WO2024005864A1 - Compositions, systèmes et procédés d'édition génomique - Google Patents

Compositions, systèmes et procédés d'édition génomique Download PDF

Info

Publication number
WO2024005864A1
WO2024005864A1 PCT/US2022/073286 US2022073286W WO2024005864A1 WO 2024005864 A1 WO2024005864 A1 WO 2024005864A1 US 2022073286 W US2022073286 W US 2022073286W WO 2024005864 A1 WO2024005864 A1 WO 2024005864A1
Authority
WO
WIPO (PCT)
Prior art keywords
casl2a
sequence
tracrrna
nuclease
dna
Prior art date
Application number
PCT/US2022/073286
Other languages
English (en)
Inventor
Adam Patrick JOYCE
Michael Andreas Kock
Hannah PHAM
Original Assignee
Inari Agriculture Technology, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Inari Agriculture Technology, Inc. filed Critical Inari Agriculture Technology, Inc.
Priority to PCT/US2022/073286 priority Critical patent/WO2024005864A1/fr
Publication of WO2024005864A1 publication Critical patent/WO2024005864A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • aspects of this disclosure relate to biotechnology, in particular compositions and methods for genome editing.
  • CRISPR technology for editing the genes of eukaryotes is disclosed in U.S. Patent Application Publications 2016/0138008A1 (now U.S. Patent 10,227,11) and US2015/0344912A1, and in U.S. Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616.
  • Cpfl (Casl2a) endonucleases and corresponding guide RNAs and PAM sites are disclosed in U.S. Patent 9,790,490 and U.S.
  • Patent Application 15/566,528 (national phase of PCT Application PCT/EP2016/058442, published as WO 2016/166340), now published as U.S. Patent Application Publication 2018/0282713.
  • CRISPR nucleases useful for editing genomes include C2cl and C2c3 (see Shmakov et al. (2015) Afo/. Cell, 60:385-397) and CasX and CasY (see Burstein et al. (2016) Nature, doi:10.1038/nature21059).
  • Plant RNA promoters for expressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9 endonuclease are disclosed in U.S. Patent Application 15/120,110, published as U.S.
  • Patent Application Publication 2017/0166912 national phase application claiming priority to International Patent Application PCT/US2015/018104 (published as WO 2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700).
  • Methods of using CRISPR technology for genome editing in plants are disclosed in in U.S. Patent Application Publications U.S. 2015/0082478A1 and U.S. 2015/0059010A1 and in International Patent Application PCT/US2015/038767 Al (published as WO 2016/007347, claiming priority to U.S. Provisional Patent Application 62/023,246, with U.S. National Phase application US15/325,! 16, now published as U.S. Patent Application Publication 2017/0306349).
  • the invention provides a method of tethering a functional RNA molecule to a Casl2a crRNA, the method including hybridizing (a) a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety; and (b) a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; thereby tethering the functional RNA molecule to the Casl2a crRNA.
  • the functional RNA molecule is transcribed from a DNA molecule that encodes the Casl2a tracrRNA and the functional RNA moiety; in embodiments, DNA encoding the functional RNA moiety is inserted or integrated at a native (naturally occurring) or artificial (engineered) restriction endonuclease cleavage site that is located in or adjacent to (typically within the 3’ region of) the DNA encoding the Casl2a tracrRNA.
  • the crRNA 3’ extension includes nucleotides that when base-paired form about one helical turn, or at least 10 contiguous nucleotides.
  • the Casl2a tracrRNA further includes a 5’ extension.
  • the functional RNA moiety is characterized by one or more functions.
  • Embodiments of the RNA moiety include RNA sequences for annealing to one or more polynucleotides, RNA sequences that provide structures for binding to other molecules (including proteins or small molecule ligands) or that are catalytically active, or RNA sequences that serve as directly or indirectly detectable labels.
  • the Casl2a crRNA is complexed with, or is capable of complexing with, a Casl2a nuclease to form a Casl2a ribonucleoprotein complex containing the functional RNA molecule; thus, in embodiments, the method further includes the step of associating or complexing a Casl2a nuclease with a Casl2a crRNA that has (e.g., in a preceding or simultaneous step), or will have (e.g., in a subsequent step) the functional RNA molecule tethered to it.
  • the Casl2a nuclease is one having a naturally occurring sequence (e.g., a native sequence of a Casl2a nuclease such as, but not limited to, LbCasl2a, AsCasl2a, and FnCasl2a); in other embodiments, the Casl2a nuclease includes a modification, such as an amino acid sequence modification or a chemical modification.
  • a naturally occurring sequence e.g., a native sequence of a Casl2a nuclease such as, but not limited to, LbCasl2a, AsCasl2a, and FnCasl2a
  • the Casl2a nuclease includes a modification, such as an amino acid sequence modification or a chemical modification.
  • the invention provides a modified Casl2a ribonucleoprotein complex including: (a) a Casl2a nuclease; (b) a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety; and (c) a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule.
  • the Casl2a nuclease is one having a naturally occurring sequence (e.g., a native sequence of a Casl2a nuclease such as, but not limited to, LbCasl2a, AsCasl2a, and FnCasl2a); in other embodiments, the Casl2a nuclease includes a modification, such as an amino acid sequence modification or a chemical modification. In a preferred embodiment the expression and/or presence of the Casl2a tracrRNA enhances the genome editing capability of the Casl2a nuclease.
  • a naturally occurring sequence e.g., a native sequence of a Casl2a nuclease such as, but not limited to, LbCasl2a, AsCasl2a, and FnCasl2a
  • the Casl2a nuclease includes a modification, such as an amino acid sequence modification or a chemical modification.
  • the invention provides a method of inserting a nucleotide sequence encoded by a donor polynucleotide at a specific locus in a target DNA, the method including: (a) annealing a donor polynucleotide to the modified Casl2a ribonucleoprotein complex of claim 12, wherein the functional RNA moiety includes an RNA sequence for annealing to the donor polynucleotide, and wherein the Casl2a crRNA includes a spacer sequence that corresponds to a specific target locus in a target DNA, thus forming a donor:RNP complex; and (b) contacting the target DNA with the donor:RNP complex; whereby the nucleotide sequence encoded by the donor polynucleotide is inserted at the specific target locus in the target DNA.
  • the donor polynucleotide includes single-stranded DNA, optionally including chemical modifications. In embodiments, the donor polynucleotide includes double-stranded DNA, optionally including chemical modifications. In embodiments, the donor polynucleotide encodes coding sequence, non-coding sequence (e.g. , a noncoding sequence or regulatory element that modifies expression of a target gene located at or near the target locus), or both coding and non-coding sequence.
  • the target DNA is genomic DNA, e.g., genomic DNA or nuclear DNA of a eukaryote. In embodiments, the target DNA is mitochondrial or plastid DNA.
  • the invention provides a DNA expression system including (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter a DNA sequence encoding a first RNA molecule including a Casl2a tracrRNA and including a restriction endonuclease cleavage site (native or engineered) that is suitable for insertion of a sequence coding for a functional RNA moiety.
  • the DNA expression system further includes: (c) optionally, a DNA sequence for a second promoter; (d) operably linked and heterologous to the first promoter or the second promoter, a DNA encoding a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the first RNA molecule; and (e) optionally, a terminator; in embodiments, the first promoter and the DNA encoding the first RNA molecule, and the DNA encoding a Casl2a crRNA that includes a 3’ extension are contained in a single construct, and in other embodiments, the first promoter and the DNA encoding the first RNA molecule are contained in a first construct, and the second promoter and the DNA encoding a Casl2a crRNA that includes a 3’ extension are contained in a second construct.
  • the DNA expression system is provided in a first construct and a second construct, these can be provided in a single vector or in separate vectors.
  • the first promoter drives expression of both the DNA encoding the first RNA molecule and the DNA encoding the Casl2a crRNA.
  • the Casl2a tracrRNA further includes a 5’ extension.
  • the Casl2a crRNA is capable of complexing with a Casl2a nuclease to form a Casl2a ribonucleoprotein complex.
  • the expression and/or presence of the Casl2a tracrRNA enhances the genome editing capability of the Casl2a nuclease.
  • the invention provides a DNA expression system including: (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter, a DNA sequence encoding a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety.
  • the functional RNA moiety is characterized by one or more functions.
  • Embodiments of the RNA moiety include RNA sequences for annealing to one or more polynucleotides, RNA sequences that provide structures for binding to other molecules (including proteins or small molecule ligands) or that are catalytically active, or RNA sequences that serve as directly or indirectly detectable labels.
  • the DNA expression system further includes: (c) optionally, a DNA sequence for a second promoter; (d) operably linked and heterologous to the first promoter or the second promoter, a DNA encoding a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; and (e) optionally, a terminator.
  • the DNA expression system includes the DNA encoding the Casl2a crRNA
  • the DNA expression system can be provided in a single construct (e.g., where the first promoter drives expression of both the DNA encoding the functional RNA molecule and the DNA encoding the Casl2a crRNA).
  • the second promoter can be operably linked to the DNA encoding the crRNA (e.g., in a second construct). In embodiments where the DNA expression system is provided in two constructs, these can be in a single vector or in separate vectors.
  • the Casl2a tracrRNA further includes a 5’ extension.
  • the Casl2a crRNA is capable of complexing with, a Casl2a nuclease to form a Casl2a ribonucleoprotein complex containing the functional RNA molecule.
  • the Casl2a nuclease is one having a naturally occurring sequence (e.g., a native sequence of a Casl2a nuclease such as, but not limited to, LbCasl2a, AsCasl2a, and FnCasl2a); in other embodiments, the Casl2a nuclease includes a modification, such as an amino acid sequence modification or a chemical modification. In a preferred embodiment the expression and/or presence of the Casl2a tracrRNA enhances the genome editing capability of the Casl2a nuclease.
  • a naturally occurring sequence e.g., a native sequence of a Casl2a nuclease such as, but not limited to, LbCasl2a, AsCasl2a, and FnCasl2a
  • the Casl2a nuclease includes a modification, such as an amino acid sequence modification or a chemical modification.
  • the invention provides an engineered system including: (a) a Casl2a nuclease; and (b) at least one engineered Casl2a crRNA, or a polynucleotide encoding the engineered Casl2a crRNA, wherein the engineered Casl2a crRNA includes (i) at least one direct repeat sequence or fragment thereof that is capable of associating with the Casl2a nuclease, and (ii) a spacer sequence that is designed to hybridize with a target sequence in a eukaryotic cell, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 5’ to the spacer sequence; and (c) a Casl2a tracrRNA including a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of the at least one direct repeat of the engineered Casl2a crRNA.
  • the invention further provides an engineered system including: (a) a Casl2a nuclease; and (b) at least one engineered Casl2a crRNA, or a polynucleotide encoding the engineered Casl2a crRNA, wherein the engineered Casl2a crRNA includes (i) at least one direct repeat sequence or fragment thereof that is capable of associating with the Casl2a nuclease, and (ii) a spacer sequence that is designed to hybridize with a target sequence in a eukaryotic cell, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 5’ to the spacer sequence; and (c) a Casl2a tracrRNA including a naturally occurring putative Casl2a tracrRNA sequence that is identified from the same genomic region as the Casl2a nuclease by at least the following steps: (i) identifying in the genomic region flanking the DNA encoding the Casl2a nuclea
  • the invention provides an engineered system including: (a) one or more nucleotide sequences encoding a Casl2a nuclease; and (b) one or more nucleotide sequences encoding at least one engineered Casl2a crR A that is designed to form a complex with the Casl2a nuclease and that includes a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell; and (c) one or more nucleotide sequences encoding at least one Casl2a tracrRNA.
  • the invention provides an engineered system including: (a) a Casl2a nuclease, or one or more nucleotide sequences encoding the Casl2a nuclease; and (b) at least one engineered Casl2a crRNA designed to form a complex with the Casl2a nuclease and including a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell, or one or more nucleotide sequences encoding the at least one engineered Casl2a crRNA polynucleotide; and (c) at least one tracrRNA, or one or more nucleotide sequences encoding the at least one Casl2a tracrRNA.
  • the engineered Casl2a crRNA, or the polynucleotide (such as a pre-processed Casl2a crRNA precursor transcript) encoding the engineered Casl2a crRNA further includes at least one direct repeat sequence or fragment thereof capable of associating with the Casl2a nuclease, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 3’ to the spacer sequence.
  • the engineered Casl2a crRNA further includes a 3’ extension adjacent to the spacer sequence.
  • the eukaryotic cell is a non-human animal cell (e.g., a cell of an animal selected from the group consisting of invertebrates, vertebrates, insects, arthropods, mollusks, fish, reptiles, amphibians, birds, mammals, primates, and non-human primates), a human cell, a plant cell, or a fungal cell.
  • the eukaryotic cell is in vitro, ex vivo, or in vivo.
  • the invention provides an engineered system including one or more vectors including: (a) a first regulatory element (such as a promoter) that is heterologous to and operably linked to one or more nucleotide sequences encoding a Casl2a nuclease; and (b) a second regulatory element (such as a promoter) that is heterologous to and operably linked to one or more nucleotide sequences encoding at least one engineered Casl2a crRNA that is designed to form a complex with the Casl2a nuclease and that includes a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell; and (c) a third regulatory element (such as a promoter) that is heterologous to and operably linked to one or more nucleotide sequences encoding at least one Casl2a tracrRNA; wherein components (a) and (b) and (c) are located on the same vector or on different vectors
  • the invention provides an engineered system including one or more vectors including: (a) a first expression cassette including at least a first promoter that is heterologous to and operably linked to one or more nucleotide sequences encoding a Casl2a nuclease and - optionally - a first transcription terminator sequence; and (b) a second expression cassette including at least a second promoter that is heterologous to operably linked to one or more nucleotide sequences encoding at least one engineered Casl2a crRNA that is designed to form a complex with the Casl2a nuclease and that includes a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell and - optionally - a second transcription terminator sequence; and (c) a third expression cassette including at least a third promoter that is heterologous to operably linked to one or more nucleotide sequences encoding at least one Casl2a tracrRNA
  • compositions e.g., a delivery particle or a delivery liquid; compositions for treating a disease or infection; and/or compositions for the manufacture of a medicament
  • Embodiments of the compositions include those wherein the Casl2a tracrRNA is present in a molar excess, relative to the amount of Casl2a nuclease.
  • Specific embodiments of the compositions include those wherein the Casl2a tracrRNA is present in a molar amount that is at least 10-fold greater than the molar amount of the Casl2a nuclease.
  • Additional aspects of the invention include a method of modifying a locus of interest having a target sequence of a eukaryotic cell including delivering one or more of the engineered systems to the locus of interest, wherein the spacer sequence hybridizes with the target sequence, whereby modification of the locus of interest occurs.
  • the locus of interest is within a eukaryotic cell.
  • the eukaryotic cell is a non-human animal cell (e.g.
  • a cell of an animal selected from the group consisting of invertebrates, vertebrates, insects, arthropods, mollusks, fish, reptiles, amphibians, birds, mammals, primates, and non-human primates), a human cell, a plant cell, or a fungal cell.
  • the eukaryotic cell is in vitro, ex vivo, or in vivo.
  • the modification of the locus of interest is an insertion of at least one nucleotide, a deletion of at least one nucleotide, a change of at least one nucleotide (such as creation of a point mutation in the locus), non-homologous end-joining (NHEJ), or homology-dependent repair (HDR).
  • NHEJ non-homologous end-joining
  • HDR homology-dependent repair
  • the invention provides a method of editing a genetic locus with a Casl2a nuclease, including contacting DNA that includes the genetic locus with: (a) a Casl2a nuclease; and (b) an engineered Casl2a crRNA that includes a spacer sequence that is designed to hybridize with a target sequence in the genetic locus; and (c) a Casl2a tracrRNA, thereby editing the genetic locus; wherein the efficiency of editing of the genetic locus is increased compared to a control method in which the DNA that includes the genetic locus is contacted with the Casl2a nuclease and the Casl2a crRNA, but not the Casl2a tracrRNA.
  • the efficiency of editing is assessed by quantifying the amount of Cast 2a nuclease required to obtain a given percentage of edited target sequences (or percentage of cells containing the edited target sequences); efficiency is increased where a given editing efficiency is achieved using a lower amount of Casl2a nuclease.
  • the Casl2a tracrRNA is provided in a molar excess, relative to the Casl2a nuclease.
  • the Casl2a tracrRNA is provided in a molar amount that is at least 10-fold greater than the molar amount of the Casl2a nuclease.
  • the invention further provides a composition for modifying a genetic locus in a eukaryotic cell, including: (a) a Casl2a nuclease, or a polynucleotide encoding the Casl2a nuclease; (b) an engineered Casl2a crRNA that includes a spacer sequence corresponding to a specific sequence in the genetic locus, or a polynucleotide encoding the Casl2a crRNA; and (c) a Casl2a tracrRNA, or a polynucleotide encoding the Casl2a tracrRNA, wherein the Casl2a tracrRNA is provided in molar excess relative to the Casl2a nuclease.
  • the composition further comprises a eukaryotic cell containing a genetic locus to be modified.
  • the Casl2a tracrRNA is identified from the same genomic region as the Casl2a nuclease by at least the following steps: (a) identifying in the genomic region flanking the DNA encoding the Casl2a nuclease at least one nucleotide sequence that includes a CRISPR array including direct repeats; (b) identifying as a putative Casl2a tracrRNA at least one nucleotide sequence in either the sense or antisense direction in the genomic region flanking the DNA encoding the Casl2a nuclease, wherein the putative Casl2a tracrRNA has a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of the direct repeat.
  • the Casl2a tracrRNA is present in a molar excess, relative to the Casl2a nuclease. In embodiments of the composition, the Casl2a tracrRNA is present in a molar amount that is at least 10-fold greater than the molar amount of the Casl2a nuclease.
  • compositions in the treatment of a genetic disease, cancer, a viral infection, a bacterial infection, a parasitic infection, a fungal infection, or a protozoan infection, wherein the composition is optionally in a pharmaceutically acceptable excipient and/or optionally delivered via particles, vesicles, or one or more viral vectors, optionally wherein the particles comprise a lipid, a sugar, a metal or a protein, optionally wherein the vesicles comprise exosomes or liposomes optionally wherein the one or more viral vectors comprise one or more of adenovirus, one or more lentivirus or one or more adeno-associated virus are provided.
  • Use of a compositions for the manufacture of a medicament for treatment of a genetic disease, cancer, a viral infection, a bacterial infection, a parasitic infection, a fungal infection, or a protozoan infection are provided.
  • Methods for producing a eukaryotic cell comprising a modification of a locus of interest comprising (i) delivering any or the aforementioned or other compositions provided herein to the locus of interest, wherein the spacer sequence hybridizes with the target sequence and whereby the composition introduces the modification into the locus of interest are also provided.
  • the DNA expression systems, engineered systems, compositions, and methods disclosed herein can be employed with Casl2a nucleases or modified variants thereof.
  • the Casl2a nuclease is any one of the Casl2a (“Cpfl”) nucleases disclosed in U.S. Patent No. 9,790,490 or in U.S. Patent Application No. 15/566,528 (national phase entry of International Application No. PCT/EP2016/058442, published as WO2016166340), or in U.S. Patent No. 9,896,696 which are specifically incorporated herein by reference.
  • the Casl2a nuclease is a Casl2a (“Cpfl”) nuclease identified from Francisella novicida V W l (FnCasl2a or “FnCpfl”), from Acidaminococcus sp.
  • Cpfl Casl2a
  • FnCasl2a or FnCpfl Francisella novicida V W l
  • BV3L6 (AsCasl2a or “AsCpfl”), or from Lachnospiraceae bacterium ND2006 (LbCasl2a or “LbCpfl”), or is an orthologue thereof, such as an orthologue having at least 50% sequence identity to FnCasl2a (“FnCpfl”); see, e.g., the FnCpfl nuclease contained in the FnCpfl locus having SEQ ID NO:211 in U.S. Patent No. 9,790,490, incorporated herein by reference.
  • a Casl2a nuclease is (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SC ADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease selected from the group consisting of (1) a variant LbCpfl that differs from a wild-type LbCpfl sequence (SEQ ID NO:135) in containing at least one point mutation selected from the group consisting of G309P, Y312F, M474
  • the Casl2a nuclease includes orthologues of LbCasl2a, AsCasl2a, or FnCasl2a.
  • the Casl2a nuclease is a deactivated Casl2a nuclease, a tagged or labelled Casl2a nuclease, or a Casl2a fusion; examples are described elsewhere in this disclosure.
  • Csm nucleases including Csm nucleases, e.g, ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g, Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wildtype ObCsml sequence (SEQ ID NO:137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • Embodiments include non-human animals and non-human animal cells, non-human animal stem cells, human cells, human stem cells, and human somatic (non- gametic) cells; isolated tissue (e.g., nervous tissue, epithelial tissue, liver, spleen, pancreas, muscle tissue, bone, connective tissue, endocrine system tissues, and tumours) or cells (e.g., blood cells, erythrocytes, leukocytes, lymphocytes, liver cells, bone cells, immune cells, T cells, B cells, dendritic cells, and tumour cells) or cell organelles (e.g., mitochondria or chloroplasts, either isolated or contained within the eukaryotic cell, tissue, or organism) obtained from a non-human animal or from a human; engineered hybrido
  • Additional aspects of the invention include progeny plants or progeny seeds obtained from a plant, plant cell, plant tissue, or seed having a genomic modification obtained by use of the polynucleotides, compositions, and methods of use disclosed herein. Also encompassed by the invention are raw plant materials, processed plant products, and commodity plant products obtained from a plant, plant cell, plant tissue, or seed (or from progeny plants or seeds thereof) having a genomic modification obtained by use of the polynucleotides, compositions, and methods of use disclosed herein. In certain optional embodiments, the methods disclosed herein are not processes for modifying the germ line genetic identity of human beings.
  • the methods disclosed herein are not processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes. In certain optional embodiments, the methods disclosed herein are not methods for treatment of the human or animal body by surgery or therapy. In a certain optional embodiments, the cells disclosed herein are not human embryos. In certain optional embodiments, the cells disclosed herein are not the human body, at the various stages of its formation and development.
  • Figure 1 illustrates schematics of the Lachnospiraceae bacterium ND2006 Casl2a (“LbCpfl”) CRISPR locus and Casl2a tracrRNA (“putative tracrRNA”) location as described in detail in Example 1.
  • the schematics are shown with (upper figure) and without (lower figure) the Cas4 and Cas2 components of the locus.
  • the direct repeats (“DR”) are indicated by upright rectangles and the spacer sequences indicated by diamonds.
  • Figure 2 illustrates a segment of the LbCpfl CRISPR locus as described in detail in
  • Example 1 showing the arrangement in a CRISPR array of the LbCpfl direct repeats (“DR”, in bold font) (SEQ ID NO: 173) flanking a spacer sequence (“spacer”, in underlined font) (SEQ ID NO: 174), and illustrating the predicted crRNA (SEQ ID NO: 175) processing from this CRISPR array.
  • the DR, spacer, and predicted crRNA sequences are provided as DNA equivalents.
  • Figure 3 illustrates the alignments of the reverse complement of the LbCpfl direct repeat (DR) sequence, ATCTACACTTAGTAGAAATTATTTAATCTTTGAAAC (SEQ ID NO:5), with the LbCpfl tracrRNA 1 (“putative tracrRNA 1”) (SEQ ID NO:2) (top, showing 52.8% identity between the two sequences) and the LbCpfl tracrRNA 2 (“putative tracrRNA 2”) (SEQ ID NO:4) (bottom, showing 50.0% identity between the two sequences), as described in detail in Example 1.
  • DR LbCpfl direct repeat
  • Figure 4 illustrates schematics of the Acidaminococcus sp. BV3L6 (“As”) Casl2a (“AsCpfl”) CRISPR locus and tracrRNA (“putative tracrRNA”) location as described in detail in Example 1.
  • the schematics are shown with (upper figure) and without (lower figure) the Cas 1, Cas2, and Cas4 components of the locus.
  • the direct repeats (“DR”) are indicated by upright rectangles and the spacer sequences are indicated by diamonds.
  • Figure 5 illustrates a segment of the AsCpfl CRISPR locus as described in detail in
  • Example 1 showing the arrangement in a CRISPR array of the AsCpfl direct repeats (“DR”, in bold font) (SEQ ID NO: 176) flanking a spacer sequence (“spacer”, in underlined font) (SEQ ID NO: 177), and illustrating the predicted crRNA (SEQ ID NO: 178) processing from this CRISPR array.
  • the DR, spacer, and predicted crRNA sequences are provided as DNA equivalents.
  • Figure 6 illustrates the alignment of the reverse complement of the AsCpfl direct repeat (DR) sequence, ATCTACAAGAGTAGAAATTAAAAAGGTCTTTTGAC (SEQ ID NO:8) with the AsCpfl tracrRNA (“putative tracrRNA”) (SEQ ID NO:7), showing 51.4% identity between the two sequences as described in detail in Example 1.
  • DR AsCpfl direct repeat
  • putative tracrRNA AsCpfl tracrRNA
  • Figure 7 schematically illustrates the position (not to scale) of some of the restriction endonuclease sites within the Lb Casl2a locus as described in detail in Example 2. Endonuclease recognition and binding sites are indicated by squares; the endonuclease cleavage sites are indicated by arrowheads. “DR” indicates a direct repeat.
  • Figure 8 schematically illustrates the position (not to scale) of some of the restriction endonuclease sites within the As Cas 12a locus as described in detail in Example 2. Endonuclease recognition and binding sites are indicated by squares; the endonuclease cleavage sites are indicated by arrowheads. “DR” indicates a direct repeat.
  • Figure 9A schematically depicts an expression cassette for expressing a Casl2a nuclease, including a promoter, coding sequence for the Casl2a nuclease, and a terminator, as described in detail in Example 3.
  • Figure 9B (left) schematically depicts a DNA expression system including: (a) DNA sequence for a first promoter; (b) operably linked and heterologous to the first promoter, a DNA encoding a first RNA molecule including a Casl2a tracrRNA or putative tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety; and (c) operably linked and heterologous to the first promoter, a DNA encoding a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the first RNA molecule, as described in detail in Example 3.
  • Figure 9B (right) schematically depicts an alternative design wherein the DNA expression system includes (a) DNA sequence for a first promoter; (b) operably linked and heterologous to the first promoter, a DNA encoding a first RNA molecule including a Casl2a tracrRNA or putative tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety; and (c) operably linked and heterologous to the first promoter, a DNA encoding a Casl2a crRNA including multiple guide RNAs (Casl2a crRNAs), and including a 3’ extension capable of hybridizing with a segment of the first RNA molecule, as described in detail in Example 3.
  • a DNA sequence for a first promoter includes a Casl2a tracrRNA or putative tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a
  • Figure 9C (left) schematically depicts a non-limiting example of a DNA expression system including: (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter, a DNA sequence encoding a first RNA molecule including a Casl2a tracrRNA or putative tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety, as described in detail in Example 3.
  • Figure 9C (right) further schematically depicts an expression construct for expressing a guide RNA (Casl2a crRNA), as described in detail in Example 3.
  • Figure 10 schematically depicts a DNA expression system, described in detail in Example 3, which includes: (a) DNA sequence for a first promoter; (b) operably linked and heterologous to the first promoter, DNA encoding a Casl2a nuclease; (c) a terminator; (d) a second promoter; (e) operably linked and heterologous to the second promoter, a DNA sequence encoding a functional RNA molecule including a Casl2a tracrRNA or putative tracrRNA and a functional RNA moiety; (f) an optional third promoter; (g) operably linked and heterologous to the second promoter (and to the optional third promoter, if present), a DNA encoding a guide RNA including a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; and (h) a second terminator; the 3 ’-most direct repeat is optional.
  • Figure 10 (bottom) schematically depicts a second DNA expression system, described in detail in Example 3, and similar to that shown in Figure 10 (top), except that the guide RNA includes multiple guide RNAs, and includes a 3’ extension capable of hybridizing with a segment of the first RNA molecule; the 3 ’-most direct repeat is optional.
  • Figure 11 schematically depicts construction of the DNA encoding the functional RNA molecule by cleavage at an HphI restriction endonuclease cleavage site (“cut site”) in or adjacent to a Lb Casl2a tracrRNA (“P-tracr”) and before the LbCasl2a crRNA with a 3’ extension (“E-crRNA”), insertion of DNA encoding multiple copies of an MS2 aptamer (“(n) x MS2”) as well as sequence for annealing (“rcEX”) to the crRNA 3 ’ extension (“EX”), and ligation to form the DNA encoding the functional RNA molecule, which can be complexed with the Casl2a nuclease to form the ribonucleoprotein depicted in Figure 11 (bottom).
  • Figure 12 depicts the results of the experiment described in Example 8 using the GD-30 crRNA.
  • the data are provided as the fraction of edited protoplasts (the number of protoplasts containing an insertion/deletion (indel) at the predicted cleavage site, divided by the total number of protoplasts treated) versus the amount in pmoles of Casl2a nuclease used.
  • Figure 13 depicts the results of the experiment using a “high-performing” Casl2a crRNA, GD-48, as described in Example 9.
  • the data are provided as the fraction of edited protoplasts (the number of protoplasts containing an insertion/deletion (indel) at the predicted cleavage site, divided by the total number of protoplasts treated) versus the amount in pmoles of Casl2a nuclease used.
  • Figure 14 depicts the results of the experiment using a “low -performing” Casl2a crRNA, GD-14, as described in Example 9.
  • the data are provided as the fraction of edited protoplasts (the number of protoplasts containing an insertion/deletion (indel) at the predicted cleavage site, divided by the total number of protoplasts treated) versus the amount in pmoles of Casl2a nuclease used.
  • nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5’ to 3’ direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as well as necessarily defines the exact complements, as is known to one of ordinary skill in the art. Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. As used herein, the terms “comprise,” comprises,” comprising,” “include,” “includes,” and “including” can be interchanged and are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.
  • in vitro axenic culture refers to a culture of a single cell type (e.g., a eukaryotic cell) in a vessel (e.g., flask, petri dish, or well).
  • a single cell type e.g., a eukaryotic cell
  • a vessel e.g., flask, petri dish, or well.
  • polynucleotide is meant a nucleic acid molecule containing multiple nucleotides and refers to “oligonucleotides” (defined here as a polynucleotide molecule of between 2 - 25 nucleotides in length) and polynucleotides of 26 or more nucleotides. Polynucleotides are generally described as single- or double-stranded. Where a polynucleotide contains double-stranded regions formed by intra- or intermolecular hybridization, the length of each double-stranded region is conveniently described in terms of the number of base pairs.
  • aspects of this invention include the use of polynucleotides or compositions containing polynucleotides; embodiments include one or more oligonucleotides or polynucleotides or a mixture of both, including single- or double-stranded RNA or single- or double-stranded DNA or double-stranded DNA/RNA hybrids or chemically modified analogues or a mixture thereof.
  • a polynucleotide includes a combination of ribonucleotides and deoxyribonucleotides (e.g., synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides), or includes non-canonical nucleotides such as inosine, thiouridine, or pseudouridine.
  • the polynucleotide includes chemically modified nucleotides (see, e.g., Verma and Eckstein (1998) Annu. Rev.
  • oligonucleotide or polynucleotide can be partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate intemucleotide linkage modifications; modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis; and oligonucleotides or polynucleotides can be labelled with a fluorescent moiety (e.g., fluorescein or rhodamine or a fluorescence resonance energy transfer or FRET pair of chromophore labels) or other label (e.g., biotin or an isotope).
  • fluorescent moiety e.g., fluorescein or rhodamine or a fluorescence resonance energy transfer or FRET pair of chromophore labels
  • other label e.g., biotin or an isotope.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas CRISPR-associated
  • a Cas endonuclease is directed to a target nucleotide sequence (e.g.
  • CRISPR loci encode both Cas endonucleases and “CRISPR arrays” of the non-coding RNA elements that determine the specificity of the CRISPR-mediated nucleic acid cleavage.
  • CRISPR systems Two classes (1 and 2) of CRISPR systems have been identified across a wide range of bacterial hosts.
  • the well characterized class 2 CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins).
  • One class 2 CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”).
  • crRNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • a “trans-activating crRNA” or “tracrRNA” is a trans-encoded small RNA that is partially homologous to repeats within a CRISPR array.
  • both a tracrRNA and a crRNA are required for the CRISPR array to be processed and for the nuclease to cleave the target DNA sequence.
  • Cas 12a type CRISPR systems have been reported to function without a tracrRNA, with the Cas 12a CRISPR arrays processed into mature crRNAs without the requirement of a tracrRNA; see Zetsche et al. (2015) Cell, 163:759-771 and U.S. Patent No. 9,790,490.
  • the Cas9 crRNA contains a “spacer sequence”, typically an RNA sequence of about 20 nucleotides (in various embodiments this is 20, 21, 22, 23, 24, 25, or up to about 30 contiguous nucleotides in length) that corresponds to (e.g., is identical or nearly identical to, or alternatively is complementary or nearly complementary to) a target DNA sequence of about equivalent length.
  • the Cas9 crRNA also contains a region that binds to the Cas9 tracrRNA to form a partially double-stranded structure which is cleaved by RNase III, resulting in a crRNA:tracrR A hybrid or duplex.
  • the crRNA: tracrRNA hybrid then directs the Cas9 endonuclease to recognize and cleave the target DNA sequence; in some examples, a tracrRNA and crRNA (e.g., a crRNA including a spacer sequence) can be included in a chimeric nucleic acid referred to as a “single guide RNA” (sgRNA).
  • sgRNA single guide RNA
  • the Casl2a (“Cpfl”) CRISPR system includes the type V endonuclease Casl2a (also known as “Cpfl”).
  • Casl2a nucleases are characterized as having only a RuvC nuclease domain, in contrast to Cas9 nucleases which have both RuvC and HNH nuclease domains.
  • Casl2a nucleases are generally smaller proteins than Cas9 nucleases, and can function with a smaller guide RNA (e.g., a crRNA having at least one spacer flanked by direct repeats), which are practical advantages in that the nuclease and guide RNAs are more economical to produce and potentially more easily delivered to a cell.
  • Casl2a nucleases examples include AsCasl2a or “AsCpfl” (from Acidaminococcus sp.) and LbCasl2a or “LbCpfl” (from Lachnospiraceae bacteria).
  • Casl2a-associated (“Cpfl”-associated) CRISPR arrays have been reported to be processed into mature crRNAs without the requirement of a tracrRNA, i.e., the naturally occurring Casl2a (Cpfl) CRISPR system was reported to require only the Casl2a (Cpfl) nuclease and a Casl2a crRNA to cleave the target DNA sequence; see Zetsche et al. (2015) Cell, 163:759-771; U.S. Patent No. 9,790,490.
  • tracrRNA sequence or analogous terms includes any polynucleotide sequence that has sufficient complementarity with a crRNA sequence to hybridize.
  • Embodiments of tracrRNA sequences useful in the compositions, engineered systems, and methods provided by this disclosure include those described as a “putative transactivating crRNA” or “putative tracrRNA” in U.S. Patent 9,490,490 (e.g., in Figures 46, 48 - 51, and 54 of U.S. Patent 9,490,490, and SEQ IDS NO: 28, 31, 34, 40, 43, 44, 47, and 50 of U.S. Patent 9,490,490) and its priority documents, all of which are incorporated by reference herein.
  • Embodiments of methods of identifying orthologues of CRISPR-Cas system enzymes may involve identifying tracrRNA sequences in genomes of interest.
  • Casl2a tracrRNA as used herein means a naturally occurring Casl2a tracrRNA or an engineered derivative thereof.
  • the term “Casl2a tracrRNA” refers to a naturally occurring Casl2a tracrRNA has a nucleotide sequence that includes or comprises:
  • a nucleotide sequence or region of about 60 to about 100 contiguous nucleotides that has more than 50% identity to the direct repeat (DR) or tracr mate sequence in the Casl2a crRNA preferably also has
  • a naturally occurring Casl2a tracrRNA has a nucleotide sequence that includes or comprises: (a) a nucleotide sequence or region of about 60 to about 100 contiguous nucleotides that has more than 50% identity to the direct repeat (DR) or tracr mate sequence in the Casl2a crRNA, and preferably also has (b) secondary structure, but is not associated with a transcriptional terminator.
  • the Casl2a tracrRNA is physically associated with the corresponding Casl2a crRNA and/or with the corresponding Casl2a nuclease. In other embodiments, the Casl2a tracrRNA is not physically associated with the corresponding Casl2a crRNA and/or with the corresponding Casl2a nuclease.
  • a naturally occurring tracrRNA sequence can be identified from a genome as DNA sequence encoding a RNA molecule (a “putative tracrRNA”) that is predicted to be or putatively transcribed (or that may be determined to be transcribed, e.g., by RNA analysis) from a CRISPR genomic region, specifically from a locus that flanks the DNA encoding the CRISPR nuclease and that includes a predicted transcriptional terminator in the direction of transcription; is about 60 to about 100 nucleotides in length, e.g, 60-80 nucleotides, 60-85 nucleotides, 60-90 nucleotides, or 65-95 nucleotides; and is not a direct repeat in the CRISPR array, but rather has a nucleotide sequence of sufficient complementarity with a crRNA sequence to hybridize, e.g., of at least 50% identity to or at least 50% complementarity to the direct repeat in a CRISPR array.
  • RNA sequencing can be used to further characterize such sequences identified as potential or putative tracrRNAs.
  • an engineered Casl2a tracrRNA is used in place of or as a substitute for a naturally occurring tracrRNA or putative tracrRNA identified from a genome.
  • an engineered Casl2a tracrRNA is designed based on guidelines similar to those employed for identifying a naturally occurring tracrRNA or putative tracrRNA identified from a genome.
  • an engineered Casl2a tracrRNA is designed to have a sequence of about 60 to about 100 ribonucleotides and to have at least 50% complementarity to the sequence of the direct repeat of the Casl2a crRNA with which the engineered Casl2a tracrRNA is meant to be used.
  • Engineered Casl2a tracrRNAs can be expressed with any of the systems described in this disclosure, e.g, in an expression construct using any of the promoters, terminator, or other expression elements described herein.
  • the genomic DNA sequence targeted for editing or modification must generally be adjacent to a “protospacer adjacent motif’ (“PAM”) that is specific for a given Cas endonuclease; however, PAM sequences are short and relatively non-specific, appearing throughout a given genome.
  • PAM protospacer adjacent motif
  • CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5’-NGG (Streptococcus pyogenes), 5’-NNAGAA (Streptococcus thermophilus CRISPR1), 5’-NGGNG (Streptococcus thermophilus CRISPR3), 5’- NNGRRT or 5’-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5’-NNNGATT (Neisseria meningitidis).
  • 5’-NGG Streptococcus pyogenes
  • 5’-NNAGAA Streptococcus thermophilus CRISPR1
  • 5’-NGGNG Streptococcus thermophilus CRISPR3
  • 5’- NNGRRT or 5’-NNGRR Staphylococcus aureus Cas9, SaCas9
  • 5’-NNNGATT Neisseria
  • Cas9 endonucleases are associated with G-rich PAM sites, e.g., 5’-NGG, and perform blunt-end cleaving of the target DNA at a location three nucleotides upstream from (5’ from) the PAM site.
  • Casl2a (Cpfl) CRISPR systems cleave the target DNA adjacent to a short T-rich PAM sequence, e.g., 5’-TTN, in contrast to the G-rich PAM sequences identified for Cas9 systems.
  • Examples of Casl2a PAM sequences include those for the naturally occurring Acidaminococcus sp.
  • Casl2a can also recognize a 5’-CTA PAM motif.
  • Other examples of potential Casl2a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as any nucleotide).
  • a PAM sequence can be identified using a PAM depletion assay.
  • Casl2a cleaves the target DNA by introducing an offset or staggered doublestrand break with a 4- or 5-nucleotide 5’ overhang, for example, cleaving a target DNA with a 5- nucleotide offset or staggered cut located 18 nucleotides downstream from (3’ from) from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759-771.
  • CRISPR arrays can be designed to contain one or multiple guide RNAs designed to target a DNA sequence for editing, where the guide RNA includes at least one spacer sequence that corresponds to a specific locus of about equivalent length in the target DNA; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308.
  • guide RNA refers to a nucleic acid that comprises or includes a nucleotide sequence (sometimes referred to a “spacer sequence”) that corresponds to (e.g., is identical or nearly identical to, or alternatively is complementary or nearly complementary to) a target DNA sequence (e.g., a contiguous nucleotide sequence that is to be modified) in a genome; the guide RNA functions in part to direct the CRISPR nuclease to a specific location on the genome.
  • a gRNA is a CRISPR RNA (“crRNA”), such as the engineered Casl2a crRNAs described in this disclosure.
  • the gRNA can be a tracrRNA:crRNA hybrid or duplex, or can be provided as a single guide RNA (sgRNA).
  • At least 16 or 17 nucleotides of gRNA sequence corresponding to a target DNA sequence are required by Cas9 for DNA cleavage to occur; for Casl2a (Cpfl) at least 16 nucleotides of gRNA sequence corresponding to a target DNA sequence are needed to achieve detectable DNA cleavage and at least 18 nucleotides of gRNA sequence corresponding to a target DNA sequence were reported necessary for efficient DNA cleavage in vitro', see Zetsche et al. (2015) Cell, 163:759-771. Casl2a (Cpfl) endonuclease and corresponding guide RNAs and PAM sites are disclosed in U.S.
  • Patent Number 9,790,490 which is incorporated herein by reference in its entirety and particularly for its disclosure of DNA encoding Casl2a (Cpfl) endonucleases and guide RNAs and PAM sites.
  • guide RNA sequences are generally designed to contain a spacer sequence of between 17-24 contiguous nucleotides (frequently 19, 20, or 21 nucleotides) with exact complementarity (e.g., perfect base-pairing) to the targeted gene or nucleic acid sequence; guide RNAs having spacers with less than 100% complementarity to the target sequence can be used (e.g., a gRNA with a spacer having a length of 20 nucleotides and between 1-4 mismatches to the target sequence), but this can increase the potential for off-target effects.
  • CRISPR-type genome editing has value in various aspects of agriculture research and development.
  • CRISPR elements e.g., CRISPR endonucleases and CRISPR single-guide RNAs, are useful in effecting genome editing without remnants of the CRISPR elements or selective genetic markers occurring in progeny.
  • genome-inserted CRISPR elements are useful in plant lines adapted for multiplex genetic screening and breeding.
  • a plant species can be created to express one or more of a CRISPR endonuclease such as a Casl2a-type endonuclease or combinations with unique PAM recognition sites.
  • CRISPR guide RNAs that interact with CRISPR endonucleases integrated into a plant genome or otherwise provided to a plant is useful for genetic editing for providing desired phenotypes or traits, for trait screening, or for trait introgression.
  • Multiple endonucleases can be provided in expression cassettes with the appropriate promoters to allow multiple genome editing in a spatially or temporally separated fashion in either in chromosome DNA or episome DNA.
  • CRISPR technology for editing the genes of eukaryotes is disclosed in U.S. Patent Application Publications 2016/0138008A1 and US2015/0344912A1, and in U.S. Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616.
  • Casl2a (Cpfl) endonuclease and corresponding guide RNAs and PAM sites are disclosed in U.S. Patent No. 9,790,490 and U.S.
  • Patent Application 15/566,528 (national phase of PCT Application PCT/EP2016/058442, published as WO 2016/166340).
  • Plant RNA promoters for expressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9 endonuclease are disclosed in U.S. Patent Application 15/120,110, published as U.S. Patent Application Publication 2017/0166912, national phase application claiming priority to International Patent Application PCT/US2015/018104 (published as WO 2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700).
  • Methods of using CRISPR technology for genome editing in plants are disclosed in in U.S. Patent Application Publications U.S. 2015/0082478A1 and U.S.
  • an endonuclease is provided as a ribonucleoprotein (RNP) complex, e.g, a preassembled RNP that includes the endonuclease complexed with a polynucleotide including the gRNA or encoding a gRNA, or a preassembled RNP that includes a polynucleotide that encodes the endonuclease (and optionally encodes the gRNA, or is provided with a separate polynucleotide including the gRNA or encoding a gRNA), complexed with a protein.
  • RNP ribonucleoprotein
  • the endonuclease is a fusion protein, i.e., wherein the endonuclease is covalently bound through a peptide bond to a cellpenetrating peptide, a nuclear localization signal peptide, a chloroplast transit peptide, or a mitochondrial targeting peptide; such fusion proteins are conveniently encoded in a single nucleotide sequence, optionally including codons for linking amino acids.
  • the endonuclease or a polynucleotide that encodes the endonuclease is provided as a complex with a cell-penetrating peptide or other transfecting agent.
  • the endonuclease or a polynucleotide that encodes the endonuclease is complexed with, or covalently or non-covalently bound to, a further element, e.g. , a carrier molecule, an antibody, an antigen, a viral movement protein, a polymer, a detectable label (e.g., a moiety detectable by fluorescence, radioactivity, or enzymatic or immunochemical reaction), a quantum dot, or a particulate or nanoparticulate.
  • a further element e.g., a carrier molecule, an antibody, an antigen, a viral movement protein, a polymer, a detectable label (e.g., a moiety detectable by fluorescence, radioactivity, or enzymatic or immunochemical reaction), a quantum dot, or a particulate or nanoparticulate.
  • the endonuclease or a polynucleotide that encodes the endonuclease is provided in a solution, or is provided in a liposome, micelle, emulsion, reverse emulsion, suspension, or other mixed-phase composition.
  • editing complexes such as those disclosed herein (or compositions including such editing complexes) can be provided in combination with further elements (e.g., a carrier molecule, an antibody, an antigen, a viral movement protein, a polymer, a detectable label, a quantum dot, or a particulate or nanoparticulate) or provided in a suitable form (e.g., in a solution, liposome, micelle, emulsion, reverse emulsion, suspension, or other mixed-phase composition).
  • further elements e.g., a carrier molecule, an antibody, an antigen, a viral movement protein, a polymer, a detectable label, a quantum dot, or a particulate or nanoparticulate
  • a suitable form e.g., in a solution, liposome, micelle, emulsion, reverse emulsion, suspension, or other mixed-phase composition.
  • heterologous sequence integration or insertion of one or more nucleotides, resulting in a sequence (including the inserted nucleotide(s) as well as at least some adjacent nucleotides of the genomic sequence flanking the site of insertion at a double-strand break effected by a CRISPR endonuclease) that is heterologous (e.g., would not otherwise or does not normally occur at the site of insertion).
  • heterologous is also used to refer to a given sequence in relationship to another, e.g., the sequence of a donor DNA is heterologous to the sequence at the site of the double-strand break wherein the donor DNA is integrated.
  • heterologous is also used to refer to components of a synthetic polynucleotide construct, where the components do not naturally occur together; for example, an expression cassette or construct can include a promoter to drive expression of a downstream sequence, where the promoter is heterologous to that downstream sequence.
  • heterologous promoter refers to discrete genetic sequences such as promoters and other expression-related genetic elements (e.g., promoters, enhancers, introns, terminators, silencers, and insulators), that are not normally associated with a particular nucleic acid in nature.
  • promoters promoters, enhancers, introns, terminators, silencers, and insulators
  • a “promoter that is heterologous to a coding region” is a promoter that is not normally associated with the coding region in nature.
  • oligonucleotide or polynucleotide capable of specifically binding to a target nucleic acid is complementary to the target nucleic acid. However, exact complementarity is not required for specific binding.
  • capable of hybridizing refers to the ability of two polynucleotides to hybridize or “anneal”, i.e., to form Watson-Crick base pairs; one of skill in the art would understand that the ability to hybridize requires a sufficient degree of complementarity between the two polynucleotides under given conditions (e.g., temperature) and that the degree of complementarity can be estimated using algorithms in the art.
  • oligonucleotide or polynucleotide is at least 80% complementary to the target, for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target.
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available at ncbi[dot]nlm[dot]nih[dot]gov/BLAST. See, e.g., Altschul et al. (1990) Mol. Biol., 215:403-410.
  • FAST A is FAST A, available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • GCG Genetics Computing Group
  • Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA.
  • alignment programs that permit gaps in the sequence.
  • the Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol., 70: 173-187 (1997).
  • the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See Mol. Biol., 48: 443-453 (1970).
  • operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • nucleic acid By “native” is meant naturally occurring in a genome, such as a prokaryotic or eukaryotic genome.
  • a “native” restriction endonuclease cleavage site means a restriction endonuclease cleavage site that is found in a naturally existing genome, such as a restriction endonuclease cleavage site that is naturally found within genomic DNA encoding the tracrRNA, in contrast to an artificial or engineered restriction endonuclease cleavage site that is created through recombinant nucleic acid technology.
  • engineered By “engineered” is meant artificial, synthetic, or not occurring in nature. For example, a polynucleotide that includes two DNA sequences that are heterologous to each other can be engineered or synthesized by recombinant nucleic acid techniques.
  • a complex is meant two or more associated components, such as two or more associated nucleic acids and/or proteins.
  • a complex may include two or more covalently linked nucleic acids and/or proteins, two or more non-covalently linked nucleic acids and/or proteins, or a combination thereof.
  • a complex includes a nuclease (such as a Casl2a nuclease) and an appropriate crRNA; such a complex can optionally include one or more polynucleotides, such as the tracrRNA molecules described herein, donor polynucleotides, and functional RNA moieties.
  • a complex in some examples, includes a gRNA (such as a crRNA) and a donor polynucleotide (or a template for production of a donor polynucleotide), which may be covalently or non-covalently linked.
  • a complex includes a nuclease and a donor DNA, which may be covalently linked.
  • a complex includes one or more CRISPR nuclease polypeptides, a gRNA, and a donor DNA (or a template for production of donor DNA).
  • covalently linked is meant that two elements (e.g., a gRNA and a tether or a gRNA and a donor DNA or a tracrRNA and an engineered crRNA) are joined by a covalent bond, for example, an internucleotide linkage such as a phosphodiester bond, a phosphorothioate bond, a phosphothioate bond, or a peptide bond.
  • a covalent bond for example, an internucleotide linkage such as a phosphodiester bond, a phosphorothioate bond, a phosphothioate bond, or a peptide bond.
  • non-covalently linked is meant that two elements (such as two discrete polynucleotides, or a polypeptide and a polynucleotide) interact non-covalently, for example by hydrogen bonding, such as Watson-Crick base pairing.
  • Other non-covalent interactions include non-Watson-Crick pairing, electrostatic interactions, van der Waals forces, 7t-effects, and hydrophobic effects.
  • in vivo' is meant that the systems, compositions, and methods of this disclosure are applied to or within a living organism, such as a living intact non-human animal, a living human, or a living plant or seed.
  • in vitro is meant that the systems, compositions, and methods of this disclosure are applied to cells or tissue maintained alive independently of an intact multicellular organism, such as a living intact non-human animal, a living intact human, or a living intact plant or seed; examples of in vitro embodiments include those where the systems, compositions, and methods of this disclosure are applied to stable or transient animal or plant cell lines in culture and tissue samples in culture. In vitro embodiments further include prokaryotic cell cultures.
  • ex vivo is meant occurring in cells, tissue, or organs (e.g. , blood, blood cells, T cells, liver cells or tissue, skin, bone, muscle, eggs or ovules, sperm, pollen, etc.) removed from a living non-human animal, human, or plant and at least temporarily maintained alive in culture.
  • Ex vivo cells or tissues may be returned to a living intact multicellular organism, such as the living non-human animal, human, or plant from which the cells or tissue were originally removed.
  • the systems, compositions, and methods of this disclosure are applied to human T-cells for the purpose of generating chimeric antigen receptor T cells (“CAR-T cells”), which may be delivered to a human as a therapeutic; see, e.g., Liu et al., Frontiers Immunol., 10:456, available at doi: 10.3389/fimmu.2019.00456.
  • CAR-T cells chimeric antigen receptor T cells
  • Ex vivo cells or tissues may be grown into differentiated tissue or even differentiated organs, or undifferentiated tissue or callus.
  • Ex vivo plant cells or plant tissues may be grown into intact plants.
  • treatment when used in the contest of a medical or veterinary use, refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • An aspect of this invention provides a method of tethering a functional RNA molecule to a Casl2a crRNA, optionally to a Casl2a ribonucleoprotein including a Casl2a nuclease and a Casl2a crRNA.
  • the method includes the step of hybridizing (a) a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety; and (b) a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; thereby tethering the functional RNA molecule to the Casl2a crRNA.
  • the method includes the step of hybridizing (a) a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety; and (b) a naturally occurring Casl2a crRNA that optionally includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; thereby tethering the functional RNA molecule to the Casl2a crRNA.
  • the method includes the step of hybridizing (a) a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety; and (b) an engineered Casl2a crRNA that optionally includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; thereby tethering the functional RNA molecule to the Casl2a crRNA.
  • Embodiments of a “Casl2a tracrRNA” include an RNA molecule having the nucleotide sequence of a naturally occurring tracrRNA identified from a native Casl2a locus.
  • a naturally occurring Casl2a tracrRNA can be identified from a genome using the techniques described by Zetsche et al. (2015) Cell, 163:759-771, and in U.S. Patent No. 9,790,490.
  • a naturally occurring Casl2a tracrRNA can be identified from a genome as DNA sequence encoding a RNA molecule that: is predicted to be or putatively transcribed (or that may be determined to be transcribed, e.g., by RNA analysis) from a Casl2a CRISPR genomic region, specifically from a locus that flanks the DNA encoding the Casl2a CRISPR nuclease; that preferably but not necessarily includes a predicted transcriptional terminator in the direction of transcription; that is about 60 to about 100 nucleotides in length, e.g., 60-80 nucleotides, 60-85 nucleotides, 60-90 nucleotides, or 65-95 nucleotides; and that is not a direct repeat in the Casl2a CRISPR array, but rather has a nucleotide sequence that permits the transcribed tracrRNA to hybridize to the crRNA (i.e., the transcribed tracrRNA has sufficient complementar
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Casl2a nuclease.
  • there is additional genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Casl2a tracrRNA locus and the DNA encoding the Casl2a nuclease; see, e.g., Figures 1 and 4.
  • the transcribed tracrRNA has a nucleotide sequence having at least 50% identity (e.g., at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, or at least 60% sequence identity) to the sequence of the direct repeat.
  • the Cas 12a tracrRNA has a sequence that is selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • Casl2a tracrRNA includes an RNA molecule having or including a nucleotide sequence based on the nucleotide sequence of a naturally occurring tracrRNA identified from a native Casl2a locus.
  • RNA molecules that include the nucleotide sequence of a tracrRNA (or putative tracrRNA) identified from a native Cas 12a locus but that further include one or more modifications such as (a) additional nucleotides, e.g., an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g, addition of a detectable label.
  • modifications such as (a) additional nucleotides, e.g., an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g, addition of a detectable label.
  • the functional RNA molecule is provided by transcription of a DNA molecule including DNA encoding the Cas 12a tracrRNA and DNA encoding the functional RNA moiety, wherein the DNA encoding the functional RNA moiety is inserted at a restriction endonuclease cleavage site in or adjacent to the DNA encoding the Cas 12a tracrRNA.
  • the restriction endonuclease cleavage site is located within the 3’ region of the DNA encoding the Cas 12a tracrRNA, or within a short distance (e.g., within about 40, about 30, about 20, or about 10 nucleotides) of the 3’ end of the Cas 12a tracrRNA.
  • the restriction endonuclease cleavage site occurs natively in the Cas 12a tracrRNA, that is to say, occurs in a naturally occurring DNA sequence that encodes a predicted or putative Cas 12a tracrRNA.
  • the restriction endonuclease cleavage site is artificial or engineered; a non-limiting example includes a naturally occurring DNA sequence that encodes a predicted or putative Cas 12a tracrRNA and further includes at least one nucleotide that is artificially added, deleted, or substituted to provide an engineered restriction endonuclease cleavage site located in or adjacent to the sequence encoding the tracrRNA.
  • the crRNA 3’ extension includes nucleotides that when base-paired form about one helical turn. In embodiments, the crRNA 3’ extension includes at least 10 contiguous nucleotides. In embodiments, the crRNA 3’ extension increases hybridization of the Cas 12a crRNA to the Cas 12a tracrRNA. In some embodiments, the Cas 12a tracrRNA further includes a 5’ extension, such as the 5’ extensions disclosed by Park et al. (2016) Nature Communications, doi: 10.1038/s41467-018-05641-3.
  • the functional RNA molecule includes a Cas 12a tracrRNA and at least one functional
  • RNA moiety is a nucleotide sequence inserted at a restriction endonuclease cleavage site in or adjacent to the DNA encoding the Cas 12a tracrRNA.
  • functional RNA moieties include: (a) an RNA sequence for annealing to a donor polynucleotide (e.g., a single- or double-stranded DNA containing a nucleotide template for HDR- mediated editing, or a single- or double-stranded polynucleotide including a nucleotide sequence to be integrated by an NHEJ mechanism at a double-stranded break effected, for example, by the Casl2a nuclease); (b) an RNA sequence for annealing to the crRNA 3’ extension; (c) a terminator sequence; (d) an RNA aptamer; (e) a ribozyme or catalytically active
  • a donor polynucleotide e.
  • the functional RNA moiety includes RNA forming at least partially doublestranded RNA capable of silencing a gene (e.g., RNA that forms one or more stem -loops where the stem includes double-stranded RNA of at least about 18 base-pairs designed to silence a target gene, or RNA that has the secondary structure of a microRNA precursor and is processed to a mature microRNA designed to silence a target gene).
  • the functional RNA moiety is a terminator, e.g., a terminator functional in eukaryotic cells, such as a U6 poly-T terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, a synthetic terminator functional in a eukaryotic cell, a 3’ element from an Agrobacterium sp. gene, a 3’ element from a non-human animal gene, a 3’ element from a human gene, and a 3’ element from a plant gene, wherein the 3’ element terminate transcription of an RNA transcript located immediately 5’ to the 3’ element.
  • a terminator functional in eukaryotic cells such as a U6 poly-T terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, a synthetic terminator functional in a eukaryotic cell, a 3’ element from an Agrobacterium sp. gene
  • RNA aptamers include an MS2 aptamer (see Parrott et al. (2000) Nucleic Acids Res. , 28(2):489 ⁇ 197), an ARiBo tag (see Di Tomasso et al. (2011) Nucleic Acids Res., 39(3):el8; doi: 10.1093/nar/gkql084), a streptavidin-binding aptamer (see Leppek and Stoecklin (2014) Nucleic Acids Res. ,452(2):el3; doi: 10.1093/nar/gkt956), a Csy4 aptamer (see Lee et al. (2013) Proc. Natl. Acad. Sci.
  • aptamers are useful for affinity purification and tagging; for example, a two-element affinity tag useful for preparing large quantities of RNA includes a variant of the hepatitis delta virus (H5V) ribozyme that is activated by imidazole and a hairpin loop from a thermostable SRP RNA that forms a high-affinity and kinetically stable complex with the Thermotoga maritima Ffh-M domain protein (see Keift and Batey (2004) RNA, 10(6): 988-995; doi: 10.1261/ma.7040604).
  • H5V hepatitis delta virus
  • the method includes the step of hybridizing in vitro (a) a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety; and (b) a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule.
  • the method includes the step of hybridizing in vitro (a) a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety; and (b) a naturally occurring or an engineered Casl2a crRNA that optionally includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule.
  • the Casl2a crRNA is complexed with, or is capable of complexing with, a Casl2a nuclease to form a Casl2a ribonucleoprotein complex containing the functional RNA molecule. Complexing of the Casl2a crRNA with the Casl2a nuclease can occur prior to, concurrently with, or after hybridization of the functional RNA molecule with the Casl2a crRNA.
  • the method includes the step of hybridizing (a) a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety; and (b) a naturally occurring Casl2a crRNA that optionally includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; following this hybridization, the Casl2a nuclease is added to form the Casl2a ribonucleoprotein.
  • the method includes the step of hybridizing (a) a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety; and (b) an engineered Casl2a crRNA that optionally includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; following this hybridization, the Casl2a nuclease is added to form the Casl2a ribonucleoprotein.
  • a related aspect of the invention is therefore a modified Casl2a ribonucleoprotein complex including (a) a Casl2a nuclease; (b) a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety; and (c) a naturally occurring or an engineered Casl2a crRNA that optionally includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule.
  • the Casl2a nuclease is any one of the Casl2a (“Cpfl”) nucleases disclosed in U.S. Patent No. 9,790,490 or in U.S. Patent Application No. 15/566,528 (national phase entry of International Application No. PCT/EP2016/058442, published as WO2016166340), or in U.S. Patent No. 9,896,696 which are specifically incorporated herein by reference.
  • Cpfl Casl2a
  • the Casl2a nuclease is a Casl2a (“Cpfl”) nuclease identified from Francisella novicida U112 (FnCasl2a or “FnCpfl”), from Acidaminococcus sp.
  • Cpfl Casl2a
  • FnCasl2a or FnCpfl Francisella novicida U112
  • BV3L6 (AsCasl2a or “AsCpfl”), or from Lachnospiraceae bacterium ND2006 (LbCasl2a or “LbCpfl”), or is an orthologue thereof, such as an orthologue having at least 50% sequence identity to FnCasl2a (“FnCpfl”); see, e.g., the FnCpfl nuclease contained in the FnCpfl locus having SEQ ID NO:211 in U.S. Patent No. 9,790,490, incorporated herein by reference.
  • a Casl2a nuclease is (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SC ADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease selected from the group consisting of (1) a variant LbCpfl that differs from a wild-type LbCpfl sequence (SEQ ID NO:135) in containing at least one point mutation selected from the group consisting of G309P, Y312F, M474
  • the Casl2a nuclease includes orthologues of LbCasl2a, AsCasl2a, or FnCasl2a. In embodiments, the Casl2a nuclease is deactivated.
  • the Casl2a nuclease includes at least one modification selected from the group consisting of: (a) a localization or signal peptide (e.g., a nuclear localization signal (NLS), a chloroplast or plastid transit peptide (CTP), or a mitochondrial targeting peptide (MTP)); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization or signal peptide e.g., a nuclear localization signal (NLS), a chloroplast or plastid transit peptide (CTP), or a mitochondrial targeting peptide (MTP)
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-penetrating peptide
  • the Casl2a nuclease includes at least one modification selected from a fluorescent protein (e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, mScarlett), a histidine tag (e.g., a 6XHis tag), a hemagglutinin (HA) tag, a FLAG tag, a Myc tag, horseradish peroxidase (HRP), alkaline phosphatase (AP), fteta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, />eta-7V-acetylghicosaminidase, fteta-glucuronidase, invertase, xanthine oxidase (XO), firefly luciferase (LUC), and glucose oxidase (GO).
  • a fluorescent protein e.g., green fluorescent
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM 12007) (see, e.g., Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild-type ObCsml sequence (SEQ ID NO: 137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • Another aspect of the invention provides a method of integrating or inserting a nucleotide sequence encoded by a donor polynucleotide at a specific locus in a target DNA, wherein the method includes the steps of: (1) annealing a donor polynucleotide to a modified Casl2a ribonucleoprotein complex, wherein the modified Cas 12a ribonucleoprotein complex includes: (a) a Cas 12a nuclease; (b) a functional RNA molecule including a Cas 12a tracrRNA and a functional RNA moiety; and (c) a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; wherein the functional RNA moiety includes an RNA sequence for annealing to the donor polynucleotide; and wherein the Casl2a crRNA includes a spacer sequence that corresponds to a specific target locus in a target DNA,
  • the Casl2a nuclease that forms part of the modified Casl2a ribonucleoprotein complex is any one of the Casl2a (“Cpfl”) nucleases disclosed in U.S. Patent No. 9,790,490 or in U.S. Patent Application No. 15/566,528 (national phase entry of International Application No. PCT/EP2016/058442, published as WO2016166340), or in U.S. Patent No. 9,896,696 which are specifically incorporated herein by reference.
  • Cpfl Casl2a
  • the Casl2a nuclease is a Casl2a (“Cpfl”) nuclease identified from Francisella novicida U112 (FnCasl2a or “FnCpfl”), from Acidaminococcus sp.
  • Cpfl Casl2a
  • FnCasl2a or FnCpfl Francisella novicida U112
  • BV3L6 (AsCasl2a or “AsCpfl”), or from Lachnospiraceae bacterium ND2006 (LbCasl2a or “LbCpfl”), or is an orthologue thereof, such as an orthologue having at least 50% sequence identity to FnCasl2a (“FnCpfl”); see, e.g., the FnCpfl nuclease contained in the FnCpfl locus having SEQ ID NO:211 in U.S. Patent No. 9,790,490, incorporated herein by reference.
  • a Casl2a nuclease is (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SC ADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens,Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:ll, 107 - 138, 172, 204 - 221, and 222; or (d) a Casl2a nuclease selected from the
  • the Cas 12a nuclease is deactivated.
  • the Cas 12a nuclease includes at least one modification selected from the group consisting of: (a) a localization or signal peptide (e.g., a nuclear localization signal (NLS), a chloroplast or plastid transit peptide (CTP), or a mitochondrial targeting peptide (MTP)); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization or signal peptide e.g., a nuclear localization signal (NLS), a chloroplast or plastid transit peptide (CTP), or a mitochondrial targeting peptide (MTP)
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-pen
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild-type ObCsml sequence (SEQ ID NO:137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • Embodiments of a “Cas 12a tracrRNA” include an RNA molecule having the nucleotide sequence of a naturally occurring tracrRNA identified from a native Casl2a locus.
  • a naturally occurring Cas 12a tracrRNA can be identified from a genome using the techniques described by Zetsche et al. (2015) Cell, 163:759-771, and in U.S. Patent No. 9,790,490.
  • a naturally occurring Casl2a tracrRNA can be identified from a genome as DNA sequence encoding a RNA molecule that: is predicted to be or putatively transcribed (or that may be determined to be transcribed, e.g., by RNA analysis) from a Cas 12a CRISPR genomic region, specifically from a locus that flanks the DNA encoding the Casl2a CRISPR nuclease; that preferably but not necessarily includes a predicted transcriptional terminator in the direction of transcription; that is about 60 to about 100 nucleotides in length, e.g., 60-80 nucleotides, 60-85 nucleotides, 60-90 nucleotides, or 65-95 nucleotides; and that is not a direct repeat in the Casl2a CRISPR array, but rather has a nucleotide sequence that permits the transcribed tracrRNA to hybridize to the crRNA (i.e., the transcribed tracrRNA has sufficient complementarity
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Casl2a nuclease.
  • there is additional genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Casl2a tracrRNA locus and the DNA encoding the Casl2a nuclease; see, e.g., Figures 1 and 4.
  • the transcribed tracrRNA has a nucleotide sequence having at least 50% identity (e.g., at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, or at least 60% sequence identity) to the sequence of the direct repeat.
  • the Cas 12a tracrRNA has a sequence that is selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • Casl2a tracrRNA includes an RNA molecule having or including a nucleotide sequence based on the nucleotide sequence of a naturally occurring tracrRNA identified from a native Casl2a locus.
  • RNA molecules that include the nucleotide sequence of a tracrRNA (or putative tracrRNA) identified from a native Cas 12a locus but that further include one or more modifications such as (a) additional nucleotides, e.g, an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g, addition of a detectable label.
  • modifications such as (a) additional nucleotides, e.g, an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g, addition of a detectable label.
  • the functional RNA moiety includes an RNA sequence for annealing to the donor polynucleotide.
  • the RNA sequence for annealing to the donor polynucleotide has a length and degree of sequence identity that in combination are sufficient to allow hybridization of the functional RNA moiety and the donor polynucleotide at physiological conditions.
  • the RNA sequence for annealing to the donor polynucleotide is short, about 10 nucleotides in length, and has exact (100%) sequence complementarity to a segment of the donor polynucleotide of equivalent length.
  • the RNA sequence for annealing to the donor polynucleotide is greater than 10 nucleotides in length, and has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence complementarity to a segment of the donor polynucleotide of equivalent length.
  • the donor polynucleotide includes single-stranded DNA, optionally including chemical modifications. In other embodiments, the donor polynucleotide includes doublestranded DNA, optionally including chemical modifications. In some embodiments, the donor polynucleotide includes both DNA and RNA, for example as a duplex formed by a DNA strand and an RNA strand.
  • the donor polynucleotide is designed to include a template for genome editing via homology -dependent repair (HDR); the template generally includes a “core sequence” that is to replace a sequence of the genome of about the same size, as well as “homology arms” that flank the core sequence on either side and have a sequence complementary to the genomic regions flanking the genomic sequence to be replaced or edited.
  • the donor polynucleotide does not include homology arms or does not include a core sequence and homology arms, for example in embodiments where the donor polynucleotide is used to make a deletion.
  • a donor polynucleotide including a template encoding a nucleotide change over a region of less than about 50 nucleotides is conveniently provided in the form of single-stranded DNA; larger donor templates (e.g., more than 100 nucleotides) are often conveniently provided as double-stranded DNAs.
  • the donor polynucleotide is about 25 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides 80 nucleotides, 90 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 1200 nucleotides, 1500 nucleotides, 1800 nucleotides, 2000 nucleotides, 2500 nucleotides, 3000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or more (such as about 25-200 nucleotides, 50-300 nucleotides, 100-500 nucleotides, 200-800 nucleotides, 700- 2000 nucleotides, 1000-2500 nucleotides, 2000-5000 nucleo
  • the target DNA or target sequence is in many embodiments genomic DNA or chromosomal DNA of a eukaryotic cell or eukaryotic organism.
  • the target DNA is mitochondrial DNA or plastid DNA.
  • the target DNA includes recombinant DNA, for example, a transgene stably integrated in the genome of a eukaryote, such as of a plant.
  • Target sequences and eukaryotic cells are described in detail below under the section headed “Related Disclosure.”
  • Another aspect of the invention provides a DNA expression system including: (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter, a DNA sequence encoding a first RNA molecule including a Casl2a tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety.
  • the first promoter is heterologous to the DNA encoding a first RNA molecule including a Casl2a tracrRNA and including a restriction endonuclease cleavage site.
  • the first promoter is selected for operability in the context (in vitro or in vivo) in which the DNA expression system is to be used.
  • the first promoter is a promoter that functions in prokaryotic cells, such as in bacterial cells.
  • the first promoter is a promoter that functions in eukaryotic cells, such as in non-human animal (e.g, mammal) or human or plant or fungal cells; in embodiments, the first promoter is one identified from a eukaryotic genome, such as a non-human animal or human or plant or fungal genome. In embodiments, the first promoter is one identified or adapted from a viral genome, such as from a viral genome of a virus which infects eukaryotic cells (e.g., plant, mammalian, or fungal cells). In embodiments, the first promoter is a promoter selected from the group consisting of pol III promoters, pol II promoters, and pol I promoters.
  • the first promoter is a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter.
  • useful promoters include constitutive, conditional, inducible, and temporally or spatially specific promoters (e.g., a tissue specific promoter, a developmentally regulated promoter, or a cell cycle regulated promoter).
  • the promoter is a pol II promoter.
  • the promoter is a constitutive promoter that drives DNA expression in plant cells; in embodiments, the promoter drives DNA expression in the nucleus or in an organelle such as a chloroplast or mitochondrion.
  • constitutive promoters include a CaMV 35 S promoter as disclosed in U.S. Patents 5,858,742 and 5,322,938, a rice actin promoter as disclosed in U.S. Patent 5,641,876, a maize chloroplast aldolase promoter as disclosed in U.S.
  • Patent 7,151,204 and an opaline synthase (NOS) and octopine synthase (OCS) promoter from Agrobacterium tumefaciens.
  • the promoter is a promoter from figwort mosaic virus (FMV), a RUBISCO promoter, or a pyruvate phosphate dikinase (PDK) promoter, which is active in the chloroplasts of mesophyll cells.
  • FMV figwort mosaic virus
  • RUBISCO promoter
  • PDK pyruvate phosphate dikinase
  • the first promoter is a plant U6 spliceosomal RNA promoter, which can be native to the genome of the plant cell or from a different species, e.g., a U6 promoter from maize, tomato, or soybean such as those disclosed in U.S. Patent Application 15/120,110, published as U.S.
  • Patent Application Publication 2017/0166912 national phase application claiming priority to PCT/US2015/018104 (published as WO 2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700), or a homologue thereof; in an example, such a promoter is operably linked to DNA sequence encoding a first RNA molecule including a Casl2a tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety, followed by a suitable 3’ element such as a U6 poly-T terminator.
  • the first promoter is a plant U3, 7SL (signal recognition particle RNA), U2, or U5 promoter, or chimerics thereof, e.g., as described in U.S. Patent Application 15/120,110, published as U.S. Patent Application Publication 2017/0166912, national phase application claiming priority to PCT/US2015/018104 (published as WO 2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700). All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety. Additional non-limiting embodiments of promoters functional in plants are provided in Example 5.
  • Embodiments of a “Casl2a tracrRNA” include an RNA molecule having the nucleotide sequence of a naturally occurring tracrRNA identified from a native Casl2a locus.
  • a naturally occurring Casl2a tracrRNA can be identified from a genome using the techniques described by Zetsche et al. (2015) Cell, 163:759-771, and in U.S. Patent No. 9,790,490.
  • a naturally occurring Casl2a tracrRNA can be identified from a genome as DNA sequence encoding a RNA molecule that: is predicted to be or putatively transcribed (or that may be determined to be transcribed, e.g., by RNA analysis) from a Casl2a CRISPR genomic region, specifically from a locus that flanks the DNA encoding the Casl2a CRISPR nuclease; that preferably includes a predicted transcriptional terminator in the direction of transcription; that is about 60 to about 100 nucleotides in length, e.g, 60-80 nucleotides, 60-85 nucleotides, 60-90 nucleotides, or 65-95 nucleotides; and that is not a direct repeat in the Casl2a CRISPR array, but rather has a nucleotide sequence that permits the transcribed tracrRNA to hybridize to the crRNA (i.e., the transcribed tracrRNA has sufficient complementarity to a
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Casl2a nuclease.
  • there is additional genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Casl2a tracrRNA locus and the DNA encoding the Casl2a nuclease; see, e.g., Figures 1 and 4.
  • the transcribed tracrRNA has a nucleotide sequence having at least 50% identity (e.g., at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, or at least 60% sequence identity) to the sequence of the direct repeat.
  • the Cas 12a tracrRNA has a sequence that is selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • An alternative embodiment of a “Cas 12a tracrRNA” includes an RNA molecule having or including a nucleotide sequence based on the nucleotide sequence of a naturally occurring tracrRNA identified from a native Cas 12a locus.
  • RNA molecules that include the nucleotide sequence of a tracrRNA (or putative tracrRNA) identified from a native Cas 12a locus but that further include one or more modifications such as (a) additional nucleotides, e.g, an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g., addition of a detectable label.
  • the Casl2a tracrRNA further includes a 5’ extension, such as the 5’ extensions disclosed by Park et al. (2016) Nature Communications, doi: 10.1038/s41467-018-05641-3.
  • the DNA expression system is useful for expression of an RNA molecule that includes a Casl2a tracrRNA and a functional RNA moiety.
  • the DNA expression system includes a DNA sequence that encodes a first RNA molecule including a Casl2a tracrRNA, and that includes a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for an RNA moiety.
  • the restriction endonuclease cleavage site is located in or adjacent to the DNA encoding the Casl2a tracrRNA.
  • the restriction endonuclease cleavage site is located within the DNA encoding the Casl2a tracrRNA.
  • the restriction endonuclease cleavage site is located within the 3’ region of the DNA encoding the Casl2a tracrRNA, or within a short distance (e.g., within about 40, about 30, about 20, or about 10 nucleotides) of the 3’ end of the Casl2a tracrRNA.
  • the restriction endonuclease recognizes and binds to the DNA at the same location at which the endonuclease cuts the DNA.
  • the restriction endonuclease cleavage site (that is, where the restriction endonuclease cuts) can be at a location on the DNA at a short distance from where the restriction endonuclease recognizes and binds to the DNA.
  • the restriction endonuclease cleavage site occurs natively in the Casl2a tracrRNA, that is to say, occurs in a naturally occurring DNA sequence that encodes a predicted or putative Casl2a tracrRNA.
  • the restriction endonuclease cleavage site is artificial or engineered; in a non-limiting example includes a naturally occurring DNA sequence that encodes a predicted or putative Casl2a tracrRNA and further includes at least one nucleotide that is artificially added, deleted, or substituted to provide an engineered restriction endonuclease cleavage site located in or adjacent to the sequence encoding the tracrRNA.
  • the DNA expression system further includes a terminator located 3’ to the DNA sequence encoding the tracrRNA.
  • the DNA expression system further includes: (a) optionally, a DNA sequence for a second promoter; (b) operably linked and heterologous to the first promoter or the second promoter, a DNA encoding a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the first RNA molecule (wherein the segment can be within or part of the tracrRNA that is part of the first RNA molecule); and (c) optionally, a terminator.
  • the optional second promoter is selected for operability in the context (in vitro or in vivo) in which the DNA expression system is to be used.
  • the second promoter is a promoter that functions in prokaryotic cells, such as in bacterial cells.
  • the second promoter is a promoter that functions in eukaryotic cells, such as in non-human animal (e.g., mammalian, human) or human or plant or fungal cells;
  • the first promoter is one identified from a eukaryotic genome, such as a non-human animal or human or plant or fungal genome.
  • the second promoter is a promoter such as those described as suitable for the first promoter.
  • the first promoter and the second promoter, if present, are different promoters.
  • the first promoter and the DNA encoding the first RNA molecule, and the DNA encoding a Casl2a crRNA that includes a 3’ extension are contained in a single construct.
  • the DNA expression system includes the second promoter
  • the first promoter and the DNA encoding the first RNA molecule are contained in a first construct
  • the second promoter and the DNA encoding a Casl2a crRNA that includes a 3 ’ extension are contained in a second construct; in such embodiments, the first construct and the second construct can be provided (a) in a single vector, or (b) in separate vectors.
  • the first promoter drives expression of both the DNA encoding the first RNA molecule and the DNA encoding the Casl2a crRNA.
  • the terminator is functional in the cell wherein the DNA expression system is to be used.
  • terminator is meant a DNA segment near the 3' end of an expression cassette that acts as a signal to terminate transcription and directs polyadenylation of the resultant mRNA.
  • Such a 3’ element is also sometimes referred to as a “3 '-untranslated region” or “3'- UTR” or a “poly adenylation signal”.
  • Non-limiting embodiments of terminators functional in eukaryotic cells include a U6 poly-T terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, a synthetic terminator functional in a eukaryotic cell, a 3 ’ element from an Agrobacterium sp. gene, a 3’ element from a non-human animal gene, a 3’ element from a human gene, and a 3 ’ element from a plant gene, wherein the 3 ’ element terminate transcription of an RNA transcript located immediately 5’ to the 3’ element.
  • Useful 3' elements include: Agrobacterium tumefaciens nos 3', tail 3', tmr 3', tins 3', ocs 3', and tr73' elements disclosed in U.S. Pat. No. 6,090,627, incorporated herein by reference, and 3' elements from plant genes such as the heat shock protein 17, ubiquitin, and fructose- 1,6-biphosphatase genes from wheat (Triticum aestivum), and the glutelin, lactate dehydrogenase, and beta-tubulin genes from rice (Oryza sativa), disclosed in U.S. Patent Application Publication 2002/0192813 Al, incorporated herein by reference.
  • Casl2a tracrRNAs may have associated endogenous pol III terminators which can be used in the DNA expression systems disclosed herein.
  • the DNA expression system includes a heterologous promoter operably linked to a DNA sequence that encodes a first RNA molecule that includes a Casl2a tracrRNA and that includes a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for an RNA moiety, and further includes an endogenous pol III terminator (e.g., the terminator naturally associated with a putative Casl2a tracrRNA), or a heterologous terminator.
  • an endogenous pol III terminator e.g., the terminator naturally associated with a putative Casl2a tracrRNA
  • the terminator is functional in a eukaryotic cell, such as a plant cell; in embodiments, the terminator is one identified from eukaryotic genomic sequence, such as a plant genomic sequence. In embodiments, the terminator has a sequence selected from the group consisting of SEQ ID NOs:72 - 100 or comprise a U6 poly-T terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, or a synthetic terminator functional in a eukaryotic cell.
  • the Casl2a crRNA is capable of complexing with a Casl2a nuclease to form a Casl2a ribonucleoprotein complex or system.
  • the Casl2a nuclease is any one of the Casl2a (“Cpfl”) nucleases disclosed in U.S. Patent No. 9,790,490 or in U.S. Patent Application No. 15/566,528 (national phase entry of International Application No. PCT/EP2016/058442, published as WO2016166340), or in U.S. Patent No. 9,896,696 which are specifically incorporated herein by reference.
  • the Casl2a nuclease is a Casl2a (“Cpfl”) nuclease identified from Francisella novicida U112 (FnCasl2a or “FnCpfl”), from Acidaminococcus sp.
  • Cpfl Casl2a
  • FnCasl2a or FnCpfl Francisella novicida U112
  • BV3L6 (AsCasl2a or “AsCpfl”), or from Lachnospiraceae bacterium ND2006 (LbCasl2a or “LbCpfl”), or is an orthologue thereof, such as an orthologue having at least 50% sequence identity to FnCasl2a (“FnCpfl”); see, e.g., the FnCpfl nuclease contained in the FnCpfl locus having SEQ ID NO:211 in U.S. Patent No. 9,790,490, incorporated herein by reference.
  • a Casl2a nuclease is (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SC ADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens,Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:ll, 107 - 138, 172, 204 - 221, and 222; or (d) a Casl2a nuclease selected from the
  • the Cas 12a nuclease is deactivated.
  • the Cas 12a nuclease includes at least one modification selected from the group consisting of: (a) a localization signal (e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization signal e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-penetrating peptide
  • an endosomal escape peptide e.
  • the first promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter and the Casl2a tracrRNA has a nucleotide sequence including a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • the first promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter
  • the Casl2a tracrRNA has a nucleotide sequence including a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241, and the optional first terminator has a sequence selected from the group consisting of SEQ ID NOs:72 - 100 or comprise a
  • the systems including a tracrRNA as disclosed herein can also be employed with related Cas nucleases, including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild-type ObCsml sequence (SEQ ID NO:137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • DNA expression system including: (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter, a DNA sequence encoding a functional RNA molecule including a Cas 12a tracrRNA and a functional RNA moiety.
  • the DNA expression system includes (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter, a DNA sequence encoding a functional RNA molecule including a Cas 12a tracrRNA and a functional RNA moiety, and (c) a terminator 3’ to and operably linked to the DNA encoding a functional RNA molecule.
  • the DNA expression system further includes: (a) optionally, a DNA sequence for a second promoter; (b) operably linked and heterologous to the first promoter or the second promoter, a DNA encoding a Cas 12a crRNA that includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; and (c) optionally, a terminator.
  • the first promoter is heterologous to the DNA sequence encoding a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety.
  • the DNA expression system further includes a DNA encoding a Cas 12a crRNA that includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule
  • the first promoter is also heterologous to this DNA.
  • the optional second promoter is heterologous to the DNA encoding a Cas 12a crRNA that includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule.
  • Each promoter is selected for operability in the context (in vitro or in vivo) in which the DNA expression system is to be used.
  • the first promoter (and the optional second promoter) is a promoter that functions in prokaryotic cells, such as in bacterial cells.
  • the first promoter is a promoter that functions in eukaryotic cells, such as in non-human animal (e.g, mammalian, human) or human or plant or fungal cells; in embodiments, the first promoter is one identified from a eukaryotic genome, such as a non-human animal or human or plant or fungal genome.
  • the first promoter is a promoter selected from the group consisting of pol III promoters, pol II promoters, and pol I promoters.
  • the first promoter is a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter.
  • the first promoter (and the optional second promoter) is a promoter that functions in eukaryotic cells, such as in plant cells.
  • useful promoters include constitutive, conditional, inducible, and temporally or spatially specific promoters (e.g., a tissue specific promoter, a developmentally regulated promoter, or a cell cycle regulated promoter).
  • the promoter is a pol II promoter.
  • the promoter is a constitutive promoter that drives DNA expression in plant cells; in embodiments, the promoter drives DNA expression in the nucleus or in an organelle such as a chloroplast or mitochondrion.
  • constitutive promoters include a CaMV 35S promoter as disclosed in U.S. Patents 5,858,742 and 5,322,938, a rice actin promoter as disclosed in U.S.
  • Patent 5,641,876, a maize chloroplast aldolase promoter as disclosed in U.S. Patent 7,151,204, and an opaline synthase (NOS) and octopine synthase (OCS) promoter from Agrobacterium tumefaciens.
  • the promoter is a promoter from figwort mosaic virus (FMV), a RUBISCO promoter, or a pyruvate phosphate dikinase (PDK) promoter, which is active in the chloroplasts of mesophyll cells.
  • FMV figwort mosaic virus
  • RUBISCO promoter
  • PDK pyruvate phosphate dikinase
  • promoters include cell-specific or tissue-specific or developmentally regulated promoters, for example, a promoter that limits the expression of the DNA expression system to germline or reproductive cells (e.g., promoters of genes encoding DNA ligases, recombinases, replicases, or other genes specifically expressed in germline or reproductive cells).
  • the first promoter is a plant U6 spliceosomal RNA promoter, which can be native to the genome of the plant cell or from a different species, e.g, a U6 promoter from maize, tomato, or soybean such as those disclosed in U.S. Patent Application 15/120,110, published as U.S.
  • Patent Application Publication 2017/0166912 national phase application claiming priority to PCT/US2015/018104 (published as WO 2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700), or a homologue thereof; in an example, such a promoter is operably linked to DNA sequence encoding a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety, followed by a suitable 3’ element such as a U6 poly-T terminator.
  • the first promoter is a plant U3, 7SL (signal recognition particle RNA), U2, or U5 promoter, or chimerics thereof, e.g., as described in U.S.
  • Patent Application 15/120,110 published as U.S. Patent Application Publication 2017/0166912, national phase application claiming priority to PCT/US2015/018104 (published as WO 2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700). All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety.
  • Embodiments of a “Casl2a tracrRNA” include an RNA molecule having the nucleotide sequence of a naturally occurring tracrRNA identified from a native Casl2a locus.
  • a naturally occurring Casl2a tracrRNA can be identified from a genome using the techniques described by Zetsche et al. (2015) Cell, 163:759-771, and in U.S. Patent No. 9,790,490.
  • a naturally occurring Casl2a tracrRNA can be identified from a genome as DNA sequence encoding a RNA molecule that: is predicted to be or putatively transcribed (or that may be determined to be transcribed, e.g., by RNA analysis) from a Casl2a CRISPR genomic region, specifically from a locus that flanks the DNA encoding the Casl2a CRISPR nuclease; that preferably includes a predicted transcriptional terminator in the direction of transcription; that is about 60 to about 100 nucleotides in length, e.g, 60-80 nucleotides, 60-85 nucleotides, 60-90 nucleotides, or 65-95 nucleotides; and that is not a direct repeat in the Casl2a CRISPR array, but rather has a nucleotide sequence that permits the transcribed tracrRNA to hybridize to the crRNA (i.e., the transcribed tracrRNA has sufficient complementarity to a
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Casl2a nuclease.
  • there is additional genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Casl2a tracrRNA locus and the DNA encoding the Casl2a nuclease; see, e.g, Figures 1 and 4.
  • the transcribed tracrRNA has a nucleotide sequence having at least 50% identity (e.g., at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, or at least 60% sequence identity) to the sequence of the direct repeat.
  • the Cas 12a tracrRNA has a sequence that is selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • An alternative embodiment of a “Cas 12a tracrRNA” includes an RNA molecule having or including a nucleotide sequence based on the nucleotide sequence of a naturally occurring tracrRNA identified from a native Cas 12a locus.
  • RNA molecules that include the nucleotide sequence of a tracrRNA (or putative tracrRNA) identified from a native Cas 12a locus but that further include one or more modifications such as (a) additional nucleotides, e.g, an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g., addition of a detectable label.
  • the Cas 12a tracrRNA further includes a 5’ extension, such as the 5’ extensions disclosed by Park et al.
  • the terminator is functional in the cell wherein the DNA expression system is to be used.
  • terminator is meant a DNA segment near the 3' end of an expression cassette that acts as a signal to terminate transcription and directs polyadenylation of the resultant mRNA.
  • Such a 3’ element is also sometimes referred to as a “3'- untranslated region” or “3'-UTR” or a “polyadenylation signal”.
  • Non-limiting embodiments of terminators functional in eukaryotic cells include a U6 poly-T terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, a synthetic terminator functional in a eukaryotic cell, a 3’ element from an Agrobacterium sp. gene, a 3’ element from a non-human animal gene, a 3’ element from a human gene, and a 3’ element from a plant gene, wherein the 3’ element terminate transcription of an RNA transcript located immediately 5’ to the 3’ element.
  • Useful 3' elements include: Agrobacterium tumefaciens nos 3', tml 3', tmr 3', tins 3', ocs 3', and tr73' elements disclosed in U.S. Pat. No. 6,090,627, incorporated herein by reference, and 3' elements from plant genes such as the heat shock protein 17, ubiquitin, and fructose-l,6-biphosphatase genes from wheat (Triticum aestivum), and the glutelin, lactate dehydrogenase, and beta-tubulin genes from rice (Oryza sativa), disclosed in U.S. Patent Application Publication 2002/0192813 Al, incorporated herein by reference.
  • Casl2a tracrRNAs may have associated endogenous pol III terminators which can be used in the DNA expression systems disclosed herein.
  • the DNA expression system includes: (a) DNA sequence for a first promoter; (b) operably linked and heterologous to the first promoter, a DNA sequence encoding a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety; and (c) an endogenous pol III terminator (e.g., the terminator naturally associated with a putative Casl2a tracrRNA), or a heterologous terminator.
  • the terminator is functional in a eukaryotic cell, such as a plant cell; in embodiments, the terminator is one identified from eukaryotic genomic sequence, such as a plant genomic sequence.
  • the first promoter and the DNA encoding the functional RNA molecule, and the DNA encoding a Casl2a crRNA that includes a 3’ extension are contained in a single construct.
  • the first promoter and the DNA encoding the functional RNA molecule are contained in a first construct
  • the second promoter and the DNA encoding a Casl2a crRNA that includes a 3 ’ extension are contained in a second construct; in such embodiments, the first construct and the second construct can be provided (a) in a single vector, or (b) in separate vectors.
  • the first promoter drives expression of both the DNA encoding the functional RNA molecule and the DNA encoding the Casl2a crRNA.
  • the DNA expression system is useful for expressing a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety.
  • functional RNA moieties include: (a) an RNA sequence for annealing to a donor polynucleotide (e.g., a single- or double -stranded DNA containing a nucleotide template for HDR-mediated editing, or a single- or doublestranded polynucleotide including a nucleotide sequence to be integrated by an NHEJ mechanism at a double-stranded break effected, for example, by the Casl2a nuclease); (b) an RNA sequence for annealing to the crRNA 3’ extension; (c) a terminator sequence; (d) an RNA aptamer; (e) a ribozyme or catalytically active RNA; (f) a detectable label, such as a fluorescent tag; (g)
  • the functional RNA moiety includes RNA forming at least partially double-stranded RNA capable of silencing a gene (e.g., RNA that forms one or more stem -loops where the stem includes double-stranded RNA of at least about 18 base-pairs designed to silence a target gene, or RNA that has the secondary structure of a microRNA precursor and is processed to a mature microRNA designed to silence a target gene).
  • RNA aptamers include an MS2 aptamer (see Parrott et al. (2000) Nucleic Acids Res. , 28(2):489-497). an ARiBo tag (see Di Tomasso et al.
  • aptamers are useful for affinity purification and tagging; for example, a two-element affinity tag useful for preparing large quantities of RNA includes a variant of the hepatitis delta virus (H5V) ribozyme that is activated by imidazole and a hairpin loop from a thermostable SRP RNA that forms a high-affinity and kinetically stable complex with the Thermotoga maritima Ffh-M domain protein (see Keift and Batey (2004) RNA, 10(6): 988-995; doi: 10.1261/ma.7040604).
  • H5V hepatitis delta virus
  • the Casl2a crRNA is capable of complexing with a Casl2a nuclease to form a Casl2a ribonucleoprotein complex containing the functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety.
  • the Casl2a nuclease is any one of the Casl2a (“Cpfl”) nucleases disclosed in U.S. Patent No. 9,790,490 or in U.S. Patent Application No. 15/566,528 (national phase entry of International Application No. PCT/EP2016/058442, published as WO2016166340), or in U.S. Patent No.
  • the Casl2a nuclease is a Casl2a (“Cpfl”) nuclease identified from Francisella novicida V Ul (FnCasl2a or “FnCpfl”), from Acidaminococcus sp.
  • Cpfl Casl2a
  • BV3L6 (AsCasl2a or “AsCpfl”), or from Lachnospiraceae bacterium ND2006 (LbCasl2a or “LbCpfl”), or is an orthologue thereof, such as an orthologue having at least 50% sequence identity to FnCasl2a (“FnCpfl”); see, e.g., the FnCpfl nuclease contained in the FnCpfl locus having SEQ ID NO:211 in U.S. Patent No. 9,790,490, specifically incorporated herein by reference.
  • a Casl2a nuclease is (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SC ADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:ll, 107 - 138, 172, 204 - 221, and 222; or (d) a Casl2a nuclease variant selected from the
  • the Casl2a nuclease is deactivated.
  • the Casl2a nuclease includes at least one modification selected from the group consisting of: (a) a localization signal (e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization signal e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-penetrating peptide
  • an endosomal escape peptide e.
  • the first promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter and the Casl2a tracrRNA has a nucleotide sequence including a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • the first promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter
  • the Casl2a tracrRNA has a nucleotide sequence including a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241, and the optional first terminator has a sequence selected from the group consisting of SEQ ID NOs:72 - 100 or comprise a
  • the systems including a tracrRNA as disclosed herein can also be employed with related Cas nucleases, including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g, Begemann et al.
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g, Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild-type ObCsml sequence (SEQ ID NO: 137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • Engineered Systems Engineered Systems
  • Another aspect of the invention provides a first engineered system including: (a) a Casl2a nuclease; and (b) at least one engineered Casl2a crRNA, or a polynucleotide encoding the engineered Casl2a crRNA, wherein the engineered Casl2a crRNA includes (i) at least one direct repeat sequence or fragment thereof that is capable of associating with the Casl2a nuclease, and (ii) a spacer sequence that is designed to hybridize with a target sequence in a eukaryotic cell, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 5’ to the spacer sequence; and (c) a Casl2a tracrRNA including a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of the at least one direct repeat of the engineered Casl2a crRNA.
  • the invention provides a second engineered system including: (a) a Casl2a nuclease; and (b) at least one engineered Casl2a crRNA, or a polynucleotide encoding the engineered Casl2a crRNA, wherein the engineered Casl2a crRNA includes (i) at least one direct repeat sequence or fragment thereof that is capable of associating with the Casl2a nuclease, and (ii) a spacer sequence that is designed to hybridize with a target sequence in a eukaryotic cell, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 5’ to the spacer sequence; and (c) a Casl2a tracrRNA including a naturally occurring putative Casl2a tracrRNA sequence that is identified from the same genomic region as the Casl2a nuclease by at least the following steps: (i) identifying in the genomic region flanking the DNA encoding the Casl
  • the Casl2a nuclease is (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SC ADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:ll, 107 - 138, 172, 204 - 221, and 222; or (d) a Casl2a nuclease variant selected from the
  • the Casl2a nuclease is deactivated.
  • the Casl2a nuclease includes at least one modification selected from the group consisting of: (a) a localization signal (e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization signal e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-penetrating peptide
  • an endosomal escape peptide e.
  • the systems including a tracrRNA as disclosed herein can also be employed with related Cas nucleases, including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild-type ObCsml sequence (SEQ ID NO:137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • the Casl2a tracrRNA includes a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of the at least one direct repeat of the engineered Casl2a crRNA; such a Casl2a tracrRNA can include a naturally occurring putative Casl2a tracrRNA sequence identified from a native Casl2a locus.
  • a naturally occurring putative Cas 12a tracrRNA sequence can be identified from a genome using the techniques described by Zetsche et al. (2015) Cell, 163:759-771, and in U.S. Patent No. 9,790,490.
  • a naturally occurring putative Cas 12a tracrRNA sequence can be identified from a genome as DNA sequence encoding a RNA molecule that: is predicted to be or putatively transcribed (or that may be determined to be transcribed, e.g, by RNA analysis) from a Cas 12a CRISPR genomic region, specifically from a locus that flanks the DNA encoding the Casl2a CRISPR nuclease; that preferably includes a predicted transcriptional terminator in the direction of transcription; that is about 60 to about 100 nucleotides in length, e.g., 60-80 nucleotides, 60-85 nucleotides, 60-90 nucleotides, or 65-95 nucleotides; and that is not a direct repeat in the Casl2a CRISPR array, but rather has a nucleotide sequence that permits the transcribed tracrRNA to hybridize to the crRNA (i.e., the naturally occurring putative Casl2a tracrRNA sequence
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Cas 12a nuclease.
  • there is additional genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Cas 12a tracrRNA locus and the DNA encoding the Cas 12a nuclease; see, e.g, Figures 1 and 4.
  • the naturally occurring putative Cas 12a tracrRNA sequence has a nucleotide sequence having at least 50% identity (e.g., at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, or at least 60% sequence identity) to the sequence of the direct repeat.
  • the Casl2a tracrRNA has a nucleotide sequence including a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • Casl2a tracrRNA includes an RNA molecule having or including a nucleotide sequence based on the nucleotide sequence of a naturally occurring putative Casl2a tracrRNA sequence identified from a native Casl2a locus.
  • RNA molecules that include the nucleotide sequence of a naturally occurring putative Casl2a tracrRNA sequence identified from a native Casl2a locus but that further include one or more modifications such as (a) additional nucleotides, e.g., an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g., addition of a detectable label.
  • the Casl2a tracrRNA further includes a 5’ extension, such as the 5’ extensions disclosed by Park et al. (2016) Nature Communications, doi: 10.1038/s41467-018-05641-3.
  • the engineered Casl2a crRNA includes a spacer sequence that is designed to hybridize with a target sequence in a eukaryotic cell.
  • Target sequences and eukaryotic cells are described in detail below under the section headed “Related Disclosure”.
  • the “polynucleotide encoding the engineered Casl2a crRNA” is a naturally occurring polynucleotide, e.g., a pre-processed transcript that includes at least the Casl2a crRNA and often includes additional nucleotides that are removed during processing.
  • the “polynucleotide encoding the engineered Casl2a crRNA” is an engineered or recombinant polynucleotide.
  • the engineered Casl2a crRNA, or the polynucleotide encoding the engineered Casl2a crRNA further includes at least one direct repeat sequence or fragment thereof capable of associating with the Casl2a nuclease, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 3’ to the spacer sequence.
  • the Casl2a tracrRNA has a nucleotide sequence that has at least about 50% or preferably greater than 50% complementarity to the at least one direct repeat sequence or fragment adjacent to and 3’ to the spacer sequence.
  • the engineered Casl2a crRNA, or the polynucleotide encoding the engineered Casl2a crRNA further includes: (a) at least one direct repeat sequence or fragment thereof capable of associating with the Casl2a nuclease, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 3’ to the spacer sequence; and (b) a 3 ’extension adjacent to the spacer sequence.
  • the 3’ extension includes nucleotides that when base-paired form about one helical turn.
  • the 3’ extension includes at least 10 contiguous nucleotides.
  • the Casl2a tracrRNA includes a nucleotide sequence having sufficient complementarity (e.g., about 50% or greater than 50%) to the engineered Casl2a crRNA’ s 3’ extension to allow hybridization between the tracrRNA and the engineered Casl2a crRNA.
  • the polynucleotide encoding the engineered Casl2a crRNA is a pre-processed Casl2a crRNA precursor transcript that includes, in 5’ to 3’ direction, a first direct repeat or fragment thereof, the spacer, and a 3 ’ extension; in some embodiments, the 3 ’ extension includes a second direct repeat or fragment thereof, and/or includes at least 10 contiguous nucleotides, and/or includes nucleotides that when base-paired form about one helical turn; in some embodiments, such a pre-processed Casl2a crRNA precursor transcript is processed (e.g., in vivo or in vitro), resulting in a processed Casl2a crRNA that has had the 3’ extension nucleotides excised.
  • a pre-processed Casl2a crRNA precursor transcript is processed (e.g., in vivo or in vitro), resulting in a processed Casl2a crRNA that has had the 3’ extension nucleot
  • the invention provides a third engineered system including: (a) one or more nucleotide sequences encoding a Casl2a nuclease; and (b) one or more nucleotide sequences encoding at least one engineered Casl2a crRNA that is designed to form a complex with the Casl2a nuclease and that includes a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell; and (c) one or more nucleotide sequences encoding at least one Casl2a tracrRNA.
  • the invention provides a fourth engineered system including: (a) a Casl2a nuclease, or one or more DNA or RNA nucleotide sequences encoding the Casl2a nuclease; and (b) at least one engineered Casl2a crRNA designed to form a complex with the Casl2a nuclease and including a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell, or one or more DNA or RNA nucleotide sequences encoding the at least one engineered Casl2a crRNA polynucleotide; and (c) at least one tracrRNA, or one or more DNA or RNA nucleotide sequences encoding the at least one Casl2a tracrRNA.
  • the Casl2a nuclease is (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SC ADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:ll, 107 - 138, 172, 204 - 221, and 222; or (d) a Casl2a nuclease variant selected from the
  • the Casl2a nuclease is deactivated.
  • the Casl2a nuclease includes at least one modification selected from the group consisting of: (a) a localization signal (e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization signal e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-penetrating peptide
  • an endosomal escape peptide e.
  • the systems including a tracrRNA as disclosed herein can also be employed with related Cas nucleases, including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild-type ObCsml sequence (SEQ ID NO:137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • the engineered Cas 12a crRNA includes a spacer sequence that is designed to hybridize with a target sequence in a eukaryotic cell.
  • Target sequences and eukaryotic cells are described in detail below under the section headed “Related Disclosure”.
  • the “polynucleotide encoding the engineered Cas 12a crRNA” is a naturally occurring polynucleotide, e.g., a pre-processed transcript that includes at least the Casl2a crRNA and often includes additional nucleotides that are removed during processing.
  • the “polynucleotide encoding the engineered Cas 12a crRNA” is an engineered or recombinant polynucleotide.
  • the at least one engineered Cas 12a crRNA, or a polynucleotide encoding the at least one engineered Cas 12a crRNA further includes at least one direct repeat sequence or fragment thereof capable of associating with the Cas 12a nuclease, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 3’ to the spacer sequence.
  • the engineered Casl2a crRNA, or a polynucleotide encoding the engineered Cas 12a crRNA further includes at least one direct repeat sequence or fragment thereof capable of associating with the Cas 12a nuclease, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 3’ to the spacer sequence.
  • the Casl2a tracrRNA has a nucleotide sequence that has at least about 50% or preferably greater than 50% complementarity to the at least one direct repeat sequence or fragment adjacent to and 3’ to the spacer sequence.
  • the engineered Casl2a crRNA further includes a 3’ extension adjacent to the spacer sequence; in some embodiments, the 3’ extension includes nucleotides that when base-paired form about one helical turn; in some embodiments, the 3’ extension includes at least 10 contiguous nucleotides.
  • the Casl2a tracrRNA has a nucleotide sequence having sufficient complementarity (e.g., about 50% or greater than 50%) to the engineered Casl2a crRNA’ s 3’ extension to allow hybridization between the Casl2a tracrRNA and the engineered Casl2a crRNA.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Casl2a tracrRNA includes a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of the at least one direct repeat of the engineered Casl2a crRNA.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Casl2a tracrRNA includes a nucleotide sequence of a naturally occurring putative Casl2a tracrRNA sequence identified from a native Casl2a locus, such as a nucleotide sequence of a naturally occurring putative Casl2a tracrRNA sequence identified from a genome using the techniques described by Zetsche et al. (2015) Cell, 163:759-771, and in U.S. Patent No. 9,790,490.
  • a naturally occurring putative Casl2a tracrRNA sequence can be identified from a genome as DNA sequence encoding a RNA molecule that: is predicted to be or putatively transcribed (or that may be determined to be transcribed, e.g, by RNA analysis) from a Casl2a CRISPR genomic region, specifically from a locus that flanks the DNA encoding the Casl2a CRISPR nuclease; that preferably includes a predicted transcriptional terminator in the direction of transcription; that is about 60 to about 100 nucleotides in length, e.g, 60-80 nucleotides, 60-85 nucleotides, 60-90 nucleotides, or 65-95 nucleotides; and that is not a direct repeat in the Casl2a CRISPR array, but rather has a nucleotide sequence that permits the transcribed tracrRNA to hybridize to the crRNA (i.e., the naturally occurring putative Casl2a tracrRNA
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Casl2a nuclease.
  • there is additional genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Casl2a tracrRNA locus and the DNA encoding the Casl2a nuclease; see, e.g., Figures 1 and 4.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Cas 12a tracrRNA has a nucleotide sequence having at least 50% identity (e.g., at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, or at least 60% sequence identity) to the sequence of the direct repeat.
  • at least 50% identity e.g., at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, or at least 60% sequence identity
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Cas 12a tracrRNA has a nucleotide sequence including a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • An alternative embodiment of a “Casl2a tracrRNA” includes an RNA molecule having or including a nucleotide sequence based on the nucleotide sequence of a naturally occurring putative Casl2a tracrRNA sequence identified from a native Casl2a locus.
  • RNA molecules that include the nucleotide sequence of a naturally occurring putative Casl2a tracrRNA sequence identified from a native Casl2a locus but that further include one or more modifications such as (a) additional nucleotides, e.g., an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g, addition of a detectable label.
  • modifications such as (a) additional nucleotides, e.g., an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g, addition of a detectable label.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Casl2a tracrRNA further includes a 5’ extension, such as the 5’ extensions disclosed by Park et al. (2016) Nature Communications, doi: 10.1038/s41467-018- 05641-3.
  • the invention provides a fifth engineered system including one or more vectors including: (a) a first regulatory element that is heterologous to and operably linked to one or more nucleotide sequences encoding a Casl2a nuclease; and (b) a second regulatory element that is heterologous to and operably linked to one or more nucleotide sequences encoding at least one engineered Casl2a crRNA that is designed to form a complex with the Casl2a nuclease and that includes a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell; and (c) a third regulatory element that is heterologous to and operably linked to one or more nucleotide sequences encoding at least one Casl2a tracrRNA; wherein components (a) and (b) and (c) are located on the same vector or on different vectors of the system.
  • the first regulatory element, the second regulatory element, and the third regulatory element each include a promoter that is functional in the eukaryotic cell.
  • Each promoter is selected for operability in the context (in vitro or in vivo) in which the DNA expression system is to be used.
  • the first regulatory element, the second regulatory element, and the third regulatory element each include a promoter that functions in prokaryotic cells, such as in bacterial cells.
  • the first regulatory element, the second regulatory element, and the third regulatory element each include a promoter that functions in eukaryotic cells, such as in non-human animal (e.g, mammalian, human) or human or plant or fungal cells; in embodiments, the first regulatory element, the second regulatory element, and the third regulatory element each include a promoter identified from a eukaryotic genome, such as a non-human animal or human or plant or fungal genome.
  • any one or more of the regulatory elements also includes other elements that regulate expression, such as, but not limited to, enhancers and introns, examples of which are provided in Table 3.
  • the first regulatory element, the second regulatory element, and the third regulatory element each include a promoter selected from the group consisting of pol III promoters, pol II promoters, and pol I promoters.
  • the first regulatory element, the second regulatory element, and the third regulatory element each include a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter a dihydrofolate reductase promoter
  • beta-actin promoter a phosphoglycerol kinase (PGK)
  • the first regulatory element, the second regulatory element, and the third regulatory element each include a promoter selected from the group consisting of pol III promoters, pol II promoters, and pol I promoters.
  • the first regulatory element, the second regulatory element, and the third regulatory element each include a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, an EFla promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter a dihydrofolate reductase promoter
  • beta-actin promoter a phosphoglycerol kinase (PGK) promote
  • the first regulatory element, the second regulatory element, and the third regulatory element each include a promoter that functions in eukaryotic cells, such as in plant cells.
  • useful promoters include constitutive, conditional, inducible, and temporally or spatially specific promoters (e.g., a tissue specific promoter, a developmentally regulated promoter, or a cell cycle regulated promoter).
  • the promoter is a pol II promoter.
  • the promoter is a constitutive promoter that drives DNA expression in plant cells; in embodiments, the promoter drives DNA expression in the nucleus or in an organelle such as a chloroplast or mitochondrion.
  • constitutive promoters examples include a CaMV 35S promoter as disclosed in U.S. Patents 5,858,742 and 5,322,938, a rice actin promoter as disclosed in U.S. Patent 5,641,876, a maize chloroplast aldolase promoter as disclosed in U.S. Patent 7,151,204, and an opaline synthase (NOS) and octapine synthase (OCS) promoter from Agrobacterium tumefaciens.
  • NOS opaline synthase
  • OCS octapine synthase
  • the promoter is a promoter from figwort mosaic virus (FMV), a RUBISCO promoter, or a pyruvate phosphate dikinase (PDK) promoter, which is active in the chloroplasts of mesophyll cells.
  • FMV figwort mosaic virus
  • RUBISCO RUBISCO promoter
  • PDK pyruvate phosphate dikinase
  • Other contemplated promoters include cell-specific or tissue-specific or developmentally regulated promoters, for example, a promoter that limits the expression of the DNA expression system to germline or reproductive cells (e.g., promoters of genes encoding DNA ligases, recombinases, replicases, or other genes specifically expressed in germline or reproductive cells).
  • the first regulatory element, the second regulatory element, and/or the third regulatory element includes a plant U6 spliceosomal RNA promoter, which can be native to the genome of the plant cell or from a different species, e.g, a U6 promoter from maize, tomato, or soybean such as those disclosed in U.S. Patent Application 15/120,110, published as U.S. Patent Application Publication 2017/0166912, national phase application claiming priority to PCT/US2015/018104 (published as WO 2015/131101 and claiming priority to U.S.
  • the first promoter is a plant U3, 7SL (signal recognition particle RNA), U2, or U5 promoter, or chimerics thereof, e.g., as described in U.S. Patent Application 15/120,110, published as U.S.
  • Patent Application Publication 2017/0166912 national phase application claiming priority to PCT/US2015/018104 (published as WO 2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700). All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety.
  • the eukaryotic cell is a plant cell
  • the third regulatory element includes a promoter having a nucleotide sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter.
  • the at least one Casl2a tracrRNA is at least one Casl2a tracrRNA having a nucleotide sequence including a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • the eukaryotic cell is a plant cell
  • the third regulatory element includes a promoter having a nucleotide sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter
  • the at least one Casl2a tracrRNA is at least one Casl2a tracrRNA having a nucleotide sequence including a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169,
  • the Casl2a nuclease is (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SC ADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:ll, 107 - 138, 172, 204 - 221, and 222; or (d) a Casl2a nuclease variant selected from the
  • the Casl2a nuclease is deactivated.
  • the Casl2a nuclease includes at least one modification selected from the group consisting of: (a) a localization signal (e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization signal e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-penetrating peptide
  • an endosomal escape peptide e.
  • the systems including a tracrRNA as disclosed herein can also be employed with related Cas nucleases, including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild-type ObCsml sequence (SEQ ID NO:137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • the at least one engineered Casl2a crRNA includes a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell. Target sequences and eukaryotic cells are described in detail below under the section headed “Related Disclosure”.
  • the at least one engineered Casl2a crRNA, or the one or more nucleotide sequences encoding the at least one engineered Cas 12a crRNA further includes at least one direct repeat sequence or fragment thereof that is capable of associating with the Casl2a nuclease and is adjacent to and 5’ to the spacer sequence.
  • the engineered Cas 12a crRNA, or a polynucleotide encoding the engineered Cas 12a crRNA further includes at least one direct repeat sequence or fragment thereof capable of associating with the Casl2a nuclease, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 3’ to the spacer sequence.
  • the Cas 12a tracrRNA has a nucleotide sequence that has at least about 50% or preferably greater than 50% complementarity to the at least one direct repeat sequence or fragment adjacent to and 3’ to the spacer sequence.
  • the engineered Cas 12a crRNA further includes a 3’ extension adjacent to the spacer sequence; in some embodiments, the 3 ’ extension includes nucleotides that when base-paired form about one helical turn; in some embodiments, the 3’ extension includes at least 10 contiguous nucleotides.
  • the Casl2a tracrRNA has a nucleotide sequence having sufficient complementarity (e.g., about 50% or greater than 50%) to the engineered Casl2a crRNA’s 3’ extension to allow hybridization between the Cas 12a tracrRNA and the engineered Cas 12a crRNA.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding at least one Cas 12a tracrRNA includes a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of a direct repeat sequence or fragment thereof that is capable of associating with the Casl2a nuclease and is adjacent to and 5’ to the spacer sequence of the engineered Casl2a crRNA.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Casl2a tracrRNA includes a nucleotide sequence of a naturally occurring putative Casl2a tracrRNA sequence identified from a native Casl2a locus, such as a nucleotide sequence of a naturally occurring putative Casl2a tracrRNA sequence identified from a genome using the techniques described by Zetsche et al. (2015) Cell, 163:759-771, and in U.S. Patent No. 9,790,490.
  • a naturally occurring putative Casl2a tracrRNA sequence can be identified from a genome as DNA sequence encoding a RNA molecule that: is predicted to be or putatively transcribed (or that may be determined to be transcribed, e.g, by RNA analysis) from a Casl2a CRISPR genomic region, specifically from a locus that flanks the DNA encoding the Casl2a CRISPR nuclease; that preferably includes a predicted transcriptional terminator in the direction of transcription; that is about 60 to about 100 nucleotides in length, e.g., 60-80 nucleotides, 60-85 nucleotides, 60-90 nucleotides, or 65-95 nucleotides; and that is not a direct repeat in the Casl2a CRISPR array, but rather has a nucleotide sequence that permits the transcribed tracrRNA to hybridize to the crRNA (i.e., the naturally occurring putative Casl2a tracr
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Casl2a nuclease.
  • there is additional genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Casl2a tracrRNA locus and the DNA encoding the Casl2a nuclease; see, e.g., Figures 1 and 4.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Cas 12a tracrRNA has a nucleotide sequence having at least 50% identity (e.g., at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, or at least 60% sequence identity) to the sequence of the direct repeat.
  • at least 50% identity e.g., at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, or at least 60% sequence identity
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Cas 12a tracrRNA has a nucleotide sequence including a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • An alternative embodiment of a “Cas 12a tracrRNA” includes an RNA molecule having or including a nucleotide sequence based on the nucleotide sequence of a naturally occurring putative Cas 12a tracrRNA sequence identified from a native Cas 12a locus.
  • RNA molecules that include the nucleotide sequence of a naturally occurring putative Cas 12a tracrRNA sequence identified from a native Cas 12a locus but that further include one or more modifications such as (a) additional nucleotides, e.g., an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g. , addition of a detectable label.
  • modifications such as (a) additional nucleotides, e.g., an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g. , addition of a detectable label.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Cas 12a tracrRNA further includes a 5’ extension, such as the 5’ extensions disclosed by Park et al. (2016) Nature Communications, doi: 10.1038/s41467-018-05641-3.
  • Another aspect of the invention provides a sixth engineered system including one or more vectors including: (a) a first expression cassette including at least a first promoter that is heterologous to and operably linked to one or more nucleotide sequences encoding a Casl2a nuclease and - optionally - a first transcription terminator sequence; and (b) a second expression cassette including at least a second promoter that is heterologous to operably linked to one or more nucleotide sequences encoding at least one engineered Casl2a crRNA that is designed to form a complex with the Casl2a nuclease and that includes a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell and - optionally - a second transcription terminator sequence; and (c) a third expression cassette including at least a third promoter that is heterologous to operably linked to one or more nucleotide sequences encoding at least one Casl2a tracr
  • the first promoter, the second promoter, and the third promoter are each a promoter that is functional in the eukaryotic cell, and wherein the first transcription terminator, the second transcription terminator, and the third transcription terminator are each a transcription terminator that is functional in the eukaryotic cell.
  • the first promoter, the second promoter, and the third promoter are each a promoter selected from the group consisting of pol III promoters, pol II promoters, and pol I promoters.
  • the first promoter, the second promoter, and the third promoter are each a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, an EFla promoter.
  • a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, an EFla promoter.
  • the first promoter, the second promoter, and the third promoter are each a promoter with a nucleotide sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a heta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter.
  • the first transcription terminator, the second transcription terminator, and the third transcription terminator are each selected from the group consisting of a U6 poly-T terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, a synthetic terminator functional in a eukaryotic cell, a 3 ’ element from an Agrobacterium sp. gene, a 3’ element from a non-human animal gene, a 3’ element from a human gene, and a 3 ’ element from a plant gene, wherein the 3 ’ element terminate transcription of an RNA transcript located immediately 5’ to the 3’ element.
  • the first transcription terminator, the second transcription terminator, and the third transcription terminator are each a transcription terminator having a sequence selected from the group consisting of SEQ ID NOs:72 - 100 or comprise a U6 poly-T terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, or a synthetic terminator functional in a eukaryotic cell.
  • the Casl2a nuclease is (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SC ADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:ll, 107 - 138, 172, 204 - 221, and 222; or (d) a Casl2a nuclease variant selected from the
  • the Casl2a nuclease is deactivated.
  • the Casl2a nuclease includes at least one modification selected from the group consisting of: (a) a localization signal (e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization signal e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-penetrating peptide
  • an endosomal escape peptide e.
  • the systems including a tracrRNA as disclosed herein can also be employed with related Cas nucleases, including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild-type ObCsml sequence (SEQ ID NO:137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding at least one Cas 12a tracrRNA includes a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of a direct repeat sequence or fragment thereof that is capable of associating with the Casl2a nuclease and is adjacent to and 5’ to the spacer sequence of the engineered Casl2a crRNA.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Casl2a tracrRNA includes a nucleotide sequence of a naturally occurring putative Casl2a tracrRNA sequence identified from a native Casl2a locus, such as a nucleotide sequence of a naturally occurring putative Casl2a tracrRNA sequence identified from a genome using the techniques described by Zetsche et al. (2015) Cell, 163:759-771, and in U.S. Patent No. 9,790,490.
  • a naturally occurring putative Casl2a tracrRNA sequence can be identified from a genome as DNA sequence encoding a RNA molecule that: is predicted to be or putatively transcribed (or that may be determined to be transcribed, e.g, by RNA analysis) from a Casl2a CRISPR genomic region, specifically from a locus that flanks the DNA encoding the Casl2a CRISPR nuclease; that preferably includes a predicted transcriptional terminator in the direction of transcription; that is about 60 to about 100 nucleotides in length, e.g., 60-80 nucleotides, 60-85 nucleotides, 60-90 nucleotides, or 65-95 nucleotides; and that is not a direct repeat in the Casl2a CRISPR array, but rather has a nucleotide sequence that permits the transcribed tracrRNA to hybridize to the crRNA (i.e., the naturally occurring putative Casl2a tracr
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Casl2a nuclease.
  • there is additional genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Casl2a tracrRNA locus and the DNA encoding the Casl2a nuclease; see, e.g., Figures 1 and 4.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Cas 12a tracrRNA has a nucleotide sequence having at least 50% identity (e.g., at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, or at least 60% sequence identity) to the sequence of the direct repeat.
  • at least 50% identity e.g., at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, or at least 60% sequence identity
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Cas 12a tracrRNA has a nucleotide sequence including a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • An alternative embodiment of a “Cas 12a tracrRNA” includes an RNA molecule having or including a nucleotide sequence based on the nucleotide sequence of a naturally occurring putative Cas 12a tracrRNA sequence identified from a native Cas 12a locus.
  • RNA molecules that include the nucleotide sequence of a naturally occurring putative Cas 12a tracrRNA sequence identified from a native Cas 12a locus but that further include one or more modifications such as (a) additional nucleotides, e.g., an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g. , addition of a detectable label.
  • modifications such as (a) additional nucleotides, e.g., an extension at the 3’ terminus, (b) insertion, deletion, or substitution of one or more nucleotides within the naturally occurring tracrRNA sequence, preferably maintaining the secondary structure of the tracrRNA, and (c) chemical modifications, e.g. , addition of a detectable label.
  • the at least one tracrRNA, or the one or more nucleotide sequences encoding the at least one Cas 12a tracrRNA further includes a 5’ extension, such as the 5’ extensions disclosed by Park et al. (2016) Nature Communications, doi: 10.1038/s41467-018-05641-3.
  • the at least one engineered Casl2a crRNA includes a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell.
  • Target sequences and eukaryotic cells are described in detail below under the section headed “Related Disclosure”.
  • the eukaryotic cell is a non-human animal cell, a human cell, a plant cell, or a fungal cell.
  • the eukaryotic cell is in vitro, ex vivo, or in vivo.
  • the Casl2a nuclease is: (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium
  • the Casl2a nuclease is deactivated.
  • the Casl2a nuclease includes at least one modification selected from the group consisting of: (a) a localization signal (e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization signal e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-penetrating peptide
  • an endosomal escape peptide e.
  • the systems including a tracrRNA as disclosed herein can also be employed with related Cas nucleases, including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g, Begemann et al.
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g, Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild- type ObCsml sequence (SEQ ID NO:137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • the at least one Casl2a tracrRNA has a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • the genomic sequence encoding the Casl2a nuclease and the genomic sequence encoding the Casl2a tracrRNA occur naturally within the same region in a genome; in embodiments, the genomic sequence encoding the Casl2a tracrRNA is located preferably within about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 kilobases of the DNA segment(s) encoding the Casl2a nuclease.
  • the target sequence is located 3' of a Protospacer Adjacent Motif (PAM) and the PAM sequence is TTN, where N is A/C7G or T and the Casl2a nuclease is FnCpfl, or the PAM sequence is TTTV, where V is A/C or G and the Casl2a nuclease is PaCpf Ip, LbCpfl or AsCpfl .
  • PAM Protospacer Adjacent Motif
  • the Casl2a tracrRNA is a putative Casl2a tracrRNA that is identified from the same genomic region as the Casl2a nuclease by at least the following steps: (a) identifying in the genomic region flanking the DNA encoding the Casl2a nuclease at least one nucleotide sequence that includes a CRISPR array including direct repeats; (b) identifying as a putative Casl2a tracrRNA at least one nucleotide sequence in either the sense or antisense direction in the genomic region flanking the DNA encoding the Casl2a nuclease, wherein the putative Casl2a tracrRNA has a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of the direct repeat.
  • the genomic sequence encoding the Casl2a tracrRNA is located preferably within about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 kilobases of the DNA segment(s) encoding the Casl2a nuclease.
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Casl2a nuclease.
  • genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Casl2a tracrRNA locus and the DNA encoding the Casl2a nuclease; see, e.g., Figures 1 and 4.
  • the Casl2a tracrRNA is provided: (a) as DNA encoding a first RNA molecule including the Cas 12a tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety; or (b) as a functional RNA molecule including the Cas 12a tracrRNA and a functional RNA moiety, or as a polynucleotide encoding the functional RNA molecule; or (c) by a DNA expression system including: (i) DNA sequence for a first promoter; and (ii) operably linked and heterologous to the first promoter, a DNA sequence encoding a first RNA molecule including a Casl2a tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety; or (b) as a functional RNA molecule including the Cas 12a trac
  • the DNA expression system includes (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter, a DNA sequence encoding a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety, and (c) a terminator 3’ to and operably linked to the DNA encoding a functional RNA molecule.
  • the Casl2a tracrRNA is provided as DNA encoding a first RNA molecule including the Casl2a tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety, and wherein the functional RNA moiety is at least one selected from the group consisting of: (a) a nucleotide sequence for annealing to a donor polynucleotide; (b) a nucleotide sequence for annealing to the crRNA 3’ extension; (c) a terminator sequence; (d) an RNA aptamer; (e) an enzymatically active RNA sequence; (f) a detectable label; and (g) an RNA sequence forming at least partially double-stranded RNA.
  • the Casl2a tracrRNA is provided as DNA encoding a first RNA molecule including the Casl2a tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety, wherein the functional RNA moiety includes RNA forming at least partially double-stranded RNA capable of silencing a gene (e.g., RNA that forms one or more stem -loops where the stem includes double-stranded RNA of at least about 18 base-pairs designed to silence a target gene, or RNA that has the secondary structure of a microRNA precursor and is processed to a mature microRNA designed to silence a target gene).
  • RNA forming at least partially double-stranded RNA capable of silencing a gene e.g., RNA that forms one or more stem -loops where the stem includes double-stranded RNA of at least about 18 base-pairs designed to silence a target gene
  • the Casl2a tracrRNA is provided as DNA encoding a first RNA molecule including the Casl2a tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety, wherein the functional RNA moiety includes an RNA sequence for annealing to a donor polynucleotide, and wherein the composition further includes the donor polynucleotide.
  • the Casl2a tracrRNA is provided as a functional RNA molecule including the Casl2a tracrRNA and a functional RNA moiety, or as a polynucleotide encoding the functional RNA molecule, and wherein the functional RNA moiety is at least one selected from the group consisting of: (a) a nucleotide sequence for annealing to a donor polynucleotide; (b) a nucleotide sequence for annealing to the crRNA 3’ extension; (c) a terminator sequence; (d) an RNA aptamer; (e) an enzymatically active RNA sequence; (f) a detectable label; and (g) an RNA sequence forming at least partially double-stranded RNA.
  • the Casl2a tracrRNA is provided as a functional RNA molecule including the Casl2a tracrRNA and a functional RNA moiety, or as a polynucleotide encoding the functional RNA molecule, and wherein the functional RNA moiety includes RNA forming at least partially double-stranded RNA capable of silencing a gene (e.g., RNA that forms one or more stem -loops where the stem includes double-stranded RNA of at least about 18 base-pairs designed to silence a target gene, or RNA that has the secondary structure of a microRNA precursor and is processed to a mature microRNA designed to silence a target gene).
  • RNA forming at least partially double-stranded RNA capable of silencing a gene e.g., RNA that forms one or more stem -loops where the stem includes double-stranded RNA of at least about 18 base-pairs designed to silence a target gene, or RNA that has the secondary structure of
  • the Casl2a tracrRNA is provided as a functional RNA molecule including the Casl2a tracrR A and a functional RNA moiety, or as a polynucleotide encoding the functional RNA molecule, and wherein the functional RNA moiety includes RNA forming at least partially double-stranded RNA capable of silencing a gene that is involved in one or more small RNA-mediated silencing pathways, e.g., RDR6, SDE3, RDR2, WEX, SGS3, DCL2-4, UPF1, UPF3, HEN1, NRPD1A, NRPD2, DRD1, HDA6, AGO1, and MOPE
  • the Casl2a tracrRNA is provided as a functional RNA molecule including the Casl2a tracrRNA and a functional RNA moiety, or as a polynucleotide encoding the functional RNA molecule, wherein the functional RNA moiety includes an RNA sequence for annealing to a donor polynucleotide, and wherein the system further includes the donor polynucleotide.
  • the donor polynucleotide is single-stranded DNA, or is blunt-ended double-stranded DNA, or is double-stranded DNA with an overhang at least at one terminus, or is an at least partially double-stranded DNA/RNA hybrid.
  • the Casl2a tracrRNA is provided in a molar excess, relative to the amount of Casl2a nuclease.
  • the Casl2a tracrRNA is provided in a molar amount that is at least 10-fold greater than the molar amount of the Casl2a nuclease.
  • a related aspect of the invention includes a delivery particle including the engineered system any one of the first, second, third, fourth, fifth, and sixth engineered systems.
  • the Casl2a nuclease is complexed with the engineered Casl2a crRNA polynucleotide, e.g, to form a Casl2a ribonucleoprotein.
  • the Casl2a tracrRNA is present in a molar excess, relative to the amount of Casl2a nuclease (or Casl2a ribonucleoprotein).
  • the Casl2a tracrRNA is present in a molar amount that is at least 10-fold greater than the molar amount of the Casl2a nuclease (or Casl2a ribonucleoprotein).
  • the Casl2a nuclease is first complexed with the engineered Casl2a crRNA polynucleotide, e.g., to form a Casl2a ribonucleoprotein, followed by provision, without an annealing step, of the Casl2a tracrRNA in a molar amount that is at least 10-fold greater than the molar amount of the Casl2a nuclease (or Casl2a ribonucleoprotein).
  • a related aspect of the invention includes a composition or a delivery liquid including the engineered system any one of the first, second, third, fourth, fifth, and sixth engineered systems.
  • the Casl2a nuclease is complexed with the engineered Casl2a crRNA polynucleotide, e.g, to form a Casl2a ribonucleoprotein.
  • the Casl2a tracrRNA is present in a molar excess, relative to the amount of Casl2a nuclease (or Casl2a ribonucleoprotein).
  • the Casl2a tracrRNA is present in a molar amount that is at least 10-fold greater than the molar amount of the Casl2a nuclease (or Casl2a ribonucleoprotein).
  • the Casl2a nuclease is first complexed with the engineered Casl2a crRNA polynucleotide, e.g., to form a Casl2a ribonucleoprotein, followed by provision, without an annealing step, of the Casl2a tracrRNA in a molar amount that is at least 10-fold greater than the molar amount of the Casl2a nuclease (or Casl2a ribonucleoprotein).
  • Yet another aspect of the invention includes a method of modifying or editing a target sequence in a locus of interest of a eukaryotic cell including delivering the engineered system according to any one of the first, second, third, fourth, fifth, and sixth engineered systems or delivering the compositions provided herein to the locus of interest, wherein the spacer sequence hybridizes with the target sequence, whereby modification or editing of the locus of interest occurs.
  • Target sequences and eukaryotic cells are described in detail below under the section headed “Related Disclosure”.
  • the locus of interest is any specific locus in a DNA molecule, chromosome, or genome obtained from or located within a eukaryotic cell, a eukaryotic tissue, or a eukaryotic organism; in embodiments, the DNA molecule, chromosome, or genome is in vitro, ex vivo, or in vivo.
  • the eukaryotic cell includes a cell of a cell line or a cell of a multicellular organism, and the locus of interest includes a locus of interest in the genome of the eukaryotic cell.
  • the locus of interest is DNA encoding a gene of interest, and can include coding sequence, non-coding sequence, or both coding and non-coding sequence, and can include an intact functional gene (e.g., including untranscribed regions located 3 ’ or 5 ’ to DNA regions encoding a polypeptide, introns, and regulatory elements such as promoters, enhancers, polyadenylation signals, and terminators).
  • the target sequence is a segment of the locus of interest; in embodiments, the target sequence includes about 20 to about 30 contiguous nucleotides and has near perfect or perfect sequence complementarity to the nucleotide sequence of the spacer of the Casl2a crRNA.
  • the eukaryotic cell is a non-human animal cell, a human cell, a plant cell, or a fungal cell. In embodiments of the method, the eukaryotic cell is in vitro, ex vivo, or in vivo. In embodiments of the method, the locus of interest is within a eukaryotic cell, a eukaryotic tissue, or a eukaryotic organism; in embodiments, the locus of interest is within the genome of a non-human animal, a human, a plant, or a fungus, or within the genome of a eukaryotic organelle such as a mitochondrion or a plastid (e.g., a chloroplast).
  • a eukaryotic organelle such as a mitochondrion or a plastid (e.g., a chloroplast).
  • the eukaryotic cell includes a non-human animal or human stem cell or a non-human animal or human stem cell line.
  • the locus of interest is within a eukaryotic DNA molecule in vitro or in a recombinant DNA molecule or recombinant vector.
  • the eukaryotic cell includes a plant cell, and the modification of the locus of interest is correlated with a modified trait in a plant including cells containing the modification of the locus of interest.
  • the eukaryotic cell includes a plant cell, the modification of the locus of interest is correlated with a modified trait in a plant including cells containing the modification of the locus of interest, and the method further includes obtaining, growing, or regenerating a plant from the plant cell, wherein the plant includes cells containing the modification of the locus of interest, and wherein the plant exhibits the modified trait effected by the modification of the locus of interest.
  • the eukaryotic cell includes a plant cell
  • the method further includes identifying a trait of interest in a plant obtained, grown, or regenerated from the plant cell, wherein the trait of interest is effected by the modification of the locus of interest.
  • the eukaryotic cell includes a plant cell
  • the method further includes identifying a trait of interest in a plant obtained, grown, or regenerated from the plant cell, wherein the trait of interest is effected by the modification of the locus of interest
  • the method further includes (a) introducing a nucleotide sequence including the locus of interest into a plant cell, a plant cell line, or a plant germplasm, and generating from the plant cell, the plant cell line, or the plant germplasm a plant including cells containing the nucleotide sequence including the locus of interest; or (b) introducing the modified locus of interest into a plant cell, a plant cell line, or a plant germplasm, and generating from the plant cell, the plant cell line, or the plant germplasm a plant including cells containing the modified locus of interest; or (c) modifying expression of the locus of interest in a plant cell, a plant cell line, or a plant germplasm, and generating from the plant cell, the plant
  • the eukaryotic cell includes a non-human animal cell (e.g. a somatic cell or gamete of a non-human animal, in vivo, in vitro, or ex vivo), and the modification of the locus of interest is correlated with a modified trait or characteristic in a non-human animal including cells containing the modification of the locus of interest.
  • a non-human animal cell e.g. a somatic cell or gamete of a non-human animal, in vivo, in vitro, or ex vivo
  • the modification of the locus of interest is correlated with a modified trait or characteristic in a non-human animal including cells containing the modification of the locus of interest.
  • the eukaryotic cell includes a non-human animal cell
  • the modification of the locus of interest is correlated with a modified trait or characteristic in a non-human animal or in a non-human animal cell line including cells containing the modification of the locus of interest
  • the method further includes obtaining, growing, regenerating, or breeding a non-human animal or a non-human animal cell line from the non-human animal cell, wherein the non-human animal or a non-human animal cell line includes cells containing the modification of the locus of interest, and wherein the non-human animal or a non-human animal cell line exhibits the modified trait or characteristic effected by the modification of the locus of interest.
  • the eukaryotic cell includes a non-human animal cell
  • the method further includes identifying a trait or characteristic of interest in a non-human animal or a non- human animal cell line obtained, grown, regenerated, or bred from the non-human animal cell, wherein the trait or characteristic of interest is effected by the modification of the locus of interest.
  • the eukaryotic cell includes a non-human animal cell
  • the method further includes identifying a trait or characteristic of interest in a non-human animal or a non-human animal cell line obtained, grown, regenerated, or bred from the non-human animal cell, wherein the trait or characteristic of interest is effected by the modification of the locus of interest
  • the method further includes (a) introducing a nucleotide sequence including the locus of interest into a non-human animal cell, a non-human animal cell line, or a non-human animal, and obtaining, growing, regenerating, or breeding from the non-human animal cell, the non-human animal cell line, or the non-human animal a non-human animal or a non-human animal cell line including cells containing the nucleotide sequence including the locus of interest; or (b) introducing the modified locus of interest into a non-human animal cell, a non-human animal cell line, or a non-human animal, and obtaining, growing,
  • the engineered system, compositions, or a component thereof is delivered via delivery particles, delivery vesicles, delivery liquids, or one or more viral or bacterial vectors.
  • the engineered system or a component thereof is delivered via delivery particles such as delivery nanoparticles or delivery microparticles including at least one material or reagent selected from the group consisting of a lipid, a sugar, a metal, or a protein.
  • the system or a component thereof is delivered via delivery exosomes or liposomes.
  • the engineered system or a component thereof is delivered via at least one viral vector selected from the group consisting of adenoviruses, lentiviruses, adeno- associated viruses, retroviruses, geminiviruses, begomoviruses, tobamoviruses, potex viruses, comoviruses, wheat streak mosaic virus, barley stripe mosaic virus, bean yellow dwarf virus, bean pod mottle virus, cabbage leaf curl virus, beet curly top virus, tobacco yellow dwarf virus, tobacco rattle virus, potato virus X, and cowpea mosaic virus.
  • adenoviruses lentiviruses
  • adeno-associated viruses retroviruses
  • retroviruses geminiviruses
  • begomoviruses tobamoviruses
  • potex viruses comoviruses
  • wheat streak mosaic virus barley stripe mosaic virus
  • bean yellow dwarf virus bean pod mottle virus
  • cabbage leaf curl virus cabbage leaf curl virus
  • beet curly top virus tobacco yellow dwarf virus
  • the engineered system or a component thereof is delivered via at least one bacterial vector capable of transforming a plant cell and selected from the group consisting of Agrobacterium sp., Rhizobium sp., Sinorhizobium (Ensifer) sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., and Phyllobacterium sp. Additional information on delivery particles, delivery vesicles, delivery liquids, viral or bacterial vectors, and reagents and methods for their use is found in the section below headed ⁇ Delivery Methods and Delivery Agents”.
  • the Casl2a nuclease is: (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SCADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termi turn, Eubacterium eligens,Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:ll, 107 - 138, 172, 204 - 221, and 222; or (d) a Casl2a nuclease variant selected from
  • the Casl2a nuclease is deactivated.
  • the Casl2a nuclease includes at least one modification selected from the group consisting of: (a) a localization signal (e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization signal e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-penetrating peptide
  • an endosomal escape peptide e.
  • the Casl2a tracrRNA has a nucleotide sequence including a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • the tracrRNA is a putative Casl2a tracrRNA that is identified from the same genomic region as the Casl2a nuclease by at least the following steps: (a) identifying in the genomic region flanking the DNA encoding the Casl2a nuclease at least one nucleotide sequence that includes a CRISPR array including direct repeats; (b) identifying as a putative Casl2a tracrRNA at least one nucleotide sequence in either the sense or antisense direction in the genomic region flanking the DNA encoding the Casl2a nuclease, wherein the putative Casl2a tracrRNA has a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of the direct repeat.
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Casl2a nuclease.
  • there is additional genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Casl2a tracrRNA locus and the DNA encoding the Casl2a nuclease; see, e.g., Figures 1 and 4.
  • a further aspect of the invention includes a method of editing a genetic locus with a Casl2a nuclease, including contacting DNA that includes the genetic locus with: (a) a Casl2a nuclease; and (b) an engineered Cas 12a crRNA that includes a spacer sequence that is designed to hybridize with a target sequence in the genetic locus; and (c) a Casl2a tracrRNA, thereby editing the genetic locus; wherein the efficiency of editing of the genetic locus is increased compared to a control method in which the DNA that includes the genetic locus is contacted with the Casl2a nuclease and the Casl2a crRNA, but not the Casl2a tracrRNA.
  • the method enables editing a genetic locus or target sequence with a Casl2a nuclease with increased efficiency, in comparison to methods previously reported that employ a Casl2a nuclease and a Casl2a crRNA (or “guide RNA”) but do not include a Casl2a tracrRNA.
  • Efficiency of editing is increased, e.g., when the percentage of cells containing the expected edit at the genetic locus is increased compared to that seen in a control, or when a given level of editing is obtained using a lower amount of Casl2a nuclease, compared to a control.
  • a related aspect is a method of increasing the efficiency of editing a genetic locus by a Casl2a ribonucleoprotein that includes a Casl2a nuclease and a Casl2a crRNA, by providing together with the Casl2a ribonucleoprotein a Casl2a tracrRNA that is capable of hybridizing to the Casl2a crRNA (e.g., a Casl2a tracrRNA of between about 60 to about 100 nucleotides in length that has at least 50% complementarity to the sequence of the Casl2a crRNA), whereby the efficiency of the editing of the genetic locus is increased, compared to that obtained in a control method where the Casl2a ribonucleoprotein is provided without the Casl2a tracrRNA; in embodiments of the method, the Casl2a tracrRNA is provided in molar excess of the Casl2a ribonucleoprotein.
  • the Casl2a nuclease and the engineered Casl2a crRNA are provided as a ribonucleoprotein complex.
  • the Casl2a nuclease is provided as a polynucleotide encoding the Casl2a nuclease.
  • the Casl2a nuclease is: (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SC ADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:ll, 107 - 138, 172, 204 - 221, and 222; or (d) a Casl2a nuclease variant selected from the
  • the Casl2a nuclease is deactivated.
  • the Casl2a nuclease includes at least one modification selected from the group consisting of: (a) a localization signal (e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization signal e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-penetrating peptide
  • an endosomal escape peptide e.
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild-type ObCsml sequence (SEQ ID NO:137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • the Casl2a tracrRNA is provided: (a) as DNA encoding a first RNA molecule including the Cas 12a tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety; or (b) as a functional RNA molecule including the Cas 12a tracrRNA and a functional RNA moiety, or as a polynucleotide encoding the functional RNA molecule; or (c) by a DNA expression system comprising (i) DNA sequence for a first promoter; and (ii) operably linked and heterologous to the first promoter, DNA encoding a first RNA molecule comprising a Cas 12a tracrRNA and optionally comprising a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety; and (iii) optionally,
  • the Casl2a tracrRNA is provided as a synthetic Casl2a tracrRNA, e.g, a Casl2a tracrRNA synthesized in vitro from an expression construct including a heterologous promoter operably linked to DNA encoding the Cas 12a tracrRNA and optionally including an operably linked terminator.
  • the Casl2a tracrRNA is provided as a Casl2a tracrRNA obtained from a naturally occurring source, e.g., isolated from a Casl2a tracrRNA that naturally occurs in genomic DNA, or amplified from a Cas 12a tracrRNA that naturally occurs in genomic DNA.
  • the Cas 12a tracrRNA is a putative tracrRNA.
  • the putative Cas 12a tracrRNA is identified from the same genomic region as the Cas 12a nuclease by at least the following steps: (a) identifying in the genomic region flanking the DNA encoding the Cas 12a nuclease at least one nucleotide sequence that includes a CRISPR array including direct repeats; (b) identifying as a putative Casl2a tracrRNA at least one nucleotide sequence in either the sense or antisense direction in the genomic region flanking the DNA encoding the Cast 2a nuclease, wherein the putative Cast 2a tracrRNA has a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of the direct repeat.
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Casl2a nuclease.
  • there is additional genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Casl2a tracrRNA locus and the DNA encoding the Casl2a nuclease; see, e.g., Figures 1 and 4.
  • the Cas 12a tracrRNA is provided in a molar excess, relative to the Casl2a nuclease.
  • the Casl2a tracrRNA is provided in a molar amount that is at least 10-fold (e.g., at least 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 90-fold, 100-fold, 150- fold, or 200-fold) greater than the molar amount of the Casl2a nuclease.
  • the Casl2a tracrRNA is added in a molar excess (relative to the Cas 12a nuclease) to a composition containing the Casl2a ribonucleoprotein including the Casl2a nuclease and the Casl2a crRNA.
  • the Casl2a tracrRNA has a nucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • the engineered Cas 12a crRNA includes a spacer sequence that is designed to hybridize with a target sequence in the genetic locus.
  • the target sequence is a segment of a genetic locus or a gene of interest.
  • the target sequence includes about 20 to about 30 contiguous nucleotides and has near perfect or perfect sequence complementarity to the nucleotide sequence of the spacer of the Cas 12a crRNA.
  • the engineered Cas 12a crRNA, or a polynucleotide encoding the engineered Cas 12a crRNA further includes at least one direct repeat sequence or fragment thereof capable of associating with the Cas 12a nuclease, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 3’ to the spacer sequence.
  • the engineered Casl2a crRNA further includes a 3’ extension adjacent to the spacer sequence.
  • the 3’ extension includes nucleotides that when base-paired form about one helical turn.
  • the 3’ extension includes at least 10 contiguous nucleotides.
  • the Cas 12a tracrRNA has a nucleotide sequence having sufficient complementarity to the engineered Cas 12a crRNA’ s 3’ extension to allow hybridization between the tracrRNA and the engineered Casl2a crRNA.
  • the Casl2a tracrRNA is covalently linked to the engineered Cas 12a crRNA.
  • the editing of the genetic locus is effecting an indel in the genetic locus.
  • the editing of the genetic locus is effecting non-homologous end joining (NHEJ) in the genetic locus.
  • NHEJ non-homologous end joining
  • the editing of the genetic locus is effecting homology -dependent repair (HDR) in the genetic locus; in such embodiments, the method can further comprise providing a donor polynucleotide encoding a sequence to be integrated at a double-stranded break that is effected by the method in the genetic locus; examples of suitable donor polynucleotides are described above under the heading “A Method of Integrating a Sequence Encoded by a Donor Polynucleotide at a Specific Locus”.
  • the DNA that includes the genetic locus is genomic DNA.
  • the genetic locus is genomic DNA of a eukaryotic nucleus, or mitochondrial DNA, or plastid DNA.
  • the DNA that includes the genetic locus is in a eukaryotic DNA molecule in vitro.
  • the DNA that includes the genetic locus is in a eukaryotic cell; in embodiments, the eukaryotic cell is an isolated eukaryotic cell, or a eukaryotic cell in culture, or a eukaryotic cell located in a tissue or organ of a eukaryotic organism.
  • the eukaryotic cell is a cell of a non-human animal, an invertebrate, a vertebrate, a mollusk, an arthropod, an insect, a fish, a reptile, an amphibian, a bird, a mammal, a primate, a non-human primate, a human, a plant, or a fungus.
  • the eukaryotic cell includes a cell of a cell line or a cell of a multicellular organism, and wherein the genetic locus to be modified includes a locus of interest in the genome of the eukaryotic cell.
  • the eukaryotic cell is in vitro, ex vivo, or in vivo.
  • the eukaryotic cell includes a non-human animal or human stem cell or a non-human animal or human stem cell line. Target sequences and eukaryotic cells are described in detail below under the section headed “Related Disclosure”.
  • compositions for modifying a genetic locus in a eukaryotic cell including: (a) a Casl2a nuclease, or a polynucleotide encoding the Casl2a nuclease; (b) an engineered Casl2a crRNA that includes a spacer sequence that is designed to hybridize with a target sequence in the genetic locus, or a polynucleotide encoding the Casl2a crRNA; and (c) a Casl2a tracrRNA, or a polynucleotide encoding the Casl2a tracrRNA, wherein the Casl2a tracrRNA is provided in molar excess relative to the Casl2a nuclease.
  • the compositions further comprise a eukaryotic cell containing the genetic locus to be modified.
  • the compositions are in a eukaryotic cell containing the genetic locus to be modified.
  • the composition further includes: (e) a donor polynucleotide encoding a sequence to be integrated at a double-stranded break in the genetic locus; examples of suitable donor polynucleotides are described above under the heading “A Method of Integrating a Sequence Encoded by a Donor Polynucleotide at a Specific Locus”.
  • the Casl2a tracrRNA is identified from the same genomic region as the Casl2a nuclease by at least the following steps: (a) identifying in the genomic region flanking the DNA encoding the Casl2a nuclease at least one nucleotide sequence that includes a CRISPR array including direct repeats; (b) identifying as a putative Casl2a tracrRNA at least one nucleotide sequence in either the sense or antisense direction in the genomic region flanking the DNA encoding the Casl2a nuclease, wherein the putative Casl2a tracrRNA has a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of the direct repeat.
  • the Casl2a tracrRNA locus is directly adjacent to the DNA encoding the Casl2a nuclease.
  • there is additional genomic sequence such as DNA sequence encoding other Cas nucleases, located between the Casl2a tracrRNA locus and the DNA encoding the Casl2a nuclease; see, e.g., Figures 1 and 4.
  • the Casl2a tracrRNA is present in a molar excess, relative to the Casl2a nuclease.
  • the Casl2a tracrRNA is present in a molar amount that is at least 10-fold (e.g., at least 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 90-fold, 100-fold, 150-fold, or 200-fold) greater than the molar amount of the Casl2a nuclease.
  • the eukaryotic cell is a cell of a non-human animal, an invertebrate, a vertebrate, a mollusk, an arthropod, an insect, a fish, a reptile, an amphibian, a bird, a mammal, a primate, a non-human primate, a human, a plant, or a fungus.
  • the eukaryotic cell includes a cell of a cell line or a cell of a multicellular organism, and wherein the genetic locus to be modified includes a locus of interest in the genome of the eukaryotic cell.
  • the eukaryotic cell is in vitro, ex vivo, or in vivo.
  • the eukaryotic cell includes a non-human animal or human stem cell or a non-human animal or human stem cell line.
  • the Casl2a nuclease and the engineered Casl2a crRNA are provided as a ribonucleoprotein complex.
  • the Casl2a nuclease is provided as a polynucleotide encoding the Casl2a nuclease.
  • the Casl2a nuclease is: (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SC ADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae', or (b) a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or (c) a Casl2a nuclease having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:ll, 107 - 138, 172, 204 - 221, and 222; or (d) a Casl2a nuclease variant selected from the
  • the Casl2a nuclease is deactivated.
  • the Casl2a nuclease includes at least one modification selected from the group consisting of: (a) a localization signal (e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag.
  • a localization signal e.g., a nuclear localization signal, a mitochondrial localization signal, or a plastid localization signal
  • a detectable label e.g., a fluorescent dye
  • a cell-penetrating peptide e.g., a cell-penetrating peptide
  • an endosomal escape peptide e.
  • compositions including a tracrRNA as disclosed herein can also be employed with related Cas nucleases, including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g., Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild-type ObCsml sequence (SEQ ID NO:137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • the engineered Cas 12a crRNA includes a spacer sequence that is designed to hybridize with a target sequence in the genetic locus.
  • the target sequence is a segment of a genetic locus or a gene of interest; in embodiments, the target sequence includes about 20 to about 30 contiguous nucleotides and has near perfect or perfect sequence complementarity to the nucleotide sequence of the spacer of the Cas 12a crRNA.
  • the engineered Casl2a crRNA, or a polynucleotide encoding the engineered Casl2a crRNA further includes at least one direct repeat sequence or fragment thereof capable of associating with the Cas 12a nuclease, wherein the at least one direct repeat sequence or fragment thereof is adjacent to and 3’ to the spacer sequence.
  • the engineered Casl2a crRNA further includes a 3’ extension adjacent to the spacer sequence.
  • the 3’ extension includes nucleotides that when base-paired form about one helical turn.
  • the 3’ extension includes at least 10 contiguous nucleotides.
  • the Cas 12a tracrRNA has a nucleotide sequence having sufficient complementarity to the engineered Cas 12a crRNA’ s 3’ extension to allow hybridization between the tracrRNA and the engineered Casl2a crRNA.
  • the Casl2a tracrRNA is covalently linked to the engineered Cas 12a crRNA.
  • the Cas 12a tracrRNA is provided: (a) as DNA encoding a first RNA molecule including the Cas 12a tracrRNA; or (b) as a functional RNA molecule including the Cas 12a tracrRNA and a functional RNA moiety, or as a polynucleotide encoding the functional RNA molecule; or (c) by a DNA expression system comprising (i) DNA sequence for a first promoter; and (ii) operably linked and heterologous to the first promoter, DNA encoding a first RNA molecule comprising a Casl2a tracrRNA; and (iii) optionally, a terminator 3’ to and operably linked to the DNA encoding the first RNA molecule; or (d) by a DNA expression system comprising: (i) DNA sequence for a first promoter; and (ii) operably linked and heterologous to the first promoter, a DNA encoding a functional RNA
  • the Cas 12a tracrRNA is provided as DNA encoding a first RNA molecule including the Cas 12a tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety, and wherein the functional RNA moiety is at least one selected from the group consisting of: (a) a nucleotide sequence for annealing to a donor polynucleotide; (b) a nucleotide sequence for annealing to the crRNA 3’ extension; (c) a terminator sequence; (d) an RNA aptamer; (e) an enzymatically active RNA sequence; (f) a detectable label; and (g) an RNA sequence forming at least partially double-stranded RNA.
  • the Casl2a tracrRNA is provided as DNA encoding a first RNA molecule including the Casl2a tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety, wherein the functional RNA moiety includes RNA forming at least partially double-stranded RNA capable of silencing a gene (e.g., RNA that forms one or more stem-loops where the stem includes double-stranded RNA of at least about 18 base-pairs designed to silence a target gene, or RNA that has the secondary structure of a microRNA precursor and is processed to a mature microRNA designed to silence a target gene).
  • RNA forming at least partially double-stranded RNA capable of silencing a gene e.g., RNA that forms one or more stem-loops where the stem includes double-stranded RNA of at least about 18 base-pairs designed to silence a target gene, or
  • the Casl2a tracrRNA is provided as DNA encoding a first RNA molecule including the Casl2a tracrRNA and including a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety, wherein the functional RNA moiety includes an RNA sequence for annealing to a donor polynucleotide, and wherein the composition further includes the donor polynucleotide.
  • the Casl2a tracrRNA is provided as a functional RNA molecule including the Casl2a tracrRNA and a functional RNA moiety, or as a polynucleotide encoding the functional RNA molecule, and wherein the functional RNA moiety is at least one selected from the group consisting of: (a) a nucleotide sequence for annealing to a donor polynucleotide; (b) a nucleotide sequence for annealing to the crRNA 3’ extension; (c) a terminator sequence; (d) an RNA aptamer; (e) an enzymatically active RNA sequence; (f) a detectable label; and (g) an RNA sequence forming at least partially double-stranded RNA.
  • the Casl2a tracrRNA is provided as a functional RNA molecule including the Casl2a tracrRNA and a functional RNA moiety, or as a polynucleotide encoding the functional RNA molecule, and wherein the functional RNA moiety includes RNA forming at least partially double-stranded RNA capable of silencing a gene (e.g., RNA that forms one or more stem -loops where the stem includes double-stranded RNA of at least about 18 base-pairs designed to silence a target gene, or RNA that has the secondary structure of a microRNA precursor and is processed to a mature microRNA designed to silence a target gene).
  • RNA forming at least partially double-stranded RNA capable of silencing a gene e.g., RNA that forms one or more stem -loops where the stem includes double-stranded RNA of at least about 18 base-pairs designed to silence a target gene, or RNA that has the secondary structure of
  • the Casl2a tracrRNA is provided as a functional RNA molecule including the Casl2a tracrRNA and a functional RNA moiety, or as a polynucleotide encoding the functional RNA molecule, wherein the functional RNA moiety includes an RNA sequence for annealing to a donor polynucleotide, and wherein the composition further includes the donor polynucleotide.
  • the genetic locus in a eukaryotic cell to be modified is genomic DNA.
  • the genetic locus is in genomic DNA of a eukaryotic nucleus, or mitochondrial DNA, or plastid DNA.
  • the genetic locus is in a eukaryotic DNA molecule in vitro.
  • the genetic locus is in a eukaryotic cell; in embodiments, the eukaryotic cell is an isolated eukaryotic cell, or a eukaryotic cell in culture, or a eukaryotic cell located in a tissue or organ of a eukaryotic organism.
  • the eukaryotic cell is a cell of a non-human animal, an invertebrate, a vertebrate, a mollusk, an arthropod, an insect, a fish, a reptile, an amphibian, a bird, a mammal, a primate, a non-human primate, a human, a plant, or a fungus.
  • the eukaryotic cell includes a cell of a cell line or a cell of a multicellular organism, and wherein the genetic locus to be modified includes a locus of interest in the genome of the eukaryotic cell.
  • the eukaryotic cell is in vitro, ex vivo, or in vivo.
  • the eukaryotic cell includes a non- human animal or human stem cell or a non-human animal or human stem cell line. Target sequences and eukaryotic cells are described in detail below under the section headed “Related Disclosure.”
  • compositions disclosed herein or compositions comprising any of the engineered systems disclosed herein can further comprise one or more pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients include those that serve as a vehicle or medium for the compositions described herein (e.g., any one of the inactive ingredients approved by the United States Food and Drug Administration (FDA) and listed in the Inactive Ingredient Database).
  • Compositions of the present disclosure include compositions which are sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents include those described in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
  • Non-limiting examples of pharmaceutically acceptable excipients include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifying agents, preservatives, polymers, peptides, proteins, cells, hyaluronidases, dispersing agents, granulating agents, disintegrating agents, binding agents, buffering agents (e.g., phosphate buffered saline (PBS)), lubricating agents, oils, and mixtures thereof.
  • PBS phosphate buffered saline
  • compositions disclosed herein for use in a treatments or manufacture of a medicament are suitable for administration to humans or any other animal (e.g., to non-human animals or non-human mammals).
  • the compositions or engineered systems delivered via particles (e.g., delivery particles, vesicles, or one or more viral vectors, optionally wherein the particles comprise a lipid, a sugar, a metal or a protein, optionally wherein the vesicles comprise exosomes or liposomes optionally wherein the one or more viral vectors comprise one or more of adenovirus, one or more lentivirus or one or more adeno- associated virus.
  • compositions include humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • the genetic editing is of a genetic locus involved in a genetic condition or disease.
  • the disease or disorder is caused by a mutation in an enzyme.
  • the genetic condition is a metabolic disorder.
  • Exemplary diseases and target genes include Amyloid neuropathy (TTR, PALB);Amyloidosis (APOA1, APP, AAA, CVAP, ADI, GSN, FGA, LYZ, TTR, PALB); Cirrhosis (KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988); Cystic fibrosis (CFTR, ABCC7,CF, MRP7); Glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM); Hepatic adenoma, 142330 (TCF1, HNF1A, MODY3), Hepatic failure, early onset, and neurologic disorder (SCOD1, SCO1), Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancer and carcinomas
  • TTR Amyloid neuropathy
  • PALB Amy
  • Other preferred targets include any one or more of include one or more of: PCSK9; Hmgcr; SERPINA1; ApoB; LDL; Huntington disease (Huntington), Hemochromatosis (HEF), Duchenne muscular dystrophy (Dystrophin), Sickle cell anemia (Beta Globin), and Tay-Sachs (hexosaminidase A).
  • the method or use may be ex vivo or in vitro, for instance in a cell culture or in an ex vivo or in vitro model (such as an organoid or 'animal or plant cell on a chip').
  • the method may be in vivo, in which case it may also include isolating the first population of cells from the subject, and transplanting the second population of cells (back) into the subject.
  • Gene perturbation may be for one or more, or two or more, or three or more, or four or more genes.
  • Target Sequences or Target Genes Embodiments of the polynucleotides, compositions, engineered systems, and methods disclosed herein are useful in editing or effecting a sequence-specific modification of a target DNA sequence or target gene in a DNA molecule, a chromosome, or a genome.
  • the target sequence or target gene includes coding sequence (DNA encoding a polypeptide, such as a structural protein or an enzyme), non-coding sequence, or both coding and noncoding sequence.
  • the target sequence or target gene is a gene of a pest or pathogen of a non-human animal, a human, or a plant; important target genes include genes of invertebrate pests (e.g., arthropods, nematodes, and mollusks) of plants, and invertebrate parasites of animals.
  • important target genes include genes of invertebrate pests (e.g., arthropods, nematodes, and mollusks) of plants, and invertebrate parasites of animals.
  • the target sequence or target gene includes regulatory sequence or translatable (coding) sequence for genes encoding transcription factors and genes encoding enzymes involved in the biosynthesis or catabolism of molecules of interest (such as, but not limited to, amino acids, fatty acids and other lipids, sugars and other carbohydrates, biological polymers, and secondary metabolites including alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin).
  • the target gene is an endogenous gene of a eukaryotic cell; in other embodiments, the target gene is an exogenous gene (e.g. , a transgene) in a eukaryotic cell.
  • the target sequence or target gene is a gene that is involved in one or more small RNA- mediated (sRNA-mediated) silencing pathways, i.e., a gene involved in RNA-mediated sequence-specific inhibition of gene expression wherein silencing may occur at transcription, post-transcription, or translation, and wherein the sRNA-mediated silencing pathways can be classified by the biosynthesis, biogenesis, and recognition of types of regulatory sRNAs, e.g., short interfering RNAs (siRNAs), microRNAs (miRNAs), and phased sRNAs; possible target sequences include DNA encoding a regulatory RNA (e.g., DNA encoding a pre-miRNA or miRNA precursor, or DNA encoding a recognition site for a mature miRNA or an siRNA) and DNA encoding any of the participating sRNA silencing pathway proteins (e.g., RDR6, SDE3, RDR2, WEX, SGS3, DCL2-4, UPF1, UPF3, HEN
  • Non-coding sequences include DNA encoding long noncoding RNAs (IncRNAs), DNA encoding microRNAs (miRNAs), DNA encoding tRNAs or ribosomal RNAs (rRNAs), DNA encoding untranscribed regions that are located 5 ’ to or 3 ’ to DNA encoding a polypeptide, and DNA encoding regulatory elements (e.g., promoters, enhancers, introns, terminators, silencers, insulators).
  • the target DNA is genomic DNA, e.g., genomic DNA or nuclear DNA of a eukaryote.
  • the target DNA is mitochondrial or plastid DNA, e.g., mitochondrial DNA of a non-human animal, an animal, or a plant, or plastid DNA of a plant or eukaryotic alga.
  • Embodiments of the polynucleotides, compositions, engineered systems, and methods disclosed herein are useful in genome editing of eukaryotes, cells of eukaryotes, and DNA obtained from eukaryotes.
  • the polynucleotides, compositions, engineered systems, and methods disclosed herein are useful in genome editing of non-human animals and non-human animal cells, non-human animal stem cells, human cells, human stem cells, and human somatic (non-gametic) cells; isolated tissue (e.g., nervous tissue, epithelial tissue, liver, spleen, pancreas, muscle tissue, bone, connective tissue, endocrine system tissues, and tumours) or cells (e.g., blood cells, erythrocytes, leukocytes, lymphocytes, liver cells, bone cells, immune cells, T cells, B cells, dendritic cells, and tumour cells) or cell organelles (e.g., mitochondria or chloroplasts, either isolated or contained within the eukaryotic cell, tissue, or organism) obtained from a non-human animal or from a human; engineered hybridoma cells; fungi, fungal cells, plants, plant tissue, plant cells, and seeds.
  • isolated tissue e.g., nervous tissue
  • the polynucleotides, compositions, engineered systems, and methods disclosed herein are useful in genome editing of a non-human animal cell (e.g., a cell of an animal selected from the group consisting of invertebrates, vertebrates, insects, arthropods, mollusks, fish, reptiles, amphibians, birds, mammals, primates, and non-human primates), a human cell, a plant cell, or a fungal cell.
  • a non-human animal cell e.g., a cell of an animal selected from the group consisting of invertebrates, vertebrates, insects, arthropods, mollusks, fish, reptiles, amphibians, birds, mammals, primates, and non-human primates
  • a human cell e.g., a plant cell, or a fungal cell.
  • the eukaryotic cell, tissue, or cell organelle is obtained from an economically or agriculturally important animal species, such as, but not limited to, bees, fruit flies, shrimp, oysters, clams, mussels, salmon, trout, carp, tilapia, catfish, barramundi, striped bass, pigeons, chickens, turkeys, ducks, geese, goats, sheep, cattle, water buffalo, horses, pigs, dogs, cats, rabbits, mice, and rats.
  • the eukaryotic cell is in vitro, ex vivo, or in vivo.
  • the eukaryotic cell is a non-human animal stem cell or a non-human animal gametic cell, from which a nonanimal tissue or even an intact animal can be developed.
  • the polynucleotides, compositions, engineered systems, and methods disclosed herein are useful in editing of eukaryotic DNA (e.g., genomic DNA) in vitro.
  • the polynucleotides encoding the Casl2a nuclease and Casl2a nuclease variants described herein are codon-optimized, in whole or in part, for the eukaryotic species wherein the Casl2a editing system is to be employed.
  • a codon-optimized polynucleotide encoding a Casl2a nuclease contains one or more codons selected for having a high frequency for specifying a given amino acid in the eukaryotic species in which the codon-optimized polynucleotide is to be introduced or expressed. Codon usage or bias is known in the art for various eukaryotes; see, e.g., Quax et al. (2015) Molecular Cell, 59:149 - 161. See also U.S. Patent Number 6,015,891, incorporated herein by reference.
  • the polynucleotides, compositions, engineered systems, and methods disclosed herein are useful in genome editing of plants, plant tissue (including callus), plant cells, or plant protoplasts obtained from any monocot or dicot plant species of interest, for example, row crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants including ornamental flowers, shrubs, trees, groundcovers, and turf grasses.
  • plants, plant tissue, plant cells, or plant protoplasts that are useful in such genome editing include whole plant, intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, seedling, whole seed, halved seed or other seed fragment, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, callus, or plant cell or plant protoplast cultures.
  • Plants or plant tissues or cells can be haploid, inducibly haploid, haploid- inducing, diploid, or polyploid.
  • Plant species of interest include, but are not limited to, alfalfa (Medicago saliva), almonds (Prunus dulcis), apples (Malus x domestica), apricots (Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P.
  • sibirica asparagus (Asparagus officinalis), bananas (Musa spp.), barley (Plordeum vulgare), beans (Phaseolus spp.), blueberries and cranberries (Vaccinium spp.), cacao (Theobroma cacao), canola and rapeseed or oilseed rape, (Brassica napus), carnation (Dianthus caryophyllus), carrots (Daucus carota sativus), cassava (Manihot esculentum), cherry (Prunus avium), chickpea (Cider arietinum), chicory (Cichorium intybus), chili peppers and other capsicum peppers (Capsicum annuum, C.
  • Coffea arabica and Coffea canephora including Coffea arabica and Coffea canephora), cotton (Gossypium hirsutum L.), cowpea (Vigna unguiculata), cucumber (Cucumis sativus), currants and gooseberries (Ribes spp.), eggplant or aubergine (Solanum melongena), eucalyptus (Eucalyptus spp.), flax (Linum usitatissumum L.), geraniums (Pelargonium spp.), grapefruit (Citrus x parodist), grapes (Vitus spp.) including wine grapes (Vitus vinifera), guava (Psidium guajava), irises (Iris spp.), lemon (Citrus limori), lettuce (Lactuca sativa), limes (Citrus spp.), maize (Zea mays L.), mango (M
  • the Casl2a nuclease is a fusion protein, e.g., wherein the Casl2a nuclease is covalently bound through a peptide bond to a cellpenetrating peptide, a nuclear localization signal peptide, a chloroplast transit peptide, or a mitochondrial targeting peptide; such fusion proteins are conveniently encoded in a single nucleotide sequence, optionally including codons for linking amino acids.
  • the Casl2a nuclease is a fusion protein, wherein the Casl2a nuclease is covalently bound to another polypeptide via a linker polypeptide, for example, via a glycine polymer (G) n , a glycine-serine polymer, a glycine-alanine polymer, and an alanine-serine polymer.
  • the Casl2a nuclease is chemically conjugated to another polypeptide, e.g. , via a non-peptide covalent bond.
  • the Casl2a nuclease is associated with another polypeptide via a non-covalent bond.
  • the Casl2a nuclease is a fusion protein, wherein the Casl2a nuclease is covalently bound to a protein (or a domain from a protein) that inhibits transcription (e.g. , a protein that represses transcription, recruits transcription inhibitor proteins, modifies a target DNA through a process such as methylation, recruits a DNA modifier, modulates histones associated with a target DNA, recruits a histone modifier such as those that modify acetylation and/or methylation of histones, or similar functions).
  • a protein that represses transcription recruits transcription inhibitor proteins
  • modifies a target DNA through a process such as methylation
  • recruits a DNA modifier modulates histones associated with a target DNA
  • recruits a histone modifier such as those that modify acetylation and/or methylation of histones, or similar functions.
  • proteins (or fragments thereof) that can be used in decrease transcription include but are not limited to: transcriptional repressors such as the Kriippel associated box (KRAB or SKD); KOX1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as Pr-SET7/8, SUV4-20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4,
  • the Casl2a nuclease is a fusion protein, wherein the Casl2a nuclease is covalently bound to a protein (or a domain from a protein) that increases transcription (e.g., a protein that activates transcription , recruits transcription activator proteins, modifies a target DNA through a process such as demethylation, recruits a DNA modifier, modulates histones associated with a target DNA, recruits a histone modifier such as those that modify acetylation and/or methylation of histones, or similar functions).
  • a protein that activates transcription recruits transcription activator proteins, modifies a target DNA through a process such as demethylation, recruits a DNA modifier, modulates histones associated with a target DNA, recruits a histone modifier such as those that modify acetylation and/or methylation of histones, or similar functions.
  • proteins (or fragments thereof) that can be used in increase transcription include but are not limited to: transcriptional activators such as VP 16, VP64, VP48, VP 160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and/or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, and the like; histone lysine demethylases such as JHDM2a/b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, SRC1, ACTR, P160, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1
  • the Casl2a nuclease is a fusion protein, wherein the Casl2a nuclease is covalently bound to a heterologous polypeptide that has enzymatic activity that modifies a target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA), for example, nuclease activity such as that provided by a restriction enzyme (e.g., Fokl nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase, M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), MET1, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylase activity such as that
  • the Casl2a nuclease is a fusion protein, wherein the Casl2a nuclease is covalently bound to a heterologous polypeptide that has enzymatic activity that modifies a polypeptide (e.g.
  • a histone associated with a target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA), for example, methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMTIA), Vietnamese histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET/SETDB 1 , and the like, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, D0T1L, Pr-SET7/8, SUV4-20H1, EZH2, RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), J
  • HMT
  • the Casl2a nuclease is a fusion protein, wherein the Casl2a nuclease is covalently bound to a dihydrofolate reductase (DHFR) destabilization domain (e.g., to generate a chemically controllable chimeric Casl2a nuclease).
  • DHFR dihydrofolate reductase
  • the Casl2a nuclease or a polynucleotide that encodes the Casl2a nuclease or a ribonucleoprotein including the Casl2a nuclease is provided as a complex with a cellpenetrating peptide or other transfecting agent.
  • the Casl2a nuclease or a polynucleotide that encodes the Casl2a nuclease or a ribonucleoprotein including the Casl2a nuclease is complexed with, or covalently or non-covalently bound to, a further element, e.g., a carrier molecule, an antibody, an antigen, a viral movement protein, a polymer, a detectable label (e.g., a moiety detectable by fluorescence, radioactivity, or enzymatic or immunochemical reaction), a quantum dot, or a particulate or nanoparticulate.
  • a further element e.g., a carrier molecule, an antibody, an antigen, a viral movement protein, a polymer, a detectable label (e.g., a moiety detectable by fluorescence, radioactivity, or enzymatic or immunochemical reaction), a quantum dot, or a particulate or nanoparticulate.
  • the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA.
  • the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA.
  • the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA.
  • the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA.
  • the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA.
  • the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA.
  • a vector or an expression cassette contained in a vector includes additional components, e.g., a polynucleotide encoding a drug resistance or herbicide gene or a polynucleotide encoding a detectable marker such as green fluorescent protein (GFP) or beta- glucuronidase (gus) to allow convenient screening or selection of cells expressing the vector.
  • a detectable marker such as green fluorescent protein (GFP) or beta- glucuronidase (gus)
  • the vector or expression cassette includes additional elements for improving delivery to a plant cell or plant protoplast or for directing or modifying expression of one or more genome-editing system elements, for example, fusing a sequence encoding a cell-penetrating peptide, localization signal, transit, or targeting peptide to the Casl2a nuclease, or adding a nucleotide sequence to stabilize a guide RNA; such fusion proteins (and the polypeptides encoding such fusion proteins) or combination polypeptides, as well as expression cassettes and vectors for their expression in a cell, are specifically claimed.
  • a Casl2a nuclease is fused to a localization signal, transit, or targeting peptide, e.g., a nuclear localization signal (NLS), a chloroplast transit peptide (CTP), or a mitochondrial targeting peptide (MTP); in a vector or an expression cassette, the nucleotide sequence encoding any of these can be located either 5’ and/or 3’ to the DNA encoding the nuclease.
  • CRISPR nuclease fusion proteins containing nuclear localization signals and codon-optimized for expression in maize are disclosed in U.S. Patent Application 15/120,110, published as U.S.
  • Patent Application Publication 2017/0166912 national phase application claiming priority to PCT/US2015/018104 (published as WO/2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700), incorporated herein by reference.
  • a chloroplast-targeting RNA is appended to the 5’ end of an mRNA encoding an endonuclease to drive the accumulation of the mRNA in chloroplasts; see Gomez, et al. (2010) Plant Signal Behav. , 5: 1517 - 1519.
  • a Cas 12a nuclease is fused to a nuclear localization signal (NLS), such as the NLS from SV40.
  • NLS nuclear localization signal
  • a Casl2a nuclease is fused to a cell-penetrating peptide (CPP), such as octa-arginine or nona-arginine or a homoarginine 12- mer oligopeptide, or a CPP disclosed in the database of cell-penetrating peptides CPPsite 2.0, publicly available at crdd[dot]osdd[dot]net/raghava/cppsite/.
  • CPP cell-penetrating peptide
  • CPP chloroplast transit peptide
  • a CTP sequence is obtained from any nuclear gene that encodes a protein that targets a chloroplast, and the isolated or synthesized CTP DNA is appended to the 5’ end of the DNA that encodes a Cas 12a nuclease targeted for use in a chloroplast.
  • Chloroplast transit peptides and their use are described in U.S. Patents 5,188,642, 5,728,925, and 8,420,888, all of which are incorporated herein by reference in their entirety.
  • the CTP nucleotide sequences provided with the sequence identifier (SEQ ID) numbers 12 - 15 and 17 - 22 of U.S. Patent 8,420,888 are incorporated herein by reference.
  • a Cas 12a nuclease is fused to a mitochondrial targeting peptide (MTP), such as a plant MTP sequence; see, e.g., Jores et al. (2016) Nature Communications, 7:12036 - 12051.
  • MTP mitochondrial targeting peptide
  • Plasmids designed for use in plants and encoding CRISPR genome editing elements are publicly available from plasmid repositories such as Addgene (Cambridge, Massachusetts; also see “addgene [dot] com”) or can be designed using publicly disclosed sequences, e.g., sequences of CRISPR nucleases.
  • such plasmids are used to co-express both Cas 12a nuclease mRNA and guide RNA(s) (Cas 12a crRNA(s)); in other embodiments, Cas 12a nuclease mRNA and guide RNA (Casl2a crRNA) are encoded on separate plasmids.
  • the plasmids are Agrobacterium TI plasmids. Materials and methods for preparing expression cassettes and vectors for CRISPR endonuclease and guide RNA for stably integrated and/or transient plant transformation are disclosed in U.S. Patent Application 15/120,110, published as U.S.
  • Patent Application Publication 2017/0166912 national phase application claiming priority to PCT/US2015/018104 (published as WO/2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700), U.S. Patent Application Publication 2015/0082478 Al, and PCT/US2015/038767 (published as WO/2017/007347 and claiming priority to U.S. Provisional Patent Application 62/023,246), all of which are incorporated herein by reference in their entirety.
  • expression cassettes are isolated linear fragments, or are part of a larger construct that includes bacterial replication elements and selectable markers; such embodiments are useful, e.g, for particle bombardment or nanoparticle delivery or protoplast transformation.
  • the expression cassette is adjacent to or located between T-DNA borders or contained within a binary vector, e.g., for Agrob acterium- mediated transformation.
  • a plasmid encoding a Casl2a nuclease is delivered to a cell (such as a plant cell or a plant protoplast) for stable integration of the Casl2a nuclease into the genome of the cell, or alternatively for transient expression of the Casl2a nuclease.
  • plasmids encoding a Casl2a nuclease are delivered to a plant cell or a plant protoplast to achieve stable or transient expression of the Casl2a nuclease, and one or multiple guide RNAs (Casl2a crRNAs) (such as a library of individual guide RNAs or multiple pooled guide RNAs) or plasmids encoding the guide RNAs are delivered to the plant cell or plant protoplast individually or in combinations, thus providing libraries or arrays of plant cells or plant protoplasts (or of plant callus or whole plants derived therefrom), in which a variety of genome edits are provided by the different guide RNAs (Casl2a crRNAs).
  • Casl2a crRNAs guide RNAs
  • the polynucleotides, ribonucleoproteins, DNA expression systems, and engineered systems are delivered to a eukaryotic cell using a viral vector.
  • Viral vectors especially useful in animal cells include viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al, Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191 ; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus; and the like.
  • a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector.
  • a recombinant expression vector of the present disclosure is a recombinant lentivirus vector.
  • a recombinant expression vector of the present disclosure is a recombinant retroviral vector.
  • Viral vectors especially useful in plant cells include geminiviruses, begomoviruses, tobamoviruses, potex viruses, comoviruses, wheat streak mosaic virus, barley stripe mosaic virus, bean yellow dwarf virus, bean pod mottle virus, cabbage leaf curl virus, beet curly top virus, tobacco yellow dwarf virus, tobacco rattle virus, potato virus X, and cowpea mosaic virus.
  • the polynucleotides, ribonucleoproteins, DNA expression systems, and engineered systems are delivered to a plant cell using a bacterial vector.
  • the engineered system or a component thereof is delivered via at least one bacterial vector capable of transforming a plant cell and selected from the group consisting of Agrobacterium sp., Rhizobium sp., Sinorhizobium (Ensifer) sp., Mesorhizobium sp., Bradyrhizobium sp.. Azohacler sp., and Phyllobacterium sp.
  • the polynucleotides, ribonucleoproteins, DNA expression systems, engineered systems, compositions, and vectors can be delivered to a eukaryotic cell, such as a plant cell, using various techniques and agents.
  • one or more treatments is employed to deliver genome editing reagents into a plant cell or plant protoplast, e.g, through barriers such as a cell wall or a plasma membrane or nuclear envelope or other lipid bilayer.
  • genome editing reagents are delivered directly, for example by direct contact of the polynucleotide composition with a plant cell or plant protoplast.
  • a genome editing reagent-containing composition in the form of a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a eukaryotic cell, such as a non-human animal cell, a human cell, a fungal cell, or a plant cell or plant protoplast (e.g. , through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection).
  • a eukaryotic cell such as a non-human animal cell, a human cell, a fungal cell, or a plant cell or plant protoplast (e.g. , through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spray
  • a eukaryotic cell such as a non-human animal cell, a human cell, a fungal cell, or a plant cell or plant protoplast is soaked in a liquid genome editing reagentcontaining composition, whereby the genome editing reagent is delivered to the plant cell or plant protoplast.
  • the genome editing reagent-containing composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure.
  • the genome editing reagent-containing composition is introduced into a eukaryotic cell, such as a non-human animal cell, a human cell, a fungal cell, or a plant cell or plant protoplast, e.g., by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of negative or positive pressure, shear forces, or treatment with a chemical or physical delivery agent such as surfactants, liposomes, or nanoparticles; see, e.g., delivery of materials to cells employing microfluidic flow through a cell-deforming constriction as described in U.S. Published Patent Application 2014/0287509, incorporated by reference in its entirety herein.
  • a eukaryotic cell such as a non-human animal cell, a human cell, a fungal cell, or a plant cell or plant protoplast, e.g., by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of
  • a eukaryotic cell such as a non-human animal cell, a human cell, a fungal cell, or a plant cell or plant protoplast
  • ultrasound or sonication vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or breakage; enzymatic cell wall or cell membrane breakage or permeabilization; abrasion or mechanical scarification (e.g., abrasion with carborundum or other particulate abrasive or scarification with a fde or sandpaper) or chemical scarification (e.g., treatment with an acid or caustic agent); and electroporation.
  • abrasion or mechanical scarification e.g., abrasion with carborundum or other particulate abrasive or scarification with a fde or sandpaper
  • chemical scarification e.g., treatment with an acid or caustic agent
  • the genome editing reagent-containing composition is provided to a plant cell or plant protoplast by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cell or plant protoplast with a polynucleotide encoding the gRNA; see, e.g., Broothaerts et al. (2005) Nature, 433:629 - 633.
  • bacterially mediated e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.
  • any of these techniques or a combination thereof are alternatively employed on the plant part or tissue or intact plant (or seed) from which a plant cell or plant protoplast is optionally subsequently obtained or isolated; in embodiments, the genome editing reagent-containing composition is delivered in a separate step after the plant cell or plant protoplast has been obtained or isolated.
  • a treatment employed in delivery of a genome editing reagent to a eukaryotic cell is carried out under a specific thermal regime, which can involve one or more appropriate temperatures, e.g., chilling or cold stress (exposure to temperatures below that at which normal growth of the eukaryotic cell occurs), or heating or heat stress (exposure to temperatures above that at which normal growth of the eukaryotic cell occurs), or treating at a combination of different temperatures.
  • a specific thermal regime which can involve one or more appropriate temperatures, e.g., chilling or cold stress (exposure to temperatures below that at which normal growth of the eukaryotic cell occurs), or heating or heat stress (exposure to temperatures above that at which normal growth of the eukaryotic cell occurs), or treating at a combination of different temperatures.
  • a specific thermal regime is carried out on the eukaryotic cell, such as a non-human animal cell, a human cell, a fungal cell, or plant cell or plant protoplast, in one or more steps separate from the genome editing reagent delivery.
  • a specific thermal regime is carried out on a plant cell or plant protoplast, or on a plant or plant part from which a plant cell or plant protoplast is subsequently obtained or isolated, in one or more steps separate from the genome editing reagent delivery.
  • a Casl2a nuclease or a polynucleotide that encodes the Casl2a nuclease or a ribonucleoprotein including the Casl2a nuclease can be provided to a cell (e.g., a plant cell) by any suitable technique.
  • the Casl2a nuclease or a polynucleotide that encodes the Casl2a nuclease or a ribonucleoprotein including the Casl2a nuclease is provided in a solution, or is provided in a liposome, micelle, emulsion, reverse emulsion, suspension, or other mixed-phase composition.
  • the Casl2a nuclease or a polynucleotide that encodes the Casl2a nuclease or a ribonucleoprotein including the Casl2a nuclease is provided by directly contacting a plant cell or plant protoplast with the Casl2a nuclease or a polynucleotide that encodes the Casl2a nuclease or a ribonucleoprotein including the Casl2a nuclease.
  • the Casl2a nuclease or a polynucleotide that encodes the Casl2a nuclease or a ribonucleoprotein including the Casl2a nuclease is provided by transporting the Casl2a nuclease or a polynucleotide that encodes the Casl2a nuclease or a ribonucleoprotein including the Casl2a nuclease into a plant cell or plant protoplast using a chemical, enzymatic, or physical agent.
  • the Casl2a nuclease is provided by bacterially mediated (e.g.
  • the Casl2a nuclease is provided by transcription in a plant cell or plant protoplast of a DNA that encodes the Casl2a nuclease and is stably integrated in the genome of the plant cell or plant protoplast or that is provided to the plant cell or plant protoplast in the form of a plasmid or expression vector (e.g, a viral vector) that encodes the Casl2a nuclease.
  • the Casl2a nuclease is provided to the plant cell or plant protoplast as a polynucleotide that encodes the Casl2a nuclease, e.g, in the form of an mRNA encoding the nuclease.
  • a whole plant or plant part or seed, or an isolated plant cell or plant protoplast, or the plant or plant part from which a plant cell or plant protoplast is obtained or isolated is treated with one or more delivery agents which can include at least one chemical, enzymatic, or physical agent, or a combination thereof.
  • a genome editing reagent-containing composition further includes one or more one chemical, enzymatic, or physical agent for delivery. Treatment with the chemical, enzymatic or physical agent can be carried out simultaneously with the genome editing reagent delivery, or in one or more separate steps that precede or follow the genome editing reagent delivery.
  • a chemical, enzymatic, or physical agent, or a combination of these is associated or complexed with a genome editing reagent composition; examples of such associations or complexes include those involving non-covalent interactions (e.g., ionic or electrostatic interactions, hydrophobic or hydrophilic interactions, formation of liposomes, micelles, or other heterogeneous composition) and covalent interactions (e.g., peptide bonds, bonds formed using cross-linking agents).
  • non-covalent interactions e.g., ionic or electrostatic interactions, hydrophobic or hydrophilic interactions, formation of liposomes, micelles, or other heterogeneous composition
  • covalent interactions e.g., peptide bonds, bonds formed using cross-linking agents
  • a genome editing reagent is provided as a liposomal complex with a cationic lipid, or as a complex with a carbon nanotube, or as a fusion protein between the nuclease and a cell-penetrating peptide.
  • agents useful for delivering a genome editing reagent include the various cationic liposomes and polymer nanoparticles reviewed by Zhang et al. (2007) J. Controlled Release, 123:1 - 10, and the cross-linked multilamellar liposomes described in U.S. Patent Application Publication 2014/0356414 Al, incorporated by reference in its entirety herein.
  • the chemical agent is at least one selected from the group consisting of:
  • solvents e.g., water, dimethylsulfoxide, dimethylformamide, acetonitrile, A-pyrrolidinc. pyridine, hexamethylphosphoramide, alcohols, alkanes, alkenes, dioxanes, polyethylene glycol, and other solvents miscible or emulsifiable with water or that will dissolve phosphonucleotides in non-aqueous systems
  • solvents e.g., water, dimethylsulfoxide, dimethylformamide, acetonitrile, A-pyrrolidinc. pyridine, hexamethylphosphoramide, alcohols, alkanes, alkenes, dioxanes, polyethylene glycol, and other solvents miscible or emulsifiable with water or that will dissolve phosphonucleotides in non-aqueous systems
  • fluorocarbons e.g., perfluorodecalin, perfluoromethyldecalin
  • glycols or polyols e.g., propylene glycol, polyethylene glycol
  • surfactants including cationic surfactants, anionic surfactants, non-ionic surfactants, and amphiphilic surfactants, e.g., alkyl or aryl sulfates, phosphates, sulfonates, or carboxylates; primary, secondary, or tertiary amines; quaternary ammonium salts; sultaines, betaines; cationic lipids; phospholipids; tallowamine; bile acids such as cholic acid; long chain alcohols; organosilicone surfactants including nonionic organosilicone surfactants such as trisiloxane ethoxylate surfactants or a silicone polyether copolymer such as a copolymer of polyalkylene oxide modified heptamethyl trisiloxane and allyloxypolypropylene glycol methylether (commercially available as SIL WET L-77TM brand surfactant having CAS Number 27306-78-1
  • surfactants include sodium lauryl sulfate, the Tween series of surfactants, Triton-XlOO, Triton-X114, CHAPS and CHAPSO, Tergitol-type NP-40, Nonidet P-40;
  • acids, bases, caustic agents e.g., peptides, proteins, or enzymes (e.g., cellulase, pectolyase, maceroenzyme, pectinase), including cell-penetrating or pore-forming peptides (e.g., (B0100)2K8, Genscript; poly-lysine, poly-arginine, or poly -homoarginine peptides; gamma zein, see U.S.
  • peptides, proteins, or enzymes e.g., cellulase, pectolyase, maceroenzyme, pectinase
  • cell-penetrating or pore-forming peptides e.g., (B0100)2K8, Genscript
  • poly-lysine, poly-arginine, or poly -homoarginine peptides gamma zein, see U.S.
  • Patent Application publication 2011/0247100 incorporated herein by reference in its entirety; transcription activator of human immunodeficiency virus type 1 (“HIV-1 Tat”) and other Tat proteins, see, e.g., www[dot]lifetein[dot]com/Cell_Penetrating_Peptides[dot]html and Jarver (2012) Mol. Therapy-Nucleic Acids, l:e27,l - 17); octa-arginine or nona-arginine; poly -homoarginine (see Unnamalai et al.
  • cationic branched or linear polymers such as chitosan, poly -lysine, DEAE-dextran, polyvinylpyrrolidone (“PVP”), or polyethylenimine (“PEI”, e.g., PEI, branched, MW 25,000, CAS# 9002-98-6; PEI, linear, MW 5000, CAS# 9002-98-6; PEI linear, MW 2500, CAS# 9002-98-6);
  • (k) counter-ions amines or polyamines (e.g., spermine, spermidine, putrescine), osmolytes, buffers, and salts (e.g., calcium phosphate, ammonium phosphate);
  • polynucleotides e.g., non-specific double-stranded DNA, salmon sperm DNA
  • transfection agents e.g., Lipofectin®, Lipofectamine®, and Oligofectamine®, and Invivofectamine® (all from Thermo Fisher Scientific, Waltham, MA), PepFect (see Ezzat et al. (2011) Nucleic Acids Res. , 39:5284 - 5298), Transit® transfection reagents (Mirus Bio, LLC, Madison, WI), and poly -lysine, poly-homoarginine, and poly -arginine molecules including octa-arginine and nonaarginine as described in Lu et al. (2010) J. Agric. Food Chem., 58:2288 - 2294);
  • antibiotics including non-specific DNA double-strand-break-inducing agents (e.g., phleomycin, bleomycin, talisomycin); and
  • antioxidants e.g., glutathione, dithiothreitol, ascorbate.
  • the chemical agent is provided simultaneously with the genome editing reagent.
  • the genome editing reagent is covalently or non-covalently linked or complexed with one or more chemical agent; for example, a polynucleotide genome editing reagent can be covalently linked to a peptide or protein (e.g., a cell-penetrating peptide or a pore-forming peptide) or non-covalently complexed with cationic lipids, poly cations (e.g., polyamines), or cationic polymers (e.g., PEI).
  • the genome editing reagent is complexed with one or more chemical agents to form, e.g, a solution, liposome, micelle, emulsion, reverse emulsion, suspension, colloid, or gel.
  • the physical agent is at least one selected from the group consisting of particles or nanoparticles (e.g., particles or nanoparticles made of materials such as carbon, silicon, silicon carbide, gold, tungsten, polymers, or ceramics) in various size ranges and shapes, magnetic particles or nanoparticles (e.g., silenceMag MagnetotransfectionTM agent, OZ Biosciences, San Diego, CA), abrasive or scarifying agents, needles or microneedles, matrices, and grids.
  • particulates and nanoparticulates are useful in delivery of the polynucleotide composition or the nuclease or both.
  • Useful particulates and nanoparticles include those made of metals (e.g., gold, silver, tungsten, iron, cerium), ceramics (e.g., aluminum oxide, silicon carbide, silicon nitride, tungsten carbide), polymers (e.g., polystyrene, poly diacetylene, and poly(3,4-ethylenedioxythiophene) hydrate), semiconductors (e.g., quantum dots), silicon (e.g., silicon carbide), carbon (e.g., graphite, graphene, graphene oxide, or carbon nanosheets, nanocomplexes, or nanotubes), and composites (e.g., polyvinylcarbazole/graphene, polystyrene/graphene, platinum/graphene, palladium/graphene nanocomposites).
  • metals e.g., gold, silver, tungsten, iron, cerium
  • ceramics e.g., aluminum oxide, silicon carbide, silicon
  • such particulates and nanoparticulates are further covalently or non-covalently functionalized, or further include modifiers or cross-linked materials such as polymers (e.g., linear or branched polyethylenimine, poly -lysine), polynucleotides (e.g., DNA or RNA), polysaccharides, lipids, poly glycols (e.g., polyethylene glycol, thiolated polyethylene glycol), polypeptides or proteins, and detectable labels (e.g., a fluorophore, an antigen, an antibody, or a quantum dot).
  • polymers e.g., linear or branched polyethylenimine, poly -lysine
  • polynucleotides e.g., DNA or RNA
  • polysaccharides e.g., DNA or RNA
  • lipids lipids
  • poly glycols e.g., polyethylene glycol, thiolated polyethylene glycol
  • Embodiments of compositions including particulates include those formulated, e.g., as liquids, colloids, dispersions, suspensions, aerosols, gels, and solids.
  • Embodiments include nanoparticles affixed to a surface or support, e.g., an array of carbon nanotubes vertically aligned on a silicon or copper wafer substrate.
  • Embodiments include polynucleotide compositions including particulates (e.g., gold or tungsten or magnetic particles) delivered by a Biolistic-type technique or with magnetic force.
  • the size of the particles used in Biolistics is generally in the “microparticle” range, for example, gold microcarriers in the 0.6, 1.0, and 1.6 micrometer size ranges (see, e.g., instruction manual for the Helios® Gene Gun System, Bio-Rad, Hercules, CA; Randolph-Anderson et al. (2015) “Sub-micron gold particles are superior to larger particles for efficient Biolistic® transformation of organelles and some cell types”, BioRad US/EG Bulletin 2015), but successful Biolistics delivery using larger (40 nanometer) nanoparticles has been reported in cultured animal cells; see O’Brian and Lummis (2011) BMC Biotechnol., 11:66 - 71.
  • nanoparticles which are generally in the nanometer (nm) size range or less than 1 micrometer, e.g, with a diameter of less than about 1 nm, less than about 3 nm, less than about 5 nm, less than about 10 nm, less than about 20 nm, less than about 40 nm, less than about 60 nm, less than about 80 nm, and less than about 100 nm.
  • nanoparticles commercially available (all from Sigma- Aldrich Corp., St.
  • Louis, MO include gold nanoparticles with diameters of 5, 10, or 15 nm; silver nanoparticles with particle sizes of 10, 20, 40, 60, or 100 nm; palladium “nanopowder” of less than 25 nm particle size; single-, double-, and multi-walled carbon nanotubes, e.g., with diameters of 0.7 - 1.1, 1.3 - 2.3, 0.7 - 0.9, or 0.7 - 1.3 nm, or with nanotube bundle dimensions of 2 - 10 nm by 1- 5 micrometers, 6 - 9 nm by 5 micrometers, 7 - 15 nm by 0.5 - 10 micrometers, 7 - 12 nm by 0.5 - 10 micrometers, 110 - 170 nm by 5 - 9 micrometers, 6 - 13 nm by 2.5 - 20 micrometers.
  • Embodiments include genome editing reagent-containing compositions including materials such as gold, silicon, cerium, or carbon, e.g., gold or gold-coated nanoparticles, silicon carbide whiskers, carborundum, porous silica nanoparticles, gelatin/silica nanoparticles, nanoceria or cerium oxide nanoparticles (CNPs), carbon nanotubes (CNTs) such as single-, double-, or multi-walled carbon nanotubes and their chemically functionalized versions (e.g.
  • materials such as gold, silicon, cerium, or carbon, e.g., gold or gold-coated nanoparticles, silicon carbide whiskers, carborundum, porous silica nanoparticles, gelatin/silica nanoparticles, nanoceria or cerium oxide nanoparticles (CNPs), carbon nanotubes (CNTs) such as single-, double-, or multi-walled carbon nanotubes and their chemically functionalized versions (e.g.
  • a genome editing reagent is delivered to plant cells or plant protoplasts prepared or obtained from a plant, plant part, or plant tissue that has been treated with the polynucleotide compositions (and optionally the nuclease).
  • one or more one chemical, enzymatic, or physical agent, separately or in combination with the genome editing reagent is provided/applied at a location in the plant or plant part other than the plant location, part, or tissue from which the plant cell or plant protoplast is obtained or isolated.
  • the genome editing reagent is applied to adjacent or distal cells or tissues and is transported (e.g., through the vascular system or by cell-to-cell movement) to the meristem from which plant cells or plant protoplasts are subsequently isolated.
  • a genome editing reagent-containing composition is applied by soaking a seed or seed fragment or zygotic or somatic embryo in the genome editing reagent-containing composition, whereby the genome editing reagent is delivered to the seed or seed fragment or zygotic or somatic embryo from which plant cells or plant protoplasts are subsequently isolated.
  • a flower bud or shoot tip is contacted with a genome editing reagent-containing composition, whereby the genome editing reagent is delivered to cells in the flower bud or shoot tip from which plant cells or plant protoplasts are subsequently isolated.
  • a genome editing reagent-containing composition is applied to the surface of a plant or of a part of a plant (e.g., a leaf surface), whereby the genome editing reagent is delivered to tissues of the plant from which plant cells or plant protoplasts are subsequently isolated.
  • a whole plant or plant tissue is subjected to particle- or nanoparticle-mediated delivery (e.g., Biolistics or carbon nanotube or nanoparticle delivery) of a genome editing reagent-containing composition, whereby the genome editing reagent is delivered to cells or tissues from which plant cells or plant protoplasts are subsequently isolated.
  • particle- or nanoparticle-mediated delivery e.g., Biolistics or carbon nanotube or nanoparticle delivery
  • This example illustrates techniques for identifying trans-activating CRISPR (tracrRNA) sequences that are useful in compositions and methods of the invention. More specifically, this example provides examples of Casl2a tracrRNA sequences identified from Lachnospiraceae bacterium ND2006 (“Lb”) and Acidaminococcus sp. BV3L6 (“As”).
  • Lb Lachnospiraceae bacterium ND2006
  • As Acidaminococcus sp. BV3L6
  • RNA sequencing can be used to further characterize such sequences identified as potential or putative tracrRNAs.
  • Similar techniques are used to identify putative tracrRNA sequences for Casl2a (Cpfl) nucleases from Lachnospiraceae bacterium ND2006 (“Lb”) and Acidaminococcus sp. BV3L6 (“As”).
  • the techniques employed include: identifying sequences that are located in the CRISPR region flanking the DNA encoding the CRISPR nuclease and that have a nucleotide sequence that permits the transcribed tracrRNA to hybridize to the crRNA (i.e., the transcribed tracrRNA has sufficient complementarity to a crRNA sequence to hybridize to the crRNA), and searching for the presence of a predicted transcriptional terminator in direction of transcription.
  • Sequences having at least 50% sequence identity to the direct repeat in a corresponding CRISPR array are identified as potential tracrRNA sequences; sequences having slightly less than 50% sequence identity to the direct repeat are also evaluated.
  • putative tracrRNA sequences are identified having lengths of between 65 to 84 nucleotides and between 50.0% to 52.8% sequence identity to the direct repeat.
  • FIG 1 illustrates schematics of the Lachnospiraceae bacterium ND2006 Casl2a (“LbCpfl”) CRISPR locus and putative tracrRNA location; the schematics are shown with (upper figure) and without (lower figure) the Cas4 and Cas2 components of the locus.
  • Figure 2 illustrates a segment of the LbCpfl CRISPR locus, showing the arrangement in a CRISPR array of the LbCpfl direct repeats (“DR”, in bold font) flanking a spacer sequence (“spacer”, in underlined font), and illustrating the predicted crRNA processing from this CRISPR array.
  • DR in bold font
  • spacer spacer
  • putative tracrRNA sequences LbCpfl putative tracrRNA 1 (84 nucleotides) having the RNA sequence of SEQ ID NO:1 encoded by the DNA sequence of SEQ ID NO:2, and LbCpfl putative tracrRNA 2 (65 nucleotides) having the RNA sequence of SEQ ID NO:3 encoded by the DNA sequence of SEQ ID NO:4.
  • Figure 3 illustrates the alignments of the reverse complement of the LbCpfl direct repeat (DR) sequence, SEQ ID NO:5, with the LbCpfl putative tracrRNA 1 (SEQ ID NO:2) (top, showing 52.8% identity between the two sequences) and the LbCpfl putative tracrRNA 2 (SEQ ID NO:4) (bottom, showing 50.0% identity between the two sequences).
  • DR direct repeat
  • FIG 4 illustrates schematics of the Acidaminococcus sp. BV3L6 (“As”) Casl2a (“AsCpfl”) CRISPR locus and putative tracrRNA location; the schematics are shown with (upper figure) and without (lower figure) the Cas 1, Cas2, and Cas4 components of the locus.
  • Figure 5 illustrates a segment of the AsCpfl CRISPR locus, showing the arrangement in a CRISPR array of the AsCpfl direct repeats (“DR”, in bold font) flanking a spacer sequence (“spacer”, in underlined font), and illustrating the predicted crRNA processing from this CRISPR array.
  • DR CRISPR array of the AsCpfl direct repeats
  • a putative AsCpfl tracrRNA (72 nucleotides) is identified as having the RNA sequence of SEQ ID NO:6 encoded by the DNA sequence of SEQ ID NO: 7.
  • Figure 6 illustrates the alignment of the reverse complement of the AsCpfl direct repeat (DR) sequence, SEQ ID NO:8, with the AsCpflputative tracrRNA (SEQ ID NO:7), showing 51.4% identity between the two sequences.
  • DR AsCpfl direct repeat
  • SEQ ID NO:7 AsCpflputative tracrRNA
  • restriction endonuclease recognition and cleavage sites in and adjacent to Casl2a tracrRNA sequences. It is understood that the recognition site to which restriction endonuclease binds may also be where cleavage occurs, or may be a short distance from where cleavage occurs.
  • Table 1 lists restriction endonuclease sites located in or adjacent to (3’ to) the DNA encoding the LbCpfl putative tracrRNA 2 (65 nucleotides) (see Example 1);
  • Figure 7 schematically illustrates the position (not to scale) of some of the restriction endonuclease sites within the Lb Casl2a locus.
  • Table 2 lists restriction endonuclease sites located in or adjacent to (3’ to) the DNA encoding the AsCpfl putative tracrRNA (72 nucleotides) (see Example 1);
  • Figure 8 schematically illustrates the position (not to scale) of some of the restriction endonuclease sites within the As Cas 12a locus.
  • FIG. 9A schematically depicts an expression cassette for expressing a Cast 2a nuclease, including a promoter, coding sequence for the Casl2a nuclease, and a terminator.
  • This nonlimiting example illustrates a typical bacterial expression vector, wherein the promoter is a T7 promoter, the Cast 2a nuclease is a fusion protein including LbCasl2a or AsCasl2a and at least one copy of a nuclear localization signal (NLS).
  • This T7 expression system can be expressed and purified from E. coli.
  • This or similar expression cassettes are useful, e.g., for expressing a Casl2a nuclease, with one or multiple copies of nuclear localization signal (NLS) for nuclear targeting located at the nuclease’s N- terminus or C-terminus or both.
  • the Cast 2a nuclease may then be supplied as a protein and assembled with a guide RNA to form a ribonucleoprotein in vitro before delivery into cells.
  • Figure 9B (left) schematically depicts a DNA expression system including: (a) DNA sequence for a first promoter; (b) operably linked and heterologous to the first promoter, a DNA sequence encoding a first RNA molecule including a Cast 2a tracrRNA (here labelled “putative tracrRNA”) and including a restriction endonuclease cleavage site (not specifically indicated) that is suitable for insertion of a sequence coding for a functional RNA moiety; and (c) operably linked and heterologous to the first promoter (and to the optional second promoter, if present), a DNA encoding a Casl2a crRNA (which includes a direct repeat, “DR” and a spacer sequence) that includes a 3’ extension capable of hybridizing with a segment of the first RNA molecule.
  • a DNA sequence for a first promoter including a Cast 2a tracrRNA (here labelled “putative tracrRNA”) and including a restriction endonucle
  • Figure 9B (right) schematically depicts an alternative design wherein the DNA expression system includes a DNA encoding a Casl2a crRNA including multiple guide R As (/.e., multiple direct repeats and multiple spacer sequences), and including a 3’ extension capable of hybridizing with a segment of the first RNA molecule.
  • Optional elements that can be used in this system include a DNA sequence for a second promoter, which can be positioned between the DNA sequence encoding a first RNA molecule including a Casl2a tracrRNA and the DNA encoding a Casl2a crRNA; and a terminator, which can be positioned 3’ to the DNA encoding a Casl2a crRNA.
  • the 3 ’-most direct repeat is also optional.
  • a phage polymerase promoter such as an SP6, T7, or T3 promoter
  • tracrRNA and a guide RNA the Casl2a crRNA
  • Figure 9C (left) schematically depicts a non-limiting example of a DNA expression system including: (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter, a DNA sequence encoding a first RNA molecule including a Casl2a tracrRNA (here labelled “putative tracrRNA”) and including a restriction endonuclease cleavage site (not specifically indicated) that is suitable for insertion of a sequence coding for a functional RNA moiety.
  • a DNA sequence for a first promoter including a Casl2a tracrRNA (here labelled “putative tracrRNA”) and including a restriction endonuclease cleavage site (not specifically indicated) that is suitable for insertion of a sequence coding for a functional RNA moiety.
  • a Casl2a tracrRNA here labelled “putative tracrRNA”
  • restriction endonuclease cleavage site
  • FIG. 9C (A similar expression system can also be used for expressing a DNA sequence encoding a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety.)
  • Figure 9C (right) further schematically depicts an expression construct for expressing a guide RNA (here, a Casl2a crRNA including a direct repeat, “DR”, and a spacer sequence); the 3 ’-most direct repeat is optional.
  • a guide RNA here, a Casl2a crRNA including a direct repeat, “DR”, and a spacer sequence
  • DR direct repeat
  • spacer sequence the 3 ’-most direct repeat is optional.
  • a phage polymerase promoter such as an SP6, T7, or T3 promoter
  • Figure 10 schematically depicts a DNA expression system including: (a) DNA sequence for a first promoter (here, an RNA polymerase II promoter, such as a maize ubiquitin promoter); (b) operably linked and heterologous to the first promoter, DNA encoding a Casl2a nuclease (here, a fusion protein including LbCasl2a or AsCasl2a and at least one copy of a nuclear localization signal (NLS)); (c) a pol II terminator; (d) a second promoter (e.g., a pol II or pol III promoter, such as a rice U6 or U3 promoter); (e) operably linked and heterologous to the second promoter, a DNA sequence encoding a functional RNA molecule including a Casl2a tracrRNA (here labelled “putative tracrRNA”) and a functional RNA moiety (not specifically indicated); (f) an RNA polymerase II
  • Figure 10 (bottom) schematically depicts a second DNA expression system similar to that shown in Figure 10 (top), except that the guide RNA includes multiple guide RNAs (i.e., multiple direct repeats and multiple spacer sequences), and including a 3’ extension capable of hybridizing with a segment of the first RNA molecule; the 3’-most direct repeat is optional. It will be apparent that multiple guide RNAs can alternatively be expressed under the control of multiple promoters.
  • multiple guide RNAs can alternatively be expressed under the control of multiple promoters.
  • Figure 10 can be designed for expression of a tracrRNA and a crRNA under either one promoter or two promoters, and that promoters are selected for the type of organism (for example, a dicot plant rather than a monocot plant), tissue, or cell where the encoded nuclease and/or RNAs are to be expressed. Any of these DNA expression systems is conveniently provided on a single plasmid. While Figure 10 depicts the component expression cassettes orientated in the same direction, it is understood that these can be orientated in opposite directions.
  • This example describes a DNA expression system including: (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter, a DNA sequence encoding a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety.
  • This example illustrates employing a Casl2a tracrRNA as a scaffold or carrier for a functional RNA moiety (in this case, multiple copies of an aptamer capable of recruiting regulatory proteins as well as RNA for annealing to the crRNA 3’ extension), allowing the functional RNA moiety to be incorporated into a Casl2a ribonucleoprotein complex.
  • this example illustrates a Casl2a ribonucleoprotein complex including a deactivated Casl2a nuclease, a guide RNA (a Casl2a crRNA having a 3 ’ extension) targeting the promoter region of the maize (Zea mays) Lc gene, and a functional RNA molecule provided by integrating multiple copies of an MS2 aptamer as well as sequence for annealing to the crRNA 3’ extension into a Casl2a tracr.
  • a guide RNA a Casl2a crRNA having a 3 ’ extension
  • a ternary system is constructed containing a deactivated Casl2a nuclease, a guide RNA (a Casl2a crRNA having a 3’ extension) targeting the promoter region of the maize Zea mays) Lc gene, and a functional RNA molecule provided by integrating multiple copies of an MS2 aptamer and sequence for hybridizing to the crRNA 3’ extension into a Casl2a tracr.
  • Figure 11 schematically depicts construction of the DNA encoding the functional RNA molecule by cleavage at an HphI restriction endonuclease cleavage site (see Example 2 and Table 1) in or adjacent to a tracrRNA identified from the native Lachnospiraceae bacterium ND2006 CRISPR Casl2a locus (see Example 1), insertion of DNA encoding multiple copies of an MS2 aptamer as well as sequence for annealing to the crRNA 3’ extension, and ligation to form the DNA encoding the functional RNA molecule, which can be complexed with the Casl2a nuclease to form the ribonucleoprotein depicted in Figure 11 (bottom).
  • RNA molecules including a Casl2a tracrRNA and a functional RNA moiety
  • the functional RNA moiety is one or more RNAs selected from the group consisting of (a) an RNA sequence for annealing to a donor polynucleotide; (b) an RNA sequence for annealing to the crRNA 3’ extension; (c) a terminator sequence; (d) an RNA aptamer; (e) a ribozyme; (f) a detectable label; (g) a bar-coding sequence and (h) an RNA sequence forming at least partially double-stranded RNA, for example, RNA forming at least partially double-stranded RNA capable of silencing a gene (e.g., RNA that forms one or more stem -loops where the stem includes double-stranded RNA of at least about 18 base-pairs designed to silence a target gene, or RNA that has the secondary structure of a microRNA precursor
  • the DNA encoding the predicted tracrRNA identified from the native Lachnospiraceae bacterium ND2006 CRISPR Casl2a locus has the sequence AATTGCAAATCTTTGAAATAATGCAGACTTAAATTTATAAATTCATGGAATAAGGTGATTTT ATTGTGAAAAAATACTCGTATTTTGTTGGAAAAACATCTTTTTGTTGTATAATATGATGATAT ACGG (SEQ ID NO:9), where the tracrRNA nucleotides are shown in underlined font, the HphI recognition and binding site located within the tracrRNA is shown in bold font, and nucleotides of the native sequence located between the 3’ end of the predicted tracrRNA and the 5’ end of the CRISPR array are shown in plain font.
  • the DNA encoding the MS2 aptamer stem-loops has the sequence GTTTTAGAGCTAGGCCAACATGAGGATCACCCATGTCTGCAGGGCCTAGCAAGTTAAAATA AGGCTAGTCCGTTATCAACTTGGCCAACATGAGGATCACCCATGTCTGCAGGGCCAAGTGG CACCGAGTCGGTGCTTTTT (SEQ ID NO:10).
  • the deactivated LbCasl2a nuclease is encoded by DNA having the sequence of SEQ ID NO:11, which is a D832A mutant of the wild-type Casl2a nuclease having NCBI reference sequence WP 051666128.1 (see www[dot]ncbi[dot]nlm[dot]nih[dot]gov/protein/WP_051666128.1/).
  • the deactivated LbCasl2a nuclease optionally includes additional modifications, for example, one or more copies of a nuclear localization signal (NLS), or an affinity tag for later purification of the nuclease, with, if needed, sequences permitting cleavage and removal of the affinity tag.
  • additional modifications for example, one or more copies of a nuclear localization signal (NLS), or an affinity tag for later purification of the nuclease, with, if needed, sequences permitting cleavage and removal of the affinity tag.
  • NLS nuclear localization signal
  • the DNA encoding the Casl2a crRNA with the 3’ extension has the sequence GTTTCAAAGATTAAATAATTTCTACTAAGTGTAGAT TGGACAGAGCTCCAAGTGACCAAAACCGTATATCAT (SEQ ID NO: 12), where the direct repeat is shown in underlined font, the spacer targeting the promoter region of the maize Zea mays) B104 Lc gene is shown in bold font, and the 3’ extension is shown in plain font.
  • the DNA targeted for editing is the maize (Zea mays) Lc gene, specifically the Zm-Lc promoter region having the sequence ATAGAGAGAAGAGGGGTCGTGTAGTAGTGCTTAAACTGTACATGAACAGCAGTAGTGTT ACAGAAGCTAAACTCAACCAGAGCTCCACCAAAGACAAAGAGGGTCTACTTCCATCACCGT CTTGCTCGGTCACTTGGAGCTCTGTCCATAAATTAAACCCATCGTGGCATATCTGTAGGCA TCTACCCCGTCTTCGTCGTCCGTTCCTCACTAGCTACCAAGAGGTCGCCATTATTGCCAACAT AGAGTGTACGTGGATGTCTATATATATGCCTACTTGCACCCATATGGCATAGGCGTTCGATC CCCTTAGCGCGGAGGAGAGCTCCTCCGGTTCTTCTCTACCCTTCGCATGGAAGTTCTTGCATT GCTTCGTTGCTTCTCTAGTTTCTTCCTTCTACGTCTTTCCAGCATACGCATGCCCCTCGTCCGCATT GCTTCGTTGCTTCTCTAGTTT
  • the DNA encoding the Lachnospiraceae bacterium ND2006 Cast 2a tracrRNA sequence (SEQ ID NO:9) contained within a plasmid vector is cleaved with Hphl or by another compatible restriction endonuclease (see Table 1, Example 2) and the DNA encoding the MS2 RNA-binding stemloops (SEQ ID NO:10) is inserted by ligation to provide DNA encoding the functional RNA molecule ( Figure 11, top); alternatively, the DNA encoding the MS2 RNA-binding stem-loops (SEQ ID NO:10) is inserted into the DNA encoding the Casl2a tracrRNA through another molecular cloning method.
  • RNA molecule is transcribed in vitro and purified.
  • a Casl2a crRNA (SEQ ID NO:12) containing a 3’ extension of 10 ribonucleotides complementary to the 3’ sequence of the Casl2a tracrRNA is similarly transcribed and purified.
  • the two RNA molecules are mixed with deactivated LbCas 12a nuclease (SEQ ID NO:11) in 10:1 molar excess in 50 millimolar HEPES buffer (300 millimolar NaCl, 1 millimolar MgC12) at room temperature for 20 minutes to provide the ternary ribonucleoprotein for use in modifying the Lc promoter.
  • Ternary RNPs are delivered to maize B104 protoplasts using PEG-mediated transformation.
  • An MS2-tagged transcriptional regulator is cotransfected for multi-copy recruitment to the targeted locus, which is predicted to result in a change in expression of the maize Lc gene.
  • An example of a suitable MS2-tagged transcriptional regulator sequence is an MS2-P65-HSF1 fusion of multiple transcriptional activators (P65 and HSF1) having the sequence of SEQ ID NO:14, which includes an MS2 region (nucleotides 1 - 390), a nuclear localization signal (NLS) (nucleotides 445 - 465), a PG5 region (nucleotides 481 - 1023), and an HSF1 region (nucleotides 1047 - 1419).
  • the viral protein-derived transcriptional activator, VP64 with the sequence of SEQ ID NO:15, can be similarly tagged with the MS2 sequence and used to activate transcription of the maize Lc gene.
  • This example illustrates non-limiting embodiments of DNA expression systems that include DNA sequence for a first promoter; and, operably linked and heterologous to the first promoter, a DNA sequence encoding either: (1) a first RNA molecule comprising a Casl2a tracrRNA and comprising a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety, or (2) a functional RNA molecule comprising a Casl2a tracrRNA and a functional RNA moiety.
  • the DNA expression system includes: (a) DNA sequence for a first promoter; and, operably linked and heterologous to the first promoter; (b) a DNA sequence encoding a first RNA molecule comprising a Casl2a tracrRNA and comprising a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety; and (c) optionally, a terminator.
  • the DNA expression system further comprises: (d) optionally, a DNA sequence for a second promoter; (e) operably linked and heterologous to the first promoter or the second promoter, a DNA encoding a Casl2a crRNA that comprises a 3’ extension capable of hybridizing with a segment of the first RNA molecule; and (f) optionally, a terminator.
  • the Casl2a crRNA is capable of complexing with a Casl2a nuclease to form a Casl2a ribonucleoprotein complex.
  • the DNA sequence encoding the first promoter and/or the optional second promoter and or the optional terminator(s) is selected according to the biological system of interest (e.g., for expression in a bacterial cell, a yeast cell, an animal cell, or a plant cell).
  • the biological system of interest e.g., for expression in a bacterial cell, a yeast cell, an animal cell, or a plant cell.
  • promoters useful in such DNA expression systems include Pol II promoters and Pol III promoters.
  • promoters useful in the DNA expression systems and engineered systems of this disclosure include promoters functional in prokaryotic cells (e.g., useful for in vitro transcription), such as, but not limited to, promoters from T7 bacteriophage (T7), T7 bacteriophage plus lac operators (T71ac), Sp6 bacteriophage (Sp6, used with SP6 RNA polymerase), arabinose metabolic operon (araBAD), E. coli tryptophan operator (trp), lac operon (lac), bacteriophage lambda (pL), and a hybrid promoter of lac and trp (Ptac).
  • T7 bacteriophage T7 bacteriophage plus lac operators
  • Sp6 bacteriophage Sp6 bacteriophage
  • Sp6 Sp6 bacteriophage
  • lac operon lac
  • bacteriophage lambda bacteriophage lambda
  • Ptac a hybrid promoter of lac and trp
  • Non-limiting examples of promoters useful especially in animal cells include promoters from P-actin (ACTB), cytomegalovirus (CMV), elongation factor-la, (EFla), phosphoglycerate kinase (PGK1), ubiquitin C (UbC), herpes simplex virus thymidine kinase (HSV-1 TK), early and late simian vacuolating virus 40 (SV40), long terminal repeats (LTRs) from retrovirus, mouse metallothionein-I, Drosophila actin 5c, baculovirus polyhedron, yeast transcription elongation factor (TEF1), alcohol dehydrogenase I (ADH1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), human polymerase III RNA promoter (Hl), human U6 small nuclear promoter (U6), an inducible tetracycline response element promoter, and a CAG hybrid (containing a C
  • Nonlimiting examples of promoters useful in such DNA expression systems especially in plant cells include a nopaline synthase (Nos) promoter and a ubiquitin promoter; specific examples include a promoter selected from the group consisting of a TaU3 promoter (SEQ ID NO:16), a Nos promoter (SEQ ID NO:17), a ZmUBIl promoter (SEQ ID NO:18), and a S1UBI10 promoter (SEQ ID NO:19). Further non-limiting examples of promoters especially useful in plant cells include SEQ ID NOs:20 - 71; see Table 3.
  • Specific embodiments include a DNA expression system wherein the first promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a />eta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter, and the Casl2a tracrRNA has the sequence of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • the Casl2a tracrRNA has the sequence of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223
  • Specific embodiments include a DNA expression system having the optional second promoter, wherein the second promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a />eta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter.
  • the second promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV
  • the first promoter and optional second promoter have different sequences; in other embodiments, the first promoter and optional second promoter have identical sequences.
  • the Casl2a crRNA is capable of complexing with a Casl2a nuclease to form a Casl2a ribonucleoprotein complex; in embodiments, the Casl2a nuclease is (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp.
  • the DNA expression system includes: (a) DNA sequence for a first promoter wherein the first promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter; and, operably linked and heterologous to the first promoter; (b) a DNA sequence encoding a first RNA molecule comprising a Casl2a tracrRNA, wherein the Casl2a tracrRNA has a sequence that is selected from the group consisting of SEQ ID NOs:
  • the DNA expression system includes: (a) DNA sequence for a first promoter; and, operably linked and heterologous to the first promoter, (b) a DNA sequence encoding a functional RNA molecule comprising a Casl2a tracrRNA and a functional RNA moiety.
  • the DNA expression system further comprises: (c) optionally, a DNA sequence for a second promoter; (d) operably linked and heterologous to the first promoter or the second promoter, a DNA encoding a Cas 12a crRNA that comprises a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; and (e) optionally, a terminator.
  • the Cas 12a crRNA is capable of complexing with a Cas 12a nuclease to form a Cas 12a ribonucleoprotein complex.
  • the DNA sequence encoding the first promoter is selected according to the biological system of interest (e.g., for expression in a bacterial cell, a yeast cell, an animal cell, or a plant cell).
  • Non-limiting examples of promoters useful in such DNA expression systems include a nopaline synthase (Nos) promoter and a ubiquitin promoter; specific examples include a promoter selected from the group consisting of a TaU3 promoter (SEQ ID NO:16), a Nos promoter (SEQ ID NO:17), a ZmUBIl promoter (SEQ ID NO:18), and a S1UBI10 promoter (SEQ ID NO:19).
  • Nos nopaline synthase
  • a ubiquitin promoter specific examples include a promoter selected from the group consisting of a TaU3 promoter (SEQ ID NO:16), a Nos promoter (SEQ ID NO:17), a ZmUBIl promoter (SEQ ID NO:18), and a S1UBI10 promoter (SEQ ID NO:19).
  • Specific embodiments include a DNA expression system wherein the first promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter, and the functional RNA molecule includes a Casl2a tracrRNA that has the sequence of SEQ ID NO:3.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter a dihydrofolate reductase promoter
  • fteta-actin promoter a phosphogly
  • Specific embodiments include a DNA expression system wherein the Casl2a crRNA is capable of complexing with a Casl2a nuclease to form a Casl2a ribonucleoprotein complex, wherein the first promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter and wherein the Casl2a nuclease is (a) a Casl2a nuclease identified from the genome of a bacterial species selected from the group consisting of
  • Embodiments of these various DNA expression systems further include additional expression elements, e.g., expression enhancing elements (“enhancers”), 3’ untranslated region elements such as terminators or polyadenylation elements, and introns.
  • expression enhancing elements e.g., expression enhancing elements (“enhancers”)
  • 3’ untranslated region elements such as terminators or polyadenylation elements
  • introns e.g
  • Terminators useful in DNA expression systems and engineered systems of this disclosure include terminators having a sequence selected from the group consisting of SEQ ID NOs:72 - 100 or comprise a U6 poly-T terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, or a synthetic terminator functional in a eukaryotic cell.
  • Other expression elements useful in DNA expression systems and engineered systems of this disclosure include expression elements having a sequence selected from the group consisting of SEQ ID NOs:101 - 106.
  • Promoters useful in plant cells include promoters especially suited to monocot plants and promoters especially suited to dicot plants.
  • Non-limiting examples of these various expression elements are provided in Table 3 and are useful for heterologous expression of Casl2a tracrRNAs, such as a tracrRNA having an RNA sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241 or the Casl2a tracrRNA sequences disclosed in U.S. Patent 9,790,490 (incorporated herein by reference).
  • This example illustrates non-limiting embodiments of Casl2a nucleases and their corresponding tracrRNA sequences.
  • Table 4 provides the protein sequences of several Casl2a nucleases and the RNA sequence of each nuclease’s associated tracrRNA.
  • Table 4 further identifies for each tracrRNA one or more native restriction endonuclease cleavage sites that are suitable, e.g., for insertion of a sequence coding for a functional RNA moiety.
  • Embodiments include a DNA expression system including (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter a DNA sequence encoding a first RNA molecule including a Casl2a tracrRNA and including a restriction endonuclease cleavage site (native or engineered) that is suitable for insertion of a sequence coding for a functional RNA moiety.
  • the functional RNA moiety is characterized by one or more functions.
  • Embodiments of the RNA moiety include RNA sequences for annealing to one or more polynucleotides, RNA sequences that provide structures for binding to other molecules (including proteins or small molecule ligands) or that are catalytically active, or RNA sequences that serve as directly or indirectly detectable labels.
  • non-limiting embodiments include a DNA expression system including (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter a DNA sequence encoding a first RNA molecule including a Casl2a tracrRNA and including a restriction endonuclease cleavage site (native or engineered) that is suitable for insertion of a sequence coding for a functional RNA moiety, wherein the Casl2a tracrRNA has an RNA sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241; in some embodiments, the first promoter includes a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegal
  • the DNA expression system includes: (a) DNA sequence for a first promoter; (b) operably linked and heterologous to the first promoter a DNA sequence encoding a first RNA molecule including a Casl2a tracrRNA and including a restriction endonuclease cleavage site (native or engineered) that is suitable for insertion of a sequence coding for a functional RNA moiety; (c) optionally, a DNA sequence for a second promoter; (d) operably linked and heterologous to the first promoter or the second promoter, a DNA encoding a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the first RNA molecule; and (e) optionally, a terminator.
  • the Casl2a tracrRNA has an RNA sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241;
  • the first promoter includes a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter;
  • the optional second promoter includes a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of S
  • the first promoter and the DNA encoding the first RNA molecule, and the DNA encoding a Casl2a crRNA that includes a 3’ extension are contained in a single construct, and in other embodiments, the first promoter and the DNA encoding the first RNA molecule are contained in a first construct, and the second promoter and the DNA encoding a Casl2a crRNA that includes a 3’ extension are contained in a second construct.
  • the DNA expression system is provided in a first construct and a second construct, these can be provided in a single vector or in separate vectors.
  • the first promoter drives expression of both the DNA encoding the first RNA molecule and the DNA encoding the Casl2a crRNA.
  • the Casl2a tracrRNA further includes a 5’ extension.
  • the Cast 2a crRNA is capable of complexing with a Casl2a nuclease to form a Casl2a ribonucleoprotein complex.
  • RNA expression system including: (a) DNA sequence for a first promoter; and (b) operably linked and heterologous to the first promoter, a DNA sequence encoding a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety.
  • the functional RNA moiety is characterized by one or more functions.
  • Embodiments of the RNA moiety include RNA sequences for annealing to one or more polynucleotides, RNA sequences that provide structures for binding to other molecules (including proteins or small molecule ligands) or that are catalytically active, or RNA sequences that serve as directly or indirectly detectable labels.
  • the DNA expression system further includes: (c) optionally, a DNA sequence for a second promoter; (d) operably linked and heterologous to the first promoter or the second promoter, a DNA encoding a Casl2a crRNA that includes a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; and (e) optionally, a terminator.
  • the functional RNA molecule includes a Casl2a tracrRNA having an RNA sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241;
  • the first promoter includes a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter;
  • the optional second promoter includes a DNA sequence selected from the group consisting of SEQ ID NOs:16
  • the DNA expression system can be provided in a single construct (e.g., where the first promoter drives expression of both the DNA encoding the functional RNA molecule and the DNA encoding the Casl2a crRNA).
  • the second promoter can be operably linked to the DNA encoding the crRNA (e.g., in a second construct).
  • the DNA expression system is provided in two constructs, these can be in a single vector or in separate vectors.
  • the Casl2a tracrRNA further includes a 5’ extension.
  • the Casl2a crRNA is capable of complexing with a Casl2a nuclease to form a Casl2a ribonucleoprotein complex.
  • the various embodiments of the DNA expression systems disclosed herein are useful for expression of a Casl2a tracrRNA that is: (1) contained in a first RNA molecule including a Casl2a tracrRNA and including a restriction endonuclease cleavage site (native or engineered) that is suitable for insertion of a sequence coding for a functional RNA moiety, or (2) contained in a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety.
  • a Casl2a nuclease is associated or complexed with a Casl2a crRNA that is previously, simultaneously, or subsequently associated with, complexed with, or tethered to the Casl2a tracrRNA that is (1) contained in a first RNA molecule including a Casl2a tracrRNA and including a restriction endonuclease cleavage site (native or engineered) that is suitable for insertion of a sequence coding for a functional RNA moiety, or (2) contained in a functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety, thus forming a Casl2a ribonucleoprotein complex.
  • the resulting Casl2a ribonucleoprotein complex contains (1) the Casl2a nuclease complexed with the first RNA molecule including a Casl2a tracrRNA and including a restriction endonuclease cleavage site (native or engineered) that is suitable for insertion of a sequence coding for a functional RNA moiety, or (2) the Casl2a nuclease complexed with the functional RNA molecule including a Casl2a tracrRNA and a functional RNA moiety.
  • the Casl2a nuclease is one having a naturally occurring sequence (e.g., a native sequence of a Casl2a nuclease such as, but not limited to, LbCasl2a, AsCasl2a, FnCasl2a, and an ObCsml); in other embodiments, the Casl2a nuclease includes a modification, such as an amino acid sequence modification (e.g., the point-mutations provided in Table 5) and/or a chemical modification such as (a) a localization or signal peptide (e.g., a nuclear localization signal (NLS), a chloroplast or plastid transit peptide (CTP), or a mitochondrial targeting peptide (MTP)); (b) a detectable label (e.g., a fluorescent dye); (c) a cell-penetrating peptide; (d) an endosomal escape peptide; and (e) an affinity tag
  • the Casl2a nuclease is one selected from the Casl2a (“Cpfl”) nucleases, Cpfl orthologues, and Cpfl variants (including the codon-optimized variants) disclosed in U.S. Patent 9,790,490; the Casl2a (“Cpfl”) nuclease sequences disclosed in U.S. Patent Application Publication 2018/0282713 (see, e.g., Figure 3 and Example 5); and Casl2a (“Cpfl”) nuclease sequences disclosed in U.S. Patent 9,896,696; all of these cited patents and patent application publications are incorporated herein by reference.
  • Cpfl Casl2a
  • the Casl2a nuclease is a Casl2a (“Cpfl”) nuclease identified from Francisella novicida U112 (FnCasl2a or “FnCpfl”), from Acidaminococcus sp.
  • Cpfl Casl2a
  • FnCasl2a or FnCpfl Francisella novicida U112
  • BV3L6 (AsCasl2a or “AsCpfl”), or from Lachnospiraceae bacterium ND2006 (LbCasl2a or “LbCpfl”), or is an orthologue thereof, such as an orthologue having at least 50% sequence identity to FnCasl2a (“FnCpfl”); see, e.g., the FnCpfl nuclease contained in the FnCpfl locus having SEQ ID NO:211 in U.S. Patent No. 9,790,490, specifically incorporated herein by reference.
  • the Casl2a nuclease is a Casl2a nuclease having Casl2a nuclease activity and having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs:ll, 107 - 138, 172, 204 - 221, and 222.
  • Table 5 provides non-limiting examples of Casl2a nucleases and sequence variants thereof that are useful with the DNA expression systems and engineered systems provided in this disclosure.
  • Point mutations are designed to increase the efficiency or efficacy of gene editing by the nuclease (in comparison to the unaltered native nuclease).
  • Increased efficiency or efficacy of gene editing includes but is not limited to: altered translation, folding, and/or stability of the Casl2a nuclease or ribonucleoprotein; altered affinity and/or specificity of target binding; altered Cast 2a nuclease efficiency on target and/or non-target strands; altered PAM recognition specificity; and altered DNA repair or modification outcomes (e.g., indel rates).
  • the Cal2a nuclease sequence variants include: (1) a variant LbCpfl that differs from a wild-type LbCpfl sequence (SEQ ID NO:135) in containing at least one point mutation selected from the group consisting of G309P, Y312F, M474I, D523N, Q531K, C930A, D937N, V954F/Q, M975L, A984E, I994L, T1006K, I1014V, V1055N/D, L1065F/Y, Y1180F, V1209G, I1229L, V801I, Y802I, D850A, E943A, and DI 198A; (2) a variant AsCpfl that differs from a wild-type AsCpfl sequence (SEQ ID NO: 134) in containing at least one point mutation selected from the group consisting of L320P, V980A, Q987N, T1004F/Q, K1035E, T10
  • Csm nucleases including Csm nucleases, e.g., ObCsml (GenBank accession number OGX23684), SmCsml (GenBank accession number KFO67988), MiCsml (GenBank accession number KKQ38176), or SuCsml (GenBank accession number KIM12007) (see, e.g, Begemann et al.
  • nucleases having a sequence selected from the group consisting of SEQ ID NOs:115, 137, 138, and variants thereof, such as a variant ObCsml that differs from a wild-type ObCsml sequence (SEQ ID NO: 137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
  • compositions and systems including a Casl2a nuclease, a Casl2a crRNA that comprises a 3’ extension, and a Casl2a tracrRNA. More specifically, this illustrates an engineered system including a Casl2a crRNA that includes an artificial 3’ ribonucleotide extension including a detectable label (a fluorophore) for in vivo identification of cells containing the Casl2a crRNA.
  • a detectable label a fluorophore
  • the Casl2a crRNA is synthesized for delivery to plant cells as a ribonucleoprotein complex with a Casl2a nuclease, and (2) cloned into a plant expression vector system for co-expression with Casl2a in vivo.
  • a ZmLc crRNA was designed with the nucleotide sequence of SEQ ID NO:179 including the direct repeat sequence of SEQ ID NO:180 and the spacer sequence of SEQ ID NO:181 for targeting the promoter of the Zea mays Lc gene (GRMZM5G822829); this ZmLc crRNA sequence was modified to contain 11 nucleotides adjacent to and 3’ to the spacer region, yielding an engineered 3’- extended ZmLc crRNA.
  • the 3 ’-extended crRNA sequence is encoded into a dsDNA template downstream of a T7 polymerase binding site and initiator for in vitro transcription using the HiScribeTM T7 High Yield RNA Synthesis Kit (catalogue number E2050S, New England BioLabs, Ipswich, MA).
  • the 3 ’-extended crRNA is chemically synthesized by IDT (Coralville, IA), optionally containing chemical modifications or non -natural components.
  • the 3 ’-extended crRNA is further modified to contain a fluorescent or fluorophore-conjugated nucleotide or nucleotide analog, such as cytidine-5'-phosphate-3'-(6-aminohexyl)phosphate conjugated to Cy5 (NU-1706-CY5, Jena Bioscience, Jena, Germany), at its 3’ end using T4 RNA ligase.
  • a fluorescent or fluorophore-conjugated nucleotide or nucleotide analog such as cytidine-5'-phosphate-3'-(6-aminohexyl)phosphate conjugated to Cy5 (NU-1706-CY5, Jena Bioscience, Jena, Germany), at its 3’ end using T4 RNA ligase.
  • the 3 ’-extended crRNA containing one or multiple fluorescent components within or following the polynucleotide extension e.g., 3' ATTOTM 590 (NHS Ester) is chemically synthesized
  • RNPs Fluorescent Casl2a ribonucleoprotein complexes
  • RNPs Fluorescent Casl2a ribonucleoprotein complexes
  • SEQ ID NO: 182 modified Casl2a nuclease
  • SEQ ID NO:3 Casl2 tracrRNA
  • compositions and engineered genome editing systems including a Casl2a nuclease, a Casl2acrRNA, and a Casl2a tracrRNA.
  • RNA molecule including a Casl2a tracrRNA for, e.g., tethering a donor polynucleotide to a Casl2a nuclease/crRNA ribonucleoprotein (RNP) complex
  • RNP Casl2a nuclease/crRNA ribonucleoprotein
  • Protoplasts were prepared from leaves of 2-week old maize (Zea mays, variety Bl 04) plants, following a protocol previously described (see Example 1 in U.S. patent application 16/480,992 (filed on 25 July 2019 as the national phase application based on PCT/US2018/015793 filed on 29 January 2018 and published as WO2018/140899), incorporated herein by reference.
  • the modified Casl2a nuclease employed in this experiment had the amino acid sequence of SEQ ID NO: 182; included in this sequence are (a) the native Casl2a nuclease sequence (amino acids positions 19 - 1246 of SEQ ID NO:182), (b) remains of a linker sequence (amino acid positions 1 - 7 of SEQ ID NO: 182), (c) two linker sequences (amino acid positions 15 - 18 and 1247 - 1250 of SEQ ID NO: 182), (d) an A-tcrminus nuclear localization signal (NLS) sequence (amino acid positions 8 - 14 of SEQ ID NO: 182), and (e) a C-terminus linker/NLS (amino acid positions 1247 - 1259 of SEQ ID NO:182).
  • NLS nuclear localization signal
  • the Casl2a nuclease (5.85 milligrams/milliliter in aqueous buffer containing 25 millimolar Tris-HCl, 300 millimolar NaCl, 0.1 millimolar EDTA, pH 7.4) was diluted with dilution buffer (25 millimolar Tris-HCl, 300 millimolar NaCl, 0.1 millimolar EDTA, 40% glycerol, pH 7.4) to a final concentration of 1.25 milligrams/milliliter nuclease and 31.5% glycerol.
  • a 30 micromolar solution of Casl2a tracrRNA with the nucleotide sequence of SEQ ID NO:3 was prepared in reaction buffer (40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5). Additional solutions of 40% polyethylene glycol 4000 (PEG), maize washing solution (0.6 molar mannitol, 4 millimolar MES pH 5.7, and 20 millimolar KC1), and PIM containing 50 millimolar CaCL (previously described in Table 25, U.S.
  • reaction buffer 40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5.
  • RNPs Ribonucleoproteins
  • Casl2a nuclease having the sequence of SEQ ID NO:182 and an ZmLc crRNA with the nucleotide sequence of SEQ ID NO:179 including the direct repeat sequence of SEQ ID NO: 180 and the spacer sequence of SEQ ID NO: 181 for targeting the promoter of the 7.ea mays Lc gene (GRMZM5G822829).
  • the ZmLc crRNA was resuspended in reaction buffer to a concentration of 25 micromolar; 1680 microliters of the diluted ZmLc crRNA was mixed with 560 microliters of the Casl2a nuclease solution and incubated 20 minutes at room temperature. To this mixture 1400 microliters of the Casl2a tracrRNA was added and mixed; this “Casl2a nuclease/ZmLc crRNA/tracrRNA editing system” was apportioned in 225 microliter aliquots into 8-strip PCR tubes.
  • Diluent 1 (Casl2a nuclease/GmSHATl-5 crRNA/tracrRNA) was prepared by mixing 420 microliters of 25 micromolar GmSHATl-5 crRNA (serving as a non-specific Casl2a crRNA, this was designed to target Glycine max SHAT1-5, see www[dot]uniprot[dot]org/uniprot/W8E7Pl) in reaction buffer (40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5) and 140 microliters of the Casl2a nuclease solution, incubating for 20 minutes at room temperature, and then adding 350 microliters of the Casl2a tracrRNA solution. This was apportioned in 225 microliter aliquots into 8-strip PCR tubes.
  • “Diluent 2” (Casl2a nuclease/GmSHATl-5 crRNA) was prepared by mixing 420 microliters of 25 micromolar GmSHAT5-l crRNA in reaction buffer (40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5) and 140 microliters of the Casl2a nuclease solution, incubating for 20 minutes at room temperature, and then adding 350 microliters of the reaction buffer (40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5). This was apportioned in 225 microliter aliquots into 8-strip PCR tubes.
  • reaction buffer 40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5
  • Diluent 3 (Casl2a tracr) was prepared by mixing 350 microliters of the Casl2a tracrRNA solution with 560 microliters of reaction buffer (40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5). This was apportioned in 225 microliter aliquots into 9-strip PCR tubes.
  • “Diluent 4” consisted of reaction buffer (40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5) only. This was apportioned in 225 microliter aliquots into 9-strip PCR tubes.
  • the titrations resulted in a dilution series of the Casl2a nuclease/ZmLc crRNA/tracrRNA editing system, as shown in Table 6, which provides the resulting ratios between the additional Casl2a tracrRNA (provided in the diluent) to Casl2a nuclease (provided in the Casl2a nuclease/ZmLc crRNA/tracrRNA editing system).
  • Transfections were performed in triplicate. For transfection, 16 microliters of a given titrated dilution was added to each protoplast-containing well, and mixed; 32 microliters of 40% PEG solution are added and mixed, and the plate incubated 5 minutes at room temperature. Sequential additions of maize washing buffer were dispensed across the plate and mixed, and the plate was removed from the automated liquid handler, covered, and centrifuged. The plate was returned to the automated liquid handler for removal of the majority of the supernatant, and the protoplasts resuspended in 120 microliters PIM containing 50 millimolar CaCF. The plate was covered and incubated at 27 degrees Celsius in the dark for 48 hours before harvesting. Genomic DNA was isolated from the protoplasts, quantified using QuantiFluor (Promega Corporation, Madison, WI), and subjected to PCR amplification followed by amplicon sequencing to detect indel edits.
  • QuantiFluor Promega Corporation, Madison, WI
  • Results given as the fraction of protoplasts containing an indel at the predicted cleavage site versus the amount of Casl2a nuclease used, are illustrated in Figure 12.
  • the data indicate that dilutions made with Diluent 2 (Casl2a nuclease/Gm SHAT 1-5 crRNA in reaction buffer) had no significant effect when compared to the control dilution with Diluent 4 (reaction buffer only).
  • compositions and engineered genome editing systems including a Casl2a nuclease, a Casl2acrRNA, and a Casl2a tracrRNA. More specifically, this example illustrates additional experiments that confirmed that addition of a molar excess of tracrRNA to an editing system including a Cast 2a nuclease/crRNA RNP unexpectedly resulted in an increase of the overall efficiency of genome editing at a given concentration of RNP.
  • Protoplasts were prepared from etiolated middle leaves of 12-day old maize (Zea mays, variety Bl 04) plants, following a protocol previously described (see Example 1 in U.S. patent application 16/480,992 (filed on 25 July 2019 as the national phase application based on PCT/US2018/015793 filed on 29 January 2018 and published as WO2018/140899), incorporated herein by reference.
  • the nucleotide sequence of both Casl2a crRNAs included, from 5’ to 3’, a direct repeat sequence (SEQ ID NO:180) and a spacer sequence having perfect complementarity to a specific target sequence in the maize B104 genome.
  • a 30 micromolar solution of Casl2a tracrRNA (SEQ ID NO:3) was prepared in reaction buffer (40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5).
  • a 1.25 milligram/milliliter solution of bovine serum albumen (BSA) was prepared in protein storage buffer (25 millimolar Tris-HCl, 300 millimolar NaCl, 0.1 millimolar EDTA, 1 millimolar dithiothreitol, 40% glycerol, pH 7.4).
  • Casl2a ribonucleoproteins were prepared with a Casl2a nuclease having the sequence of SEQ ID NO:182 and the “high-performing” Casl2a crRNA, GD-48 as well as the “low- performing” Casl2a crRNA, GD-14. Following a preferred protocol, each Casl2a crRNA was resuspended in reaction buffer to a concentration of 25 micromolar; 1680 microliters of the diluted Casl2a crRNA was mixed with 560 microliters of the Casl2a nuclease solution and incubated 20 minutes at room temperature.
  • Diluent 1 (Casl2a nuclease/GmSHATl-5 crRNA/tracrRNA) was prepared by mixing 420 microliters of 25 micromolar GmSHATl-5 crRNA in reaction buffer (40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5) and 140 microliters of the Casl2a nuclease solution, incubating for 20 minutes at room temperature, and then adding 350 microliters of the Casl2a tracrRNA solution. This was apportioned in 225 microliter aliquots into 8-strip PCR tubes.
  • reaction buffer 40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5
  • Diluent 2 was prepared by mixing 140 microliters of the BSA stock solution with 770 microliters of reaction buffer (40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5). This was apportioned in 225 microliter aliquots into 8-strip PCR tubes.
  • Diluent 3 (GmSHATl-5 crRNA/tracrRNA) was prepared by mixing 420 microliters of the GmSHATl-5 crRNA, 350 microliters of the Casl2a tracrRNA solution, and 140 microliters of reaction buffer (40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5). This was apportioned in 225 microliter aliquots into 9-strip PCR tubes.
  • reaction buffer 40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5
  • “Diluent 4” consisted of reaction buffer (40mM HEPES, 200mM NaCl, lOmM MgC12, and 0.2mM EDTA, pH 6.5) only. This was apportioned in 225 microliter aliquots into 9-strip PCR tubes.
  • Transfections were performed in triplicate. For transfection, 16 microliters of a given titrated dilution was added to each protoplast-containing well, and mixed; 32 microliters of 40% PEG solution are added and mixed, and the plate incubated 5 minutes at room temperature. Sequential additions of maize washing buffer were dispensed across the plate and mixed, and the plate was removed from the automated liquid handler, covered, and centrifuged. The plate was returned to the automated liquid handler for removal of the majority of the supernatant, and the protoplasts resuspended in 120 microliters PIM containing 50 millimolar CaCF. The plate was covered and incubated at 27 degrees Celsius in the dark for 36 hours before harvesting. Genomic DNA was isolated from the protoplasts, quantified using QuantiFluor (Promega Corporation, Madison, WI), and subjected to PCR amplification followed by amplicon sequencing to detect indel edits.
  • QuantiFluor Promega Corporation, Madison, WI
  • the indel editing frequency reached about maximum (about 90 - 95%) at much lower Casl2a nuclease concentrations with Diluent 3 than with Diluent 4 (reaction buffer control); use of this Casl2a tracrRNA-containing diluent appeared to decrease the amount of Casl2a nuclease required to achieve about 90% editing to about 2.13 pmol, i.e., about one-seventh the amount of Casl2a nuclease needed to achieve the same level of editing efficiency in the control dilution (15.58 pmol).
  • Dilutions made with Diluent 1 also appeared to increase the editing efficiency of a given amount of Casl2a nuclease; use of this diluent appeared to decrease the amount of Casl2a nuclease required to achieve about 90% editing to about 11.73 pmol, i.e., about 75% of the amount of Casl2a nuclease needed to achieve the same level of editing efficiency in the control dilution (15.58 pmol).
  • the indel editing frequency reached about maximum (about 30%) at much lower Casl2a nuclease concentrations with Diluent 3 than with Diluent 4 (the reaction buffer control). Furthermore, use of Diluent 3 resulted in a higher maximum indel editing frequency (about 30%), compared to that obtained with Diluent 4 (only about 20%).
  • Diluent 3 increased overall indel editing efficiency by about fifty percent, and reached maximum editing efficiency at a Casl2a nuclease concentration that was estimated to be at least less than half of the Casl2a nuclease concentration needed to reach the maximum editing efficiency observed in the control dilution.
  • Dilutions made with Diluent 1 (Casl2a nuclease/Gm SHAT 1-5 crRNA/tracrRNA in reaction buffer) also appeared to increase the editing efficiency of a given amount of Casl2a nuclease; the maximum editing efficiency (about 30%) was also greater than that seen with Diluent 4, and this level was reached with about 15.58 pmol of Casl2a nuclease in Diluent 1.
  • a method of tethering a functional RNA molecule to a Casl2a crRNA comprising hybridizing
  • a Casl2a crRNA that comprises a 3’ extension capable of hybridizing with a segment of the functional RNA molecule; thereby tethering the functional RNA molecule to the Casl2a crRNA.
  • the functional RNA molecule is provided by transcription of a DNA molecule comprising DNA encoding the Casl2a tracrRNA and DNA encoding the functional RNA moiety, wherein the DNA encoding the functional RNA moiety is inserted at a restriction endonuclease cleavage site in or adjacent to the DNA encoding the Casl2a tracrRNA.
  • RNA sequence forming at least partially double -stranded RNA (h) an RNA sequence forming at least partially double -stranded RNA.
  • the functional RNA moiety comprises RNA forming at least partially double-stranded RNA capable of silencing a gene.
  • the Casl2a crRNA is complexed with, or is capable of complexing with, a Casl2a nuclease to form a Casl2a ribonucleoprotein complex containing the functional RNA molecule.
  • the Casl2a nuclease is:
  • a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or
  • a Casl2a nuclease variant selected from the group consisting of (1) a variant LbCpfl that differs from a wild-type LbCpfl sequence (SEQ ID NO:135) in containing at least one point mutation selected from the group consisting of G309P, Y312F, M474I, D523N, Q531K, C930A, D937N, V954F/Q, M975L, A984E, I994L, T1006K, I1014V, V1055N/D, L1065F/Y, Y1180F, V1209G, I1229L, V801I, Y802I, D850A, E943A, and D1198A; (2) a variant AsCpfl that differs from a wild-type AsCpfl sequence (SEQ ID NO:134) in containing at least one point mutation selected from the group consisting of L320P, V980A, Q987N, T1004F/Q
  • a modified Casl2a ribonucleoprotein complex comprising
  • a Cas 12a crRNA that comprises a 3’ extension capable of hybridizing with a segment of the functional RNA molecule.
  • a Cas 12a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Cas 12a nuclease; or
  • a Casl2a nuclease variant selected from the group consisting of (1) a variant LbCpfl that differs from a wild-type LbCpfl sequence (SEQ ID NO:135) in containing at least one point mutation selected from the group consisting of G309P, Y312F, M474I, D523N, Q531K, C930A, D937N, V954F/Q, M975L, A984E, I994L, T1006K, I1014V, V1055N/D, L1065F/Y, Y1180F, V1209G, I1229L, V801I, Y802I, D850A, E943A, and D1198A; (2) a variant AsCpfl that differs from a wild-type AsCpfl sequence (SEQ ID NO:134) in containing at least one point mutation selected from the group consisting of L320P, V980A, Q987N, T1004F/Q
  • a method of integrating a nucleotide sequence encoded by a donor polynucleotide at a specific locus in a target DNA comprising:
  • RNA moiety comprises an RNA sequence for annealing to the donor polynucleotide
  • Casl2a crRNA comprises a spacer sequence that corresponds to a specific target locus in a target DNA, thus forming a donor:RNP complex
  • a terminator 3’ to and operably linked to the DNA encoding the Casl2a crRNA.
  • a terminator 3’ to and operably linked to the DNA encoding the Casl2a crRNA.
  • the DNA expression system of embodiment 27 or embodiment 34, wherein the Casl2a nuclease is:
  • a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or
  • a Casl2a nuclease variant selected from the group consisting of (1) a variant LbCpfl that differs from a wild-type LbCpfl sequence (SEQ ID NO:135) in containing at least one point mutation selected from the group consisting of G309P, Y312F, M474I, D523N, Q531K, C930A, D937N, V954F/Q, M975L, A984E, I994L, T1006K, I1014V, V1055N/D, L1065F/Y, Y1180F, V1209G, I1229L, V801I, Y802I, D850A, E943A, and D1198A; (2) a variant AsCpfl that differs from a wild-type AsCpfl sequence (SEQ ID NO:134) in containing at least one point mutation selected from the group consisting of L320P, V980A, Q987N, T1004F/Q
  • the first promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter and wherein the Cast 2a tracrRNA has a nucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • a DNA expression system comprising:
  • a terminator 3’ to and operably linked to the DNA encoding a Casl2 crRNA.
  • the DNA expression system of embodiment 37 or 38, wherein the first promoter and the DNA encoding the functional RNA molecule, and the DNA encoding a Casl2a crRNA that comprises a 3’ extension are contained in a single construct.
  • the DNA expression system of embodiment 40 wherein the first construct and the second construct are provided (a) in a single vector, or (b) in separate vectors.
  • RNA sequence forming at least partially double -stranded RNA (g) an RNA sequence forming at least partially double -stranded RNA.
  • the first promoter has a DNA sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter and wherein the Casl2a tracrRNA has a nucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or
  • a Casl2a nuclease variant selected from the group consisting of (1) a variant LbCpfl that differs from a wild-type LbCpfl sequence (SEQ ID NO:135) in containing at least one point mutation selected from the group consisting of G309P, Y312F, M474I, D523N, Q531K, C930A, D937N, V954F/Q, M975L, A984E, I994L, T1006K, I1014V, V1055N/D, L1065F/Y, Y1180F, V1209G, I1229L, V801I, Y802I, D850A, E943A, and D1198A; (2) a variant AsCpfl that differs from a wild-type AsCpfl sequence (SEQ ID NO:134) in containing at least one point mutation selected from the group consisting of L320P, V980A, Q987N, T1004F/Q
  • a Casl2a tracrRNA comprising a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of the at least one direct repeat of the engineered Casl2a crRNA.
  • a Casl2a tracrRNA comprising a naturally occurring putative Casl2a tracrRNA sequence that is identified from the same genomic region as the Casl2a nuclease by at least the following steps: (i) identifying in the genomic region flanking the DNA encoding the Casl2a nuclease at least one nucleotide sequence that comprises a CRISPR array including direct repeats, and (ii) identifying as a putative Casl2a tracrRNA at least one nucleotide sequence in either the sense or antisense direction in the genomic region flanking the DNA encoding the Casl2a nuclease, wherein the putative Casl2a tracrRNA has a nucleotide sequence that has at least 50% complementarity with, the nucleotide sequence of the direct repeat.
  • the engineered system of embodiment 49 or embodiment 50 wherein the engineered Casl2a crRNA, or the polynucleotide encoding the engineered Casl2a crRNA, further comprises at least one direct repeat sequence or fragment thereof capable of associating with the Casl2a nuclease adjacent to and 3’ to the spacer sequence.
  • the engineered system of embodiment 49 or embodiment 50, wherein the engineered Casl2a crRNA further comprises a 3’ extension adjacent to the spacer sequence.
  • the engineered system of embodiment 52, wherein the 3’ extension comprises nucleotides that when base-paired form about one helical turn.
  • the engineered system of embodiment 52, wherein the 3’ extension comprises at least 10 contiguous nucleotides.
  • the Casl2a tracrRNA has a nucleotide sequence having sufficient complementarity to the engineered Casl2a crRNA’s 3’ extension to allow hybridization between the tracrRNA and the engineered Casl2a crRNA; (ii) wherein the Casl2a tracrRNA is covalently linked to the engineered Casl2a crRNA; or (iii) wherein the polynucleotides encode an RNA molecule wherein the Casl2a tracrRNA is covalently linked to the engineered Casl2a crRNA.
  • An engineered system comprising:
  • nucleotide sequences encoding at least one engineered Casl2a crRNA that is designed to form a complex with the Casl2a nuclease and that comprises a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell;
  • At least one engineered Casl2a crRNA designed to form a complex with the Casl2a nuclease and comprising a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell, or one or more nucleotide sequences encoding the at least one engineered Casl2a crRNA polynucleotide;
  • At least one tracrRNA or one or more nucleotide sequences encoding the at least one Casl2a tracrRNA.
  • the engineered system of embodiment 56 or embodiment 57, wherein the at least one engineered Casl2a crRNA, or a polynucleotide encoding the at least one engineered Casl2a crRNA, further comprises at least one direct repeat sequence or fragment thereof that is capable of associating with the Casl2a nuclease and is adjacent to and 5’ to the spacer sequence.
  • the engineered system of embodiment 56 or embodiment 57 wherein the engineered Casl2a crRNA, or a polynucleotide encoding the engineered Casl2a crRNA, further comprises at least one direct repeat sequence or fragment thereof capable of associating with the Casl2a nuclease adjacent to and 3’ to the spacer sequence.
  • the engineered system of embodiment 56 or embodiment 57, wherein the engineered Casl2a crRNA further comprises a 3’ extension adjacent to the spacer sequence.
  • the engineered system of embodiment 60, wherein the 3’ extension comprises nucleotides that when base-paired form about one helical turn.
  • the engineered system of embodiment 60, wherein the 3’ extension comprises at least 10 contiguous nucleotides.
  • An engineered system comprising one or more vectors comprising:
  • a second regulatory element that is heterologous to and operably linked to one or more nucleotide sequences encoding at least one engineered Casl2a crRNA that is designed to form a complex with the Casl2a nuclease and that comprises a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell;
  • a third regulatory element that is heterologous to and operably linked to one or more nucleotide sequences encoding at least one Casl2a tracrRNA; wherein components (a) and (b) and (c) are located on the same vector or on different vectors of the system.
  • the engineered system of embodiment 65, wherein the first regulatory element, the second regulatory element, and the third regulatory element each comprise a promoter selected from the group consisting of pol III promoters, pol II promoters, and pol I promoters.
  • first regulatory element, the second regulatory element, and the third regulatory element each comprise a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta- actin promoter, a phosphoglycerol kinase (PGK) promoter, an EFla promoter.
  • a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta- actin promoter, a phosphoglycerol kinase (PGK) promoter, an EFla promoter.
  • the third regulatory element comprises a promoter having a nucleotide sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta- actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter.
  • An engineered system comprising one or more vectors comprising:
  • a first expression cassette comprising at least a first promoter that is heterologous to and operably linked to one or more nucleotide sequences encoding a Casl2a nuclease and - optionally - a first transcription terminator sequence
  • a second expression cassete comprising at least a second promoter that is heterologous to operably linked to one or more nucleotide sequences encoding at least one engineered Casl2a crRNA that is designed to form a complex with the Casl2a nuclease and that comprises a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell and - optionally - a second transcription terminator sequence
  • a third expression cassete comprising at least a third promoter that is heterologous to operably linked to one or more nucleotide sequences encoding at least one Casl2a tracrRNA and - optionally - a third transcription terminator sequence, wherein components (a) and (b) and (c) are located on the same vector or on different vectors of the system.
  • the engineered system of embodiment 70 wherein the first promoter, the second promoter, and the third promoter are each a promoter that is functional in the eukaryotic cell, and wherein the first transcription terminator, the second transcription terminator, and the third transcription terminator are each a transcription terminator that is functional in the eukaryotic cell.
  • the engineered system of embodiment 70 wherein the first promoter, the second promoter, and the third promoter are each a promoter selected from the group consisting of pol III promoters, pol II promoters, and pol I promoters.
  • the engineered system of embodiment 70, wherein the first promoter, the second promoter, and the third promoter are each a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, an EFla promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter a dihydrofolate reductase promoter
  • first promoter, the second promoter, and the third promoter are each a promoter with a nucleotide sequence selected from the group consisting of SEQ ID NOs:16 - 71 or comprises a promoter selected from the group consisting of a U6 promoter, an Hl promoter, a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a fteta-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EFla promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter a dihydrofolate reductase promoter
  • fteta-actin promoter a phosphoglycerol kinase (PGK) promoter
  • PGK phosphog
  • first transcription terminator, the second transcription terminator, and the third transcription terminator are each selected from the group consisting of a U6 poly-T terminator, an SV40 terminator, an hGH terminator, a BGH terminator, an rbGlob terminator, a synthetic terminator functional in a eukaryotic cell, a 3’ element from an Agrobacterium sp. gene, a 3’ element from a non-human animal gene, a 3’ element from a human gene, and a 3’ element from a plant gene, wherein the 3’ element terminate transcription of an RNA transcript located immediately 5’ to the 3’ element.
  • the engineered system of embodiment 70 wherein the first transcription terminator, the second transcription terminator, and the third transcription terminator are each a transcription terminator having a sequence selected from the group consisting of SEQ ID NOs:72 - 99, and 100.
  • the engineered system of any one of embodiments 49 - 78, wherein the Casl2a nuclease is:
  • a Casl2a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Casl2a nuclease; or
  • a Casl2a nuclease variant selected from the group consisting of (1) a variant LbCpfl that differs from a wild-type LbCpfl sequence (SEQ ID NO:135) in containing at least one point mutation selected from the group consisting of G309P, Y312F, M474I, D523N, Q531K, C930A, D937N, V954F/Q, M975L, A984E, I994L, T1006K, I1014V, V1055N/D, L1065F/Y, Y1180F, V1209G, I1229L, V801I, Y802I, D850A, E943A, and D1198A; (2) a variant AsCpfl that differs from a wild-type AsCpfl sequence (SEQ ID NO:134) in containing at least one point mutation selected from the group consisting of L320P, V980A, Q987N, T1004F/Q
  • PAM Protospacer Adjacent Motif
  • the Casl2a tracrRNA is provided as DNA encoding a first RNA molecule comprising the Casl2a tracrRNA and optionally comprising a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety, and wherein the functional RNA moiety is at least one selected from the group consisting of:
  • RNA sequence forming at least partially double -stranded RNA (g) an RNA sequence forming at least partially double -stranded RNA.
  • the Casl2a tracrRNA is provided as DNA encoding a first RNA molecule comprising the Casl2a tracrRNA and optionally comprising a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety, wherein the functional RNA moiety comprises an RNA sequence for annealing to a donor polynucleotide, and wherein the composition further comprises the donor polynucleotide.
  • RNA sequence forming at least partially double -stranded RNA (g) an RNA sequence forming at least partially double -stranded RNA.
  • a delivery particle comprising the engineered system of any one of embodiments 49, 50, 56, 57, 64, and 68.
  • the delivery particle of embodiment 94 wherein the Casl2a nuclease is complexed with the engineered Casl2a crRNA polynucleotide.
  • a delivery liquid comprising the engineered system of any one of embodiments 49, 50, 56, 57, 64, and 68.
  • the delivery liquid of embodiment 98, wherein the Casl2a tracrRNA is present in a molar amount that is at least 10-fold greater than the molar amount of the Casl2a nuclease. .
  • a method of modifying a target sequence in a locus of interest of a eukaryotic cell comprising delivering the engineered system according to any one of embodiments 49, 50, 56, 57, 64, and 68 to the locus of interest, wherein the spacer sequence hybridizes with the target sequence, whereby modification of the locus of interest occurs.
  • the method of embodiment 102 wherein the locus of interest is within a eukaryotic cell.
  • the eukaryotic cell is a plant cell.
  • the eukaryotic cell is a non-human animal cell or a human cell.
  • the method of embodiment 102 wherein the engineered system or a component thereof is delivered via delivery particles, delivery vesicles, delivery liquids, or one or more viral or bacterial vectors. .
  • the method of embodiment 102, wherein the engineered system or a component thereof is delivered via delivery exosomes or liposomes. .
  • invention 102 wherein the engineered system or a component thereof is delivered via at least one viral vector selected from the group consisting of adenoviruses, lentiviruses, adeno-associated viruses, retroviruses, geminiviruses, begomoviruses, tobamoviruses, potex viruses, comoviruses, wheat streak mosaic virus, barley stripe mosaic virus, bean yellow dwarf virus, bean pod mottle virus, cabbage leaf curl virus, beet curly top virus, tobacco yellow dwarf virus, tobacco rattle virus, potato virus X, and cowpea mosaic virus.
  • adenoviruses lentiviruses
  • adeno-associated viruses retroviruses
  • retroviruses geminiviruses
  • begomoviruses tobamoviruses
  • potex viruses comoviruses
  • wheat streak mosaic virus barley stripe mosaic virus
  • bean yellow dwarf virus bean pod mottle virus
  • cabbage leaf curl virus cabbage leaf curl virus
  • the method of embodiment 102 wherein the system or a component thereof is delivered via at least one bacterial vector capable of transforming a plant cell and selected from the group consisting of Agrobacterium sp., Rhizobium sp., Sinorhizobium (Ensifer) sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., and Phyllobacterium sp. .
  • the method of embodiment 102, wherein the Cas 12a nuclease is :
  • a Cas 12a nuclease selected from the group consisting of LbCasl2a, AsCasl2a, FnCasl2a, and a deactivated Cas 12a nuclease; or
  • a Casl2a nuclease variant selected from the group consisting of (1) a variant LbCpfl that differs from a wild-type LbCpfl sequence (SEQ ID NO:135) in containing at least one point mutation selected from the group consisting of G309P, Y312F, M474I, D523N, Q531K, C930A, D937N, V954F/Q, M975L, A984E, I994L, T1006K, I1014V, V1055N/D, L1065F/Y, Y1180F, V1209G, I1229L, V801I, Y802I, D850A, E943A, and D1198A; (2) a variant AsCpfl that differs from a wild-type AsCpfl sequence (SEQ ID NO:134) in containing at least one point mutation selected from the group consisting of L320P, V980A, Q987N, T1004F/Q
  • the Casl2a tracrRNA has a nucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOs:l, 3, 6, 139 - 146, 149 - 169, 223 - 240, and 241.
  • the tracrRNA is a putative Casl2a tracrRNA that is identified from the same genomic region as the Casl2a nuclease by at least the following steps:
  • the eukaryotic cell comprises a cell of a cell line or a cell of a multicellular organism, and wherein the locus of interest comprises a locus of interest in the genome of the eukaryotic cell.
  • the eukaryotic cell is in vitro, ex vivo, or in vivo.
  • the method of embodiment 102, wherein the eukaryotic cell comprises a non-human animal or human stem cell or a non-human animal or human stem cell line.
  • the method of embodiment 102, wherein the locus of interest is within a eukaryotic DNA molecule in vitro.
  • the eukaryotic cell comprises a plant cell, and wherein the modification of the locus of interest is correlated with a modified trait in a plant comprising cells containing the modification of the locus of interest. .
  • the method of embodiment 119 wherein the method further comprises obtaining, growing, or regenerating a plant from the plant cell, wherein the plant comprises cells containing the modification of the locus of interest, and wherein the plant exhibits the modified trait effected by the modification of the locus of interest.
  • the method of embodiment 102 wherein the eukaryotic cell comprises a plant cell, and the method further comprises identifying a trait of interest in a plant obtained, grown, or regenerated from the plant cell, wherein the trait of interest is effected by the modification of the locus of interest.
  • the method of embodiment 121 further comprising:
  • a Casl2a tracrRNA thereby editing the genetic locus; wherein the efficiency of editing of the genetic locus is increased compared to a control method in which the DNA that comprises the genetic locus is contacted with the Casl2a nuclease and the Casl2a crRNA, but not the Casl2a tracrRNA.
  • the method of embodiment 123 wherein the Casl2a nuclease and the engineered Casl2a crRNA are provided as a ribonucleoprotein complex.
  • the Casl2a nuclease is provided as a polynucleotide encoding the Casl2a nuclease.
  • the Casl2a tracrRNA is provided:
  • the eukaryotic cell is a cell of a non-human animal, an invertebrate, a vertebrate, a mollusk, an arthropod, an insect, a fish, a reptile, an amphibian, a bird, a mammal, a primate, a non-human primate, a human, a plant, or a fungus.
  • the eukaryotic cell comprises a cell of a cell line or a cell of a multicellular organism, and wherein the genetic locus to be modified comprises a locus of interest in the genome of the eukaryotic cell. .
  • the method of embodiment 129 wherein the eukaryotic cell is in vitro, ex vivo, or in vivo.
  • the method of embodiment 129 wherein the eukaryotic cell comprises a non-human animal or human stem cell or a non-human animal or human stem cell line.
  • the method of embodiment 134, wherein the putative Casl2a tracrRNA is identified from the same genomic region as the Casl2a nuclease by at least the following steps:
  • the method of embodiment 123 wherein the Casl2a tracrRNA is provided in a molar amount that is at least 10-fold greater than the molar amount of the Casl2a nuclease. .
  • the method of embodiment 123, wherein the engineered Casl2a crRNA, or a polynucleotide encoding the engineered Casl2a crRNA, further comprises at least one direct repeat sequence or fragment thereof capable of associating with the Casl2a nuclease adjacent to and 3’ to the spacer sequence.
  • the method of embodiment 123, wherein the engineered Casl2a crRNA further comprises a 3’ extension adjacent to the spacer sequence. .
  • the 3’ extension comprises nucleotides that when basepaired form about one helical turn. .
  • the Casl2a tracrRNA has a nucleotide sequence having sufficient complementarity to the engineered Casl2a crRNA’ s 3’ extension to allow hybridization between the tracrRNA and the engineered Casl2a crRNA; (ii) wherein the Casl2a tracrRNA is covalently linked to the engineered Casl2a crRNA; or (iii) wherein the DNA encodes an RNA molecule wherein the Casl2a tracrRNA is covalently linked to the engineered Casl2a crRNA.
  • a composition for modifying a genetic locus in a eukaryotic cell comprising:
  • composition of embodiment 146 wherein the Casl2a tracrRNA is identified from the same genomic region as the Casl2a nuclease by at least the following steps:
  • composition of any one of embodiments 146 to 150 wherein the eukaryotic cell comprises a cell of a cell line or a cell of a multicellular organism, and wherein the genetic locus to be modified comprises a locus of interest in the genome of the eukaryotic cell.
  • composition of any one of embodiments 146 to 153, wherein the Casl2a tracrRNA is provided: (a) as DNA encoding a first RNA molecule comprising the Casl2a tracrRNA and optionally comprising a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety; or
  • RNA sequence forming at least partially double -stranded RNA (g) an RNA sequence forming at least partially double -stranded RNA.
  • RNA aptamer (d) an RNA aptamer; (e) an enzymatically active RNA sequence;
  • RNA sequence forming at least partially double -stranded RNA (g) an RNA sequence forming at least partially double -stranded RNA.
  • compositions of any one of embodiments 146 to 160 for the manufacture of a medicament for treatment of a genetic disease, cancer, a viral infection, a bacterial infection, a parasitic infection, a fungal infection, or a protozoan infection, wherein the composition is optionally in a pharmaceutically acceptable excipient and/or optionally delivered via particles, vesicles, or one or more viral vectors, optionally wherein the particles comprise a lipid, a sugar, a metal or a protein, optionally wherein the vesicles comprise exosomes or liposomes optionally wherein the one or more viral vectors comprise one or more of adenovirus, one or more lentivirus or one or more adeno-associated virus. .
  • a method of producing a eukaryotic cell comprising a modification of a locus of interest comprising (i) delivering the composition of any one of embodiments 146 to 160 to the locus of interest, wherein the spacer sequence hybridizes with a target sequence located in the locus of interest of the eukaryotic cell and whereby the composition introduces the modification into the locus of interest, and optionally wherein either: (i) the locus of interest is genomic nuclear DNA of a non-human animal, a plant, a fungus, or DNA of a non-human eukaryotic organelle; or (ii) wherein the locus of interest is genomic nuclear human DNA, and wherein the method is not a method of treatment of the human or animal body by surgery or therapy or a method of modifying the germ line genetic identity of human beings and wherein the cell is not a human embryo.
  • the method of embodiment 163, further comprising isolating the eukaryotic cell comprising a modification of a locus of interest. .
  • the method of embodiment 163 or 164 further comprising propagating the eukaryotic cell comprising the modification of the locus of interest. .
  • the method of embodiment 163, 164, or 165 further comprising introducing the eukaryotic cell comprising a modification of a locus of interest into a host organism. .
  • the method of embodiment 163, 164, or 165 further comprising regenerating the eukaryotic cell comprising a modification of a locus of interest into an organism comprising the modification of the locus of interest. .
  • the method of embodiment 167 wherein the organism is a plant, optionally wherein the plant is a maize, soybean, wheat, rice, cotton, or Brassica sp. plant, and optionally further comprising propagating the plant or harvesting seed therefrom. .
  • the eukaryotic cell is an isolated cell an in vitro axenic culture.
  • the method of embodiment 171 wherein the isolated cell is a human cell.
  • a cell comprising the DNA expression system of any one of embodiments 18 to 48, the engineered system of any one of embodiments 49 to 93, or composition of any one of embodiments 146 to 160, wherein the cell is a eukaryotic cell.
  • the cell of embodiment 178 wherein the isolated cell is a human cell, optionally wherein the human cell is an induced pluripotent stem cell.
  • a method of modifying a target sequence in a locus of interest of a eukaryotic cell comprising delivering the engineered system according to any one of embodiments 49 to 93 or delivering the composition of any one of embodiments 146 to 160 to the locus of interest, wherein the spacer sequence hybridizes with the target sequence, whereby modification of the locus of interest occurs at a greater frequency compared to a control method, wherein a control engineered system identical to the engineered system except for the absence of the Casl2a tracrRNA is delivered to the locus of interest.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention propose des compositions comprenant un polynucléotide incluant ou codant pour un ARNtracr Cas12a, ainsi que des procédés pour leur utilisation dans l'édition génomique spécifique d'une séquence, particulièrement de séquences génomiques eucaryotes. Plus particulièrement, la présente invention concerne des compositions contenant un ARNtracr Cas12a et des procédés pour leur utilisation dans l'édition médiée par Cas12a d'une séquence cible, l'efficacité de l'édition étant accrue par comparaison avec des témoins dépourvus de l'ARNtracr Cas12a.
PCT/US2022/073286 2022-06-30 2022-06-30 Compositions, systèmes et procédés d'édition génomique WO2024005864A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2022/073286 WO2024005864A1 (fr) 2022-06-30 2022-06-30 Compositions, systèmes et procédés d'édition génomique

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2022/073286 WO2024005864A1 (fr) 2022-06-30 2022-06-30 Compositions, systèmes et procédés d'édition génomique

Publications (1)

Publication Number Publication Date
WO2024005864A1 true WO2024005864A1 (fr) 2024-01-04

Family

ID=83082030

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/073286 WO2024005864A1 (fr) 2022-06-30 2022-06-30 Compositions, systèmes et procédés d'édition génomique

Country Status (1)

Country Link
WO (1) WO2024005864A1 (fr)

Citations (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5188642A (en) 1985-08-07 1993-02-23 Monsanto Company Glyphosate-resistant plants
WO1993003769A1 (fr) 1991-08-20 1993-03-04 THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Transfert induit par adenovirus de genes vers la voie gastro-intestinale
WO1993009239A1 (fr) 1991-11-08 1993-05-13 Research Corporation Technologies, Inc. Vecteurs basaux du virus 2 adeno-apparente
WO1993019191A1 (fr) 1992-03-16 1993-09-30 Centre National De La Recherche Scientifique Adenovirus recombinants defectifs exprimant des cytokines pour traitement antitumoral
WO1994012649A2 (fr) 1992-12-03 1994-06-09 Genzyme Corporation Therapie genique de la fibrose kystique
US5322938A (en) 1987-01-13 1994-06-21 Monsanto Company DNA sequence for enhancing the efficiency of transcription
WO1994028938A1 (fr) 1993-06-07 1994-12-22 The Regents Of The University Of Michigan Vecteurs d'adenovirus pour therapie genique
WO1995000655A1 (fr) 1993-06-24 1995-01-05 Mc Master University Vecteurs a base d'adenovirus destines a la therapie genique
WO1995011984A2 (fr) 1993-10-25 1995-05-04 Canji, Inc. Vecteur recombinant d'adenovirus et procedes d'utilisation
US5641876A (en) 1990-01-05 1997-06-24 Cornell Research Foundation, Inc. Rice actin gene and promoter
US5728925A (en) 1984-12-28 1998-03-17 Plant Genetic Systems, N.V. Chimaeric gene coding for a transit peptide and a heterologous polypeptide
US5858742A (en) 1983-01-17 1999-01-12 Monsanto Company Chimeric genes for transforming plant cells using viral promoters
US6015891A (en) 1988-09-09 2000-01-18 Mycogen Plant Science, Inc. Synthetic insecticidal crystal protein gene having a modified frequency of codon usage
US6090627A (en) 1983-04-15 2000-07-18 Mycogen Plant Science, Inc. Octopine T-DNA structural genes
US20020192813A1 (en) 1999-08-18 2002-12-19 Timothy W. Conner Plant expression vectors
US7151204B2 (en) 2001-01-09 2006-12-19 Monsanto Technology Llc Maize chloroplast aldolase promoter compositions and methods for use thereof
US20100311168A1 (en) 2009-04-07 2010-12-09 Dow Agrosciences Llc Nanoparticle mediated delivery of sequence specific nucleases
US20110093982A1 (en) 2009-10-16 2011-04-21 Dow Agrosciences Llc Use of dendrimer nanotechnology for delivery of biomolecules into plant cells
US20110247100A1 (en) 2010-03-31 2011-10-06 Dow Agrosciences Llc Plant peptide gamma-zein for delivery of biomolecules into plant cells
US20120023619A1 (en) 2010-07-07 2012-01-26 Dow Agrosciences Llc Linear dna molecule delivery using pegylated quantum dots for stable trasformation in plants
US20120244569A1 (en) 2011-03-23 2012-09-27 Dow Agrosciences Llc Quantum dot carrier peptide conjugates suitable for imaging and delivery applications in plants
US8420888B2 (en) 2007-02-26 2013-04-16 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of DMO and uses thereof
US20130145488A1 (en) 2011-12-06 2013-06-06 Iowa State University Research Foundation, Inc. Mesoporous silica nanoparticles suitable for co-delivery
US20130185823A1 (en) 2012-01-16 2013-07-18 Academia Sinica Mesoporous silica nanoparticle-mediated delivery of dna into arabidopsis root
US20140096284A1 (en) 2012-10-01 2014-04-03 Iowa State University Research Foundation, Inc. Method for the delivery of molecules lyophilized onto microparticles to plant tissues
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8795965B2 (en) 2012-12-12 2014-08-05 The Broad Institute, Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US20140287509A1 (en) 2011-10-17 2014-09-25 Massