprising a microparticle (such as but not limited to of a gold particle, tungsten particle, and silicon carbide whisker particle)
  • the guide polynucleotide/Cas endonuclease complex is a complex wherein the guide RNA and Cas endonuclease protein forming the guide RNA/Cas endonuclease complex are introduced into the cell as RNA and protein, respectively.
  • the guide polynucleotide/Cas endonuclease complex is a complex wherein the guide RNA and Cas endonuclease protein and the at least one protein subunit of a Cascade forming the guide RNA/Cas endonuclease complex are introduced into the cell as RNA and proteins, respectively.
  • the guide polynucleotide/Cas endonuclease complex is a complex wherein the guide RNA and Cas endonuclease protein and the at least one protein subunit of a Cascade forming the guide RNA/Cas endonuclease complex (cleavage ready cascade) are preassembled in vitro and introduced into the cell as a ribonucleotide-protein complex.
  • Protocols for introducing polynucleotides, polypeptides or polynucleotide-protein complexes (PGEN, RGEN) into eukaryotic cells, such as plants or plant cells are known and include microinjection (Crossway et al., (1986) Biotechniques 4:320-34 and U.S. Pat. No. 6,300,543), meristem transformation (U.S. Pat. No. 5,736,369), electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-6, Agrobacterium -mediated transformation (U.S. Pat. Nos.
  • polynucleotides may be introduced into cells by contacting cells or organisms with a virus or viral nucleic acids.
  • such methods involve incorporating a polynucleotide within a viral DNA or RNA molecule.
  • a polypeptide of interest may be initially synthesized as part of a viral polyprotein, which is later processed by proteolysis in vivo or in vitro to produce the desired recombinant protein.
  • Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules are known, see, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931.
  • the polynucleotide or recombinant DNA construct can be provided to or introduced into a prokaryotic and eukaryotic cell or organism using a variety of transient transformation methods.
  • transient transformation methods include, but are not limited to, the introduction of the polynucleotide construct directly into the plant.
  • Nucleic acids and proteins can be provided to a cell by any method including methods using molecules to facilitate the uptake of anyone or all components of a guided Cas system (protein and/or nucleic acids), such as cell-penetrating peptides and nanocarriers. See also US20110035836 published 10 Feb. 2011, and EP2821486A1 published 7 Jan. 2015.
  • introducing polynucleotides into a prokaryotic and eukaryotic cell or organism or plant part can be used, including plastid transformation methods, and the methods for introducing polynucleotides into tissues from seedlings or mature seeds.
  • Stable transformation is intended to mean that the nucleotide construct introduced into an organism integrates into a genome of the organism and is capable of being inherited by the progeny thereof.
  • Transient transformation is intended to mean that a polynucleotide is introduced into the organism and does not integrate into a genome of the organism or a polypeptide is introduced into an organism. Transient transformation indicates that the introduced composition is only temporarily expressed or present in the organism.
  • a variety of methods are available to identify those cells having an altered genome at or near a target site without using a screenable marker phenotype. Such methods can be viewed as directly analyzing a target sequence to detect any change in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blots, and any combination thereof.
  • the presently disclosed polynucleotides and polypeptides can be introduced into a cell.
  • Cells include, but are not limited to, human, non-human, animal, mammalian, bacterial, protist, fungal, insect, yeast, non-conventional yeast, and plant cells, as well as plants and seeds produced by the methods described herein.
  • the cell of the organism is a reproductive cell, a somatic cell, a meiotic cell, a mitotic cell, a stem cell, or a pluripotent stem cell. Any cell from any organism may be used with the compositions and methods described herein, including monocot and dicot plants, and plant elements.
  • Animal cells can include, but are not limited to: an organism of a phylum including chordates, arthropods, mollusks, annelids, cnidarians, or echinoderms; or an organism of a class including mammals, insects, birds, amphibians, reptiles, or fishes.
  • the animal is human, mouse, C.
  • elegans rat, fruit fly ( Drosophila spp.), zebrafish, chicken, dog, cat, guinea pig, hamster, chicken, Japanese ricefish, sea lamprey, pufferfish, tree frog (e.g., Xenopus spp.), monkey, or chimpanzee.
  • Drosophila spp. Drosophila spp.
  • zebrafish chicken, dog, cat, guinea pig, hamster, chicken, Japanese ricefish, sea lamprey, pufferfish, tree frog (e.g., Xenopus spp.), monkey, or chimpanzee.
  • Particular cell types that are contemplated include haploid cells, diploid cells, reproductive cells, neurons, muscle cells, endocrine or exocrine cells, epithelial cells, muscle cells, tumor cells, embryonic cells, hematopoietic cells, bone cells, germ cells, somatic cells, stem cells, pluripotent stem cells, induced pluripotent stem cells, progenitor cells, meiotic cells, and mitotic cells.
  • a plurality of cells from an organism may be used.
  • compositions and methods described herein may be used to edit the genome of an animal cell in various ways. In one aspect, it may be desirable to delete one or more nucleotides. In another aspect, it may be desirable to insert one or more nucleotides. In one aspect, it may be desirable to replace one or more nucleotides. In another aspect, it may be desirable to modify one or more nucleotides via a covalent or non-covalent interaction with another atom or molecule.
  • Genome modification may be used to effect a genotypic and/or phenotypic change on the target organism.
  • a change is preferably related to an improved phenotype of interest or a physiologically-important characteristic, the correction of an endogenous defect, or the expression of some type of expression marker.
  • the phenotype of interest or physiologically-important characteristic is related to the overall health, fitness, or fertility of the animal, the ecological fitness of the animal, or the relationship or interaction of the animal with other organisms in its environment.
  • the phenotype of interest or physiologically-important characteristic is selected from the group consisting of: improved general health, disease reversal, disease modification, disease stabilization, disease prevention, treatment of parasitic infections, treatment of viral infections, treatment of retroviral infections, treatment of bacterial infections, treatment of neurological disorders (for example but not limited to: multiple sclerosis), correction of endogenous genetic defects (for example but not limited to: metabolic disorders, Achondroplasia, Alpha-1 Antitrypsin Deficiency, Antiphospholipid Syndrome, Autism, Autosomal Dominant Polycystic Kidney Disease, Barth syndrome, Breast cancer, Charcot-Marie-Tooth, Colon cancer, Cri du chat, Crohn's Disease, Cystic fibrosis, Dercum Disease, Down Syndrome, Duane Syndrome, Duchenne Muscular Dystrophy, Factor V Leiden Thrombophilia, Familial Hypercholesterolemia, Familial Mediterranean Fever, Fragile X Syndrome, Gaucher Disease, Hemochromatosis, Hemophil
  • Cells that have been genetically modified using the compositions or methods described herein may be transplanted to a subject for purposes such as gene therapy, e.g. to treat a disease, or as an antiviral, antipathogenic, or anticancer therapeutic, for the production of genetically modified organisms in agriculture, or for biological research.
  • gene therapy e.g. to treat a disease, or as an antiviral, antipathogenic, or anticancer therapeutic, for the production of genetically modified organisms in agriculture, or for biological research.
  • Examples of monocot plants include, but are not limited to, corn ( Zea mays ), rice ( Oryza sativa ), rye ( Secale cereale ), sorghum ( Sorghum bicolor, Sorghum vulgare ), millet (e.g., pearl millet ( Pennisetum glaucum ), proso millet ( Panicum miliaceum ), foxtail millet ( Setaria italica ), finger millet ( Eleusine coracana )), wheat ( Triticum species, for example Triticum aestivum, Triticum monococcum ), sugarcane ( Saccharum spp.), oats ( Avena ), barley ( Hordeum ), switchgrass ( Panicum virgatum ), pineapple ( Ananas comosus ), banana (Musa spp.), palm, ornamentals, turfgrasses, and other grasses.
  • corn Zea mays
  • rice Oryza sativa
  • dicot plants examples include, but are not limited to, soybean ( Glycine max ), Brassica species (for example but not limited to: oilseed rape or Canola) ( Brassica napus, B. campestris, Brassica rapa, Brassica. juncea ), alfalfa ( Medicago sativa ), tobacco ( Nicotiana tabacum ), Arabidopsis ( Arabidopsis thaliana ), sunflower ( Helianthus annuus ), cotton ( Gossypium arboreum, Gossypium barbadense ), and peanut ( Arachis hypogaea ), tomato ( Solanum lycopersicum ), potato ( Solanum tuberosum.
  • soybean Glycine max
  • Brassica species for example but not limited to: oilseed rape or Canola
  • Brassica napus Brassica napus, B. campestris, Brassica rapa, Brassica. juncea
  • alfalfa Medicago sativa
  • Additional plants that can be used include safflower ( Carthamus tinctorius ), sweet potato ( Ipomoea batatus ), cassava ( Manihot esculenta ), coffee ( Coffea spp.), coconut ( Cocos nucifera ), citrus trees ( Citrus spp.), cocoa ( Theobroma cacao ), tea ( Camellia sinensis ), banana (Musa spp.), avocado ( Persea americana ), fig ( Ficus casica ), guava ( Psidium guajava ), mango ( Mangifera indica), olive ( Olea europaea ), papaya ( Carica papaya ), cashew ( Anacardium occidentale ), macadamia ( Macadamia integrifolia ), almond ( Prunus amygdalus ), sugar beets ( Beta vulgaris ), vegetables, ornamentals, and conifers.
  • safflower Carthamus tinctorius
  • Vegetables that can be used include tomatoes ( Lycopersicon esculentum ), lettuce (e.g., Lactuca sativa ), green beans ( Phaseolus vulgaris ), lima beans ( Phaseolus limensis ), peas ( Lathyrus spp.), and members of the genus Cucumis such as cucumber ( C. sativus ), cantaloupe ( C. cantalupensis ), and musk melon ( C. melo ).
  • tomatoes Lycopersicon esculentum
  • lettuce e.g., Lactuca sativa
  • green beans Phaseolus vulgaris
  • lima beans Phaseolus limensis
  • peas Lathyrus spp.
  • members of the genus Cucumis such as cucumber ( C. sativus ), cantaloupe ( C. cantalupensis ), and musk melon ( C. melo ).
  • Ornamentals include azalea ( Rhododendron spp.), hydrangea ( Macrophylla hydrangea ), hibiscus ( Hibiscus rosasanensis ), roses ( Rosa spp.), tulips ( Tulipa spp.), daffodils ( Narcissus spp.), petunias ( Petunia hybrida ), carnation ( Dianthus caryophyllus ), poinsettia ( Euphorbia pulcherrima ), and chrysanthemum.
  • Conifers that may be used include pines such as loblolly pine ( Pinus taeda ), slash pine ( Pinus elliotii ), ponderosa pine ( Pinus ponderosa ), lodgepole pine ( Pinus contorta ), and Monterey pine ( Pinus radiata ); Douglas fir ( Pseudotsuga menziesii ); Western hemlock ( Tsuga canadensis ); Sitka spruce ( Picea glauca ); redwood ( Sequoia sempervirens ); true firs such as silver fir ( Abies amabilis ) and balsam fir ( Abies balsamea ); and cedars such as Western red cedar ( Thuja plicata ) and Alaska yellow cedar ( Chamaecyparis nootkatensis ).
  • pines such as loblolly pine ( Pinus taeda ), slash pine ( Pinus elliot
  • a fertile plant is a plant that produces viable male and female gametes and is self-fertile.
  • a self-fertile plant can produce a progeny plant without the contribution from any other plant of a gamete and the genetic material comprised therein.
  • Other embodiments of the disclosure can involve the use of a plant that is not self-fertile because the plant does not produce male gametes, or female gametes, or both, that are viable or otherwise capable of fertilization.
  • the present disclosure finds use in the breeding of plants comprising one or more introduced traits, or edited genomes.
  • a non-limiting example of how two traits can be stacked into the genome at a genetic distance of, for example, 5 cM from each other is described as follows: A first plant comprising a first transgenic target site integrated into a first DSB target site within the genomic window and not having the first genomic locus of interest is crossed to a second transgenic plant, comprising a genomic locus of interest at a different genomic insertion site within the genomic window and the second plant does not comprise the first transgenic target site. About 5% of the plant progeny from this cross will have both the first transgenic target site integrated into a first DSB target site and the first genomic locus of interest integrated at different genomic insertion sites within the genomic window.
  • Progeny plants having both sites in the defined genomic window can be further crossed with a third transgenic plant comprising a second transgenic target site integrated into a second DSB target site and/or a second genomic locus of interest within the defined genomic window and lacking the first transgenic target site and the first genomic locus of interest. Progeny are then selected having the first transgenic target site, the first genomic locus of interest and the second genomic locus of interest integrated at different genomic insertion sites within the genomic window.
  • Such methods can be used to produce a transgenic plant comprising a complex trait locus having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 19, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more transgenic target sites integrated into DSB target sites and/or genomic loci of interest integrated at different sites within the genomic window.
  • a complex trait locus having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 19, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more transgenic target sites integrated into DSB target sites and/or genomic loci of interest integrated at different sites within the genomic window.
  • various complex trait loci can be generated.
  • the invention comprises:
  • a method of increasing the frequency of homology-directed repair at double strand break of a target polynucleotide sequence comprising: (a) introducing a site-specific first double-strand break to the target polynucleotide sequence, that results in a modified target sequence; (b) providing to the target polynucleotide sequence a Cas endonuclease and a guide RNA that binds to the modified sequence, thereby creating a second double-strand break; and (c) allowing the second double-strand break to undergo repair.
  • Aspect 2 The method of Aspect 1, wherein steps (b) and (c) are repeated for 1, 2, 3, 4, 5, or more subsequent cleavage(s) and repair(s) step(s).
  • Aspect 3 The method of Aspect 1, wherein a plurality of guide RNA molecules are provided.
  • Aspect 4 The method of Aspect 3, wherein the plurality of guide RNA molecules are provided simultaneously.
  • Aspect 5 The method of Aspect 3, wherein the plurality of guide RNA molecules are provided sequentially.
  • a method of increasing the frequency of homology-directed repair at double strand break of a target polynucleotide sequence comprising: (a) introducing a first double-strand break to the first target polynucleotide sequence, that results in a second target polynucleotide sequence; (b) introducing to the second target polynucleotide sequence a Cas endonuclease and a recombinant DNA construct comprising a guide RNA DNA sequence and a spacer DNA sequence, wherein the guide RNA DNA sequence is complementary to the second target polynucleotide sequence created by the repair of the first double-strand break, creating a second double-strand break; (c) allowing the second double-strand break to undergo repair, resulting in a third target polynucleotide sequence; (d) editing the DNA sequence of the spacer of the recombinant DNA construct so that it is substantially identical to the third target polynucleotide sequence; (e) expressing from the re
  • Aspect 7 The method of Aspect 6, wherein steps (d) through (f) are repeated for 1, 2, 3, 4, 5, or more subsequent cleavage(s) and repair(s), wherein subsequent guide RNA(s) are designed in response to the previous cleavage repair mutation(s).
  • a method of increasing the frequency of homology-directed repair at double strand break of a target polynucleotide sequence comprising: (a) introducing a first double-strand break to the first target polynucleotide sequence; (b) introducing a single-strand nick to a polynucleotide adjacent to the first double-strand break; and (c) allowing the first double-strand break to undergo repair.
  • Aspect 9 The method of Aspect 8, wherein two nicks are created adjacent to, and flanking the double-strand break of (a).
  • Aspect 10 The method of Aspect 8, wherein the nick in step (b) is created within 100 basepairs of the double-strand break of (a).
  • Aspect 11 The method of Aspect 1, 6, or 8, wherein at least one Cas endonuclease is provided as a protein molecule.
  • Aspect 12 The method of Aspect 1, 6, or 8, wherein at least one guide RNA is provided as an mRNA molecule.
  • Aspect 13 The method of Aspect 1, 6, or 8, further comprising introducing a heterologous polynucleotide after step (a).
  • Aspect 14 The method of Aspect 11, wherein the heterologous polynucleotide is a template for repair of the double-strand break.
  • Aspect 15 The method of Aspect 11, wherein the heterologous polynucleotide is a donor DNA molecule for insertion at the double-strand break site.
  • Aspect 16 The method of Aspect 1, 6, or 8, wherein the first double-strand break is repaired by NHEJ.
  • Aspect 17 The method of Aspect 1, 6, or 8, wherein the frequency of HDR of subsequent double-strand-break repairs is greater than the rate of HDR of the first double-strand-break repair.
  • Aspect 18 The method of Aspect 1, 6, or 8, wherein the frequency of HDR of a double-strand-break repair at a target polynucleotide site is greater than the rate of NHEJ at that same site.
  • Aspect 19 The method of Aspect 1 or 6, wherein the ratio of HDR to NHEJ increases in at least one subsequent DSB repair step as compared to the first DSB repair step with no nicks introduced.
  • Aspect 20 The method of Aspect 1, 6, or 8, wherein the cumulative frequency of HDR is greater than that observed for single cleavage.
  • Aspect 21 The method of Aspect 1, 6, or 8, wherein the cumulative percentage of HDR is at least 10 times greater than that observed for single cleavage.
  • Aspect 22 The method of Aspect 1, 6, or 8, wherein the fraction of HR reads relative to the number of total mutant reads (NHEJ+HR) is at least 10 times greater than that observed for single cleavage.
  • Aspect 23 The method of Aspect 1, 6, or 8, wherein the percent of HR reads relative to the number of total mutant reads (NHEJ+HR) is at least 3%.
  • Aspect 24 The method of Aspect 1, 6, or 8, wherein the first double-strand break is created by a Cas endonuclease and guide RNA complex.
  • Aspect 25 The method of Aspect 1, 6, or 8, wherein the method is performed in vitro.
  • Aspect 26 The method of Aspect 1, 6, or 8, wherein the method is performed in vivo.
  • Aspect 27 The method of Aspect 1, 6, or 8, wherein the method is performed in a cell.
  • Aspect 28 The method of Aspect 26, wherein the method is performed in an animal cell.
  • Aspect 29 The method of Aspect 26, wherein the method is performed in a plant cell.
  • Aspect 30 The method of Aspect 29, wherein the plant cell is obtained or derived from a plant selected from the group consisting of: maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, switchgrass, soybean, Brassica , alfalfa, sunflower, cotton, tobacco, peanut, potato, tobacco, Arabidopsis , vegetable, and safflower.
  • Aspect 31 The method of Aspect 28 or 29, wherein the method confers a benefit to the cell or to an organism comprising said cell.
  • Aspect 32 The method of Aspect 31, wherein the benefit is selected from the group consisting of: improved health, improved growth, improved fertility, improved fecundity, improved environmental tolerance, improved vigor, improved disease resistance, improved disease tolerance, improved tolerance to a heterologous molecule, improved fitness, improved physical characteristic, greater mass, increased production of a biochemical molecule, decreased production of a biochemical molecule, upregulation of a gene, downregulation of a gene, upregulation of a biochemical pathway, downregulation of a biochemical pathway, stimulation of cell reproduction, and suppression of cell reproduction.
  • Aspect 33 The method of Aspect 31, wherein the cell is a plant cell, and wherein the benefit is a trait of agronomic interest of a plant comprising said cell or a progeny cell thereof, selected from the group consisting of: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, yield improvement, health enhancement, improved fertility, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, modulation of a metabolite, modulation of the proteome, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition; as compared to an isoline plant not comprising said target site modification or as compared to the plant prior to the modification of said target site in said plant cell.
  • Aspect 34 The method of Aspect 31, wherein the cell is an animal cell, and wherein the benefit is a phenotype of physiological interest of an organism comprising said animal cell or a progeny or progeny cell thereof, selected from the group consisting of: improved health, improved nutritional status, reduced disease impact, disease stasis, disease reversal, improved fertility, improved vigor, improved mental capacity, improved organism growth, improved weight gain, weight loss, modulation of an endocrine system, modulation of an exocrine system, reduced tumor size, reduced tumor mass, stimulated cell growth, reduced cell growth, production of a metabolite, production of a hormone, production of an immune cell, and stimulation of cell production.
  • the benefit is a phenotype of physiological interest of an organism comprising said animal cell or a progeny or progeny cell thereof, selected from the group consisting of: improved health, improved nutritional status, reduced disease impact, disease stasis, disease reversal, improved fertility, improved vigor, improved mental capacity, improved organism growth, improved weight
  • the DNA coated gold particles were pelleted by centrifugation, washed with absolute ethanol and re-dispersed by sonication. Following sonication, 10 1 1.1 of the DNA coated gold particles were loaded onto a macrocarrier and air dried.
  • biolistic transformation of 8 to 10-day-old immature maize embryos (IMEs) was performed using a PDS-1000/He Gun (Bio-Rad) with a 425 lb per square inch rupture disc. Since particle gun transformation can be highly variable, a visual marker DNA expression cassette encoding a fluorescent protein was also co-delivered to aid in the selection of evenly transformed IMEs for transient experiments.
  • Transformation of maize immature embryos using particle delivery is performed as follows. Media recipes follow below.
  • the ears are husked and surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water.
  • the immature embryos are isolated and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5-cm target zone in preparation for bombardment.
  • isolated embryos are placed on 560L (Initiation medium) and placed in the dark at temperatures ranging from 26° C. to 37° C. for 8 to 24 hours prior to placing on 560Y for 4 hours at 26° C. prior to bombardment as described above.
  • Plasmids comprising plant optimized Streptococcus pyogenes (Spy) Cas9 and guide RNA expression cassettes (Svitashev et al. (2015) Plant Physiology. 169:931-945) were co-delivered with homology-directed repair (HDR) capable DNA template with plasmids containing the developmental genes ODP2 (AP2 domain transcription factor ODP2 (Ovule development protein 2); US20090328252 A1) and Wushel (US2011/0167516) and an appropriate selectable marker.
  • HDR homology-directed repair
  • NPTII neomycin phosphotransferase
  • the plasmids and donor DNA are precipitated onto 0.6 micrometer (average diameter) gold pellets using a water-soluble cationic lipid transfection reagent as follows.
  • DNA solution is prepared on ice using 1 ⁇ g of plasmid DNA and optionally other constructs for co-bombardment such as 50 ng (0.5 ⁇ l) of each plasmid containing the developmental genes ODP2 (AP2 domain transcription factor ODP2 (Ovule development protein 2); US20090328252 A1) and Wuschel.
  • ODP2 AP2 domain transcription factor ODP2 (Ovule development protein 2); US20090328252 A1
  • Wuschel To the pre-mixed DNA, 20 ⁇ l of prepared gold particles (15 mg/ml) and 5 ⁇ l of a water-soluble cationic lipid transfection reagent is added in water and mixed carefully.
  • Gold particles are pelleted in a microfuge at 10,000 rpm for 1 min and supernatant is removed. The resulting pellet is carefully rinsed with 100 ml of 100% EtOH without resuspending the pellet and the EtOH rinse is carefully removed. 105 ⁇ l of 100% EtOH is added and the particles are resuspended by brief sonication. Then, 10 ⁇ l is spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
  • the plasmids and DNA of interest are precipitated onto 1.1 ⁇ m (average diameter) tungsten pellets using a calcium chloride (CaCl2) precipitation procedure by mixing 100 ⁇ l prepared tungsten particles in water, 10 ⁇ l (1 ⁇ g) DNA in Tris EDTA buffer (1 ⁇ g total DNA), 100 ⁇ l 2.5 M CaCl2, and 10 ⁇ l 0.1 M spermidine. Each reagent is added sequentially to the tungsten particle suspension, with mixing. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes.
  • CaCl2 calcium chloride
  • the tubes are centrifuged briefly, liquid is removed, and the particles are washed with 500 ml 100% ethanol, followed by a 30 second centrifugation. Again, the liquid is removed, and 105 ⁇ l of 100% ethanol is added to the final tungsten particle pellet.
  • the tungsten/DNA particles are briefly sonicated. 10 ⁇ l of the tungsten/DNA particles is spotted onto the center of each macrocarrier, after which the spotted particles are allowed to dry about 2 minutes before bombardment.
  • sample plates are bombarded at level #4 with a Biorad Helium Gun. All samples receive a single shot at 450 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
  • the embryos are incubated on 560P (maintenance medium) for 12 to 48 hours at temperatures ranging from 26 C to 37 C, and then placed at 26 C. After 5 to 7 days the embryos are transferred to selection medium containing an appropriate selective agent (e.g. Bialaphos), and sub-cultured every 2 weeks at 26 C. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to a lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established.
  • 560P maintenance medium
  • Plants are then transferred to inserts in flats (equivalent to a 2.5′′ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to Classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for transformation efficiency, and/or modification of regenerative capabilities.
  • Initiation medium comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000 ⁇ SIGMA-1511), 0.5 mg/l thiamine HCl, 20.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H 2 O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).
  • Maintenance medium comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000 ⁇ SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, 2.0 mg/l 2,4-D, and 0.69 g/l L-proline (brought to volume with D-I H 2 O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 0.85 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).
  • Bombardment medium comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000 ⁇ SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H 2 O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature).
  • Selection medium comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000 ⁇ SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H 2 O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).
  • Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H 2 O) (Murashige and Skoog (1962) Physiol. Plant.
  • Hormone-free medium comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H2O), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H 2 O after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-I H2O), sterilized and cooled to 60° C.
  • NHEJ non-homologous end-joining
  • HDR homology-directed repair
  • chromosomal DNA repair may be monitored transiently (Svitashev et al. (2015) Plant Physiology. 169:931-945 and Karvelis, T. et al. (2015) Genome Biology. 16:253 (Methods Section: In planta mutation detection)) and in regenerated plantlets by targeting sequencing. Briefly, 2 days after particle-mediated transformation, the 20-30 most evenly transformed immature maize embryos (IMEs) are harvested based on their immunofluorescence.
  • IMEs immature maize embryos
  • Total genomic DNA is extracted and the region surrounding the intended target site is PCR amplified with Phusion® HighFidelity PCR Master Mix (New England Biolabs, M0531L) adding on the sequences necessary for amplicon-specific barcodes and Illumina sequencing using “tailed” primers through two rounds of PCR and deep sequenced.
  • the resulting reads are then examined for the presence of NHEJ or HDR mutations at the expected site of cleavage by comparison to control experiments where the single guide RNA (sgRNA) transcriptional cassette is omitted from the transformation.
  • the fraction of HDR reads perfectly matching the changes introduced by the DNA repair template are then calculated to provide a comparison between different biological reps and treatments.
  • PCR can be used to detect a change introduced as a result of HDR (Svitashev et al. (2015) Plant Physiology. 169:931-945 and Shi et al. (2017) Plant Biotechnology Journal. 15: 2017-216).
  • one oligonucleotide is designed to be specific to the HDR modification while a second oligonucleotide is oriented in the flanking genomic DNA (outside of the region with homology to the repair template) to permit the PCR amplification of DSBs that have undergone HDR.
  • the presence of an amplification is used to detect tissue that has been repaired with the donor template.
  • qPCR quantitative PCR
  • PCR primers can also be designed to amplify across the HDR induced alteration.
  • repair using the donor template should result in a size difference (either bigger or smaller) at the DSB so that the size of the PCR amplicon can be used to detect repair with the donor template.
  • a combination of PCR-based methods can be used in combination to assess the presence, frequency, and intactness of a HDR edit.
  • HDR homology-directed repair
  • Cas CRISPR-associated
  • NHEJ non-homologous end-joining
  • Hsu homology-directed DNA repair pathways
  • Indel imprecise insertion or deletion
  • bps DNA base pairs
  • HDR homology-directed repair
  • the NHEJ pathway is typically the most prevalent DSB repair outcome making the recovery of HDR-mediated alterations infrequent (Capecchi, M. R. (1989) Science. 244:1288-1292).
  • To increase the frequency of HDR repair we devised a strategy that permits Cas9 to be programmed to repeatedly cleave a DNA target (FIG. 2 A). Since the cellular repair of Cas9 induced DSBs are non-random and reproducible (see, for example, Van Overbeek, M. et al. (2016) Mol. Cell. 63:633-646), our approach targeted not only the first intended site for cleavage but also sites comprised of the most prevalent NHEJ repair outcomes.
  • DSBs were generated in a step-wise recurrent manner (e.g. the intended target sequence was cleaved, and, once repaired to a prevalent NHEJ outcome, it was cleaved again) providing multiple opportunities for HDR-mediated DNA repair to occur. Since Cas9 has high affinity for and is slow to release its cleaved substrate (Richardson, C. et al. (2016) Nat. Biotechnol. 34:339-344), our approach also extended the cellular window of time in which DSBs occur and are repaired.
  • Target Site Zea mays sgRNA Target Sequence 3′ Name Genotype Location (AGPv4) (SEQ ID NO) PAM LIG1 Hi-Type II Chr2: 4236455-4236434 1 AGG MS26 Hi-Type II Chr1: 14704689-14704667 2 AGG MS45 Hi-Type II Chr9: 143632609-143632630 3 AGG TS45 ED85E Chr1: 15409712-15409735 4 AGG LIG1 NHEJ1 Hi-Type II N/A 29 AGG LIG1 NHEJ2 Hi-Type II N/A 30 AGG MS26 NHEJ1 Hi-Type II N/A 31 AGG MS26 NHEJ2 Hi-Type II N/A 32 AGG MS45 NHEJ1 Hi-Type II N/A 33 AGG MS45 NHEJ2 Hi-Type II N/A 34 AGG TS45 NHEJ1 ED85E N/A 35 A
  • Particle gun transformation was performed as described in Example 1 and analysis of the most prevalent mutations resulting from target cleavage and repair by NHEJ was accomplished using Illumina deep sequencing as described in Example 2. Transformations were performed in duplicate from immature embryos isolated from different sources to account for both technical and biological variation except for the MS26 site. For this target, 3 replicates were performed as the second most prevalent mutation was unclear from the first two replicates. The top 5 most prevalent NHEJ mutations for LIG1, MS26, MS45, and TS45 are shown in FIGS. 7A-D .
  • SpyCas9 single guide RNA (sgRNA) DNA plasmid expression cassettes capable of directing Cas9 cleavage at the two most prevalent NHEJ repair outcomes, NHEJ1 and NHEJ2, for LIG1, MS26, MS45, and TS45 were constructed.
  • the SpyCas9 sgRNA spacer length was adjusted to maintain a length of ⁇ 20 nt ( FIGS. 7A-D ).
  • an additional G was incorporated onto the 5′ end of the sgRNAs targeting the most prevalent NHEJs if their spacer terminated in any other nucleotide.
  • the NHEJ1 and NHEJ2 sequences targeted for cleavage are listed in Table 1.
  • LIG1 Since LIG1 exhibited a strong preference for a single NHEJ mutation, it was used as a test case. Plasmid DRTs were engineered to introduce a small change (5 bp) adjacent to the Cas9 cut-site in order to easily detect HDR by deep sequencing. DRTs with and without flanking target sites were also tested ( FIGS. 1D and 1E ). Flanking target sites were comprised of the most prevalent NHEJ mutation (NHEJ1). Particle gun transformation and analysis by Illumina deep sequencing was performed as described in Examples 1 and 2, respectively. Transformations were performed in duplicate from immature embryos isolated from different sources to account for both technical and biological variation. As shown in FIG.
  • flanking the DRT were evaluated for their impact on HDR.
  • the flanking target was homologous to the iTarget ( FIG. 9A ) while in others it comprised targets with partial homologous to the iTarget (NHEJ1) ( FIG. 9B ).
  • flanking the DRT with a target site increased the frequency of HDR and frequencies were further elevated using RC Cas9 ( FIG. 10 ).
  • HDR outcomes were the highest when RC Cas9 and a DRT with partially homologous flanking target sites were used ( FIG. 10 ).
  • HDR frequencies were assessed at two other Zea mays Cas9 sites, MS26 and MS45. Additionally, another control was added to further examine the finding that DRTs with partially homologous flanking targets (comprised of NHEJ1) provided the largest increase in HDR. This was an additional DRT that contained flanking targets that had no homology to the iTarget ( FIG. 9C ). As shown in FIGS. 11 and 12 , the combination of RC Cas9 plus the NHEJ1 flanking the DRT as described for the LIG1 consistently produced higher frequencies of HDR across all 3 target sites.
  • RC Cas9 with a repair template flanked by a site partially homologous to the iTarget provided a 28-fold increase in HDR frequencies relative to experiments when only the iTarget (without DRT flanking sequences) was cleaved ( FIG. 13 ).
  • flanking targets that contained homology with the iTarget either complete or partial homology
  • enhanced HDR with the highest frequencies being recovered for targets with partial homology (NHEJ1) to the iTarget FIG. 13 .
  • NHEJ1 partial homology
  • Hi-Type II immature embryos were regenerated as described in Example 1.
  • LIG and MS26 target sites were used and experiments included a control to establish improvements made to HDR.
  • the control comprised an experiment when only the iTarget was cleaved (without targets flanking the DRT).
  • Hi-Type II transformed callus from the MS26 experiment was sampled and examined for HDR as described in Example 2. Briefly, this was accomplished using a forward primer, designed to specifically amplify from within the HDR edit, and a reverse primer, placed adjacent to the genomic region with homology to the repair template ( FIG. 14A ).
  • the MS26 HDR edit was detectable in almost all of the transformed tissues compared to only a few instances in the control ( FIGS. 14B , C). After plant regeneration, the frequency of HDR was also significantly enhanced ( FIG. 15 ). At the MS26 site, an approximately 6-fold increase in HDR was observed. Note only plants that were likely to contain a germline HDR edit (defined as having >30% of all sequence reads with the HDR edit) were used in the calculation. For the LIG target, an even greater improvement in HDR was observed where no edits were detected in the control and approximately 6% of the plantlets exhibited HDR. Interestingly, of the plants that contained edits generated with RC Cas9, approximately 1 ⁇ 3 were predicted to contain HDR alterations on both alleles (bi-allelic) ( FIG. 15 ).
  • a DRT comprised of a DNA expression cassette capable of expressing NPTII was constructed ( FIG. 16 ).
  • particle gun transformation was performed with inbred ED85E as described in Example 1. Similar to previous experiments, the DRT was flanked with the NHEJ1 Target and the iTarget as well as the two most prevalent NHEJ mutations were targeted for cleavage. Enhancements to HDR were measured using experiments when only the iTarget was cleaved (without targets flanking the DRT).
  • the proportion of HDR edits for RC Cas9 that were determined to be bi-allelic was significantly enhanced compared to the other treatments (3.3% for RC Cas9, 0.7% for iTarget (with DRTs with iTarget sites), and 0.0% for iTarget (without sites flanking the DRT).
  • the proportion of clean plants defined as having no other DNA integrated (e.g. Cas9, sgRNA, BBM, Wus) except the NPTII expression cassette at the target site, was also elevated for RC Cas9 ( FIG. 17 ).
  • FIG. 2B shows an illustration with an example of improving frequency of HDR via recursive cleavage of a target polynucleotide site.
  • a double-strand-break as created, repaired, and recursively cleaved by any method or composition for example but not limited to a Cas endonuclease and guide RNA.
  • a DSB inducing agent e.g., Cas endonuclease and first guide RNA
  • the first guide RNA is provided as a DNA sequence on a plasmid that further comprises a spacer sequence.
  • the DNA encoding the gRNA is operably linked to a regulatory expression element.
  • a first double-strand-break is created, and repaired.
  • the composition of the repaired target polynucleotide is used as the basis of a mutation generated by Cas editing of the spacer on the plasmid comprising the gRNA DNA and spacer.
  • the mutated spacer composition directs the generation of a second gRNA that is complementary to the sequence of the repaired targeted polynucleotide of the first DSB, and a second double-strand break is induced at the target site by the Cas endonuclease and the second gRNA.
  • the cycle may then repeat, with sequence of the newly repaired second DSB then being used as a template for the composition of a third gRNA that is complementary to the sequence of the repaired second DSB polynucleotide, and so forth.
  • a loop of DSB generation and repair occurs, with each subsequent repair after the first having a higher probability of repair via HDR than NHEJ, as compared to the mechanism of the first repair.
  • the process may stop or proceed at a reduced rate by any of a number of methods, including but not limited to: titrated reagent availability, mutation induced in the region of the gRNA DNA expression construct that renders the expression cassette or transcribed gRNA to be non-functional, an external factor that may optionally be inducible or repressible, or via the introduction of another molecule.
  • a novel method achieving the enhancement of homologous recombination after the creation of a double strand break in DNA is described herein.
  • the platform was based on the induction of a double strand break at a specific target site in chromosomal maize DNA using an RNA-protein complex of a Cas endonuclease (e.g., B. laterosporus , or S. aureus Cas9) and a guide RNA that binds to the Cas endonuclease and had sequences complementary to the target chromosomal DNA.
  • a Cas endonuclease e.g., B. laterosporus , or S. aureus Cas9
  • guide RNA that binds to the Cas endonuclease and had sequences complementary to the target chromosomal DNA.
  • nickase for example, Cas molecules with their own guide RNAs that are not identical to the Cas9 that made the double strand break.
  • the specific orientation of the nicks on specific strands of the DNA was designed and to generate 3′ overhangs at the double strand break which were recombinogenic.
  • FIG. 3 describes this orientation and the current Cas9s used to generate these data.
  • specific target sites and DSB agents e.g., B. laterosporus Cas9
  • nicking agents S.
  • pyogenes Cas9 molecules that had been mutated with a D10A mutation to render the enzyme a nickase instead of a DSB agent) were used in some of these particular experiments, it is contemplated that other molecules could be used to accomplish creating the DSB and/or the flanking nick sites of the DSB site; further these results are target site-independent.
  • Spy means S. pyogenes
  • Blat means B. laterosporus
  • Sa or “ S. aureus ” means S. aureus .
  • Spyn means. S. pyogenes Cas9 nickase, etc.
  • flanking nickase guides for each target sites are listed in Table 2, along with the PAM sequences for each of the Cas9 nickases.
  • the repair templates used for each experiment are listed in Table 3.
  • the sequences for each target site are given in Table 4.
  • the first experiment tested a “Spy-Blat-Spy” nickase-DSB agent-nickase strategy at the maize MS45-BLAT2 target site ( FIG. 4A ), with three different combinations of nickase target sites.
  • Co-delivered with the BlatCas9 DSB agent were Cas9 nickases (SpynCas9), different guides according to FIG. 4A , and a double strand DNA repair template oligo of 180 bp length.
  • the repair template included insertion sequences that was observed as a specific template based change via deep sequencing of bulk DNA from the embryos. Sequences changes were detected in the target sequence.
  • FIG. 4A The repair template included insertion sequences that was observed as a specific template based change via deep sequencing of bulk DNA from the embryos. Sequences changes were detected in the target sequence.
  • FIGS. 4B and 4C show the frequencies of mutations at the target site when: BLATCas9 and SpynCas9 were delivered but no guides were provided (no guide); when BLAT and its cognate guide were used by itself (BLAT only); and when BLAT and its cognate guide was co-delivered with SpynCas9 and specified Spy guides. Mutant reads for each are shown in FIG. 4B and HDR frequencies for each are shown in FIG. 4C .
  • the next experiment tested a “Spy-Blat-Spy” nickase-DSB agent-nickase strategy, at an additional three combinations of different nickase target sites.
  • the MS45-BLAT2 target site for a double strand break via the BlatCas9 was targeted.
  • Co-delivered were Cas9 nickases (SpynCas9), different guides according to FIG. 4D , and a double strand DNA repair template oligo of 180 bp length.
  • the repair template included insertion sequences that was observed as a specific template based change via deep sequencing of bulk DNA from the embryos. Sequences changes were detected in the target sequence.
  • FIGS. 4E and 4F show the frequencies of mutations at the target site for: BlatCas9 and nSpynCas9 were delivered but no guides were provided (no guide); when Blat and its cognate guide were used by itself (Blat only); when Blat and its cognate guide were co-delivered with nickase and specified paired nick site Spyn guides (5/3 and 5/4 pairs); when SpynCas9 nickase and nick site guides (5/3 pair) and were delivered without Blat or the Blat guide; and when BlatCas9, Blat guide, and SpynGuides were delivered without co-delivering the nickase Spyn Cas9. Mutant reads for each are shown in FIG. 4E and HDR frequencies for each are shown in FIG. 4F . Enhancement of SDN2-type HDR events was observed when the nickase Spyn Cas9.
  • FIG. 5A Average mutant reads for are shown in FIG. 5B and HDR frequencies shown in FIG. 5C , for: SpyCas9 delivered with no guides; SpyCas9 delivered with its cognate gRNA; SpyCas9, its cognate gRNA, nickase Blat and its guides; and Blat nickase with its guides only, with no SpyCas9 or its cognate gRNA.
  • the Blat-Spy-Blat strategy was also performed at a different maize target site (NLB-CR8) ( FIG. 5D , SEQID NO:69) in a different germplasm line, for five replicates. Average mutant reads for are shown in FIG. 5E and HDR frequencies shown in FIG. 5F , for: SpyCas9 delivered with no guides; SpyCas9 delivered with its cognate gRNA; SpyCas9, its cognate gRNA, nickase Blat and its guides; and Blat nickase with its guides only, with no SpyCas9 or its cognate gRNA.
  • FIG. 6A Average mutant reads for are shown in FIG. 6B and HDR frequencies shown in FIG. 6C , for: SaCas9 delivered with no guides; SaCas9 delivered with its cognate gRNA; SaCas9, its cognate gRNA, nickase SpynCas9 and its guides; and SpynCas9 nickase with its guides only, with no SaCas9 or its cognate gRNA.
  • FIG. 6D SEQID NO:71
  • Average mutant reads for are shown in FIG. 6E and HDR frequencies shown in FIG. 6F , for: SpyCas9 delivered with no guides; SpyCas9 delivered with its cognate gRNA; SpyCas9, its cognate gRNA, nickase SaCas9 and its guides; and SaCas9 nickase with its guides only, with no SpyCas9 or its cognate gRNA.
  • the “Sa-Spy-Sa” strategy was performed at a different maize genomic target site (TS45) ( FIG. 6G , SEQID NO:72), for two replicates. Average mutant reads for are shown in FIG. 6H and HDR frequencies shown in FIG. 6I , for: SpyCas9 delivered with no guides; SpyCas9 delivered with its cognate gRNA; SpyCas9, its cognate gRNA, nickase SaCas9 and its guides; and SaCas9 nickase with its guides only, with no SpyCas9 or its cognate gRNA.

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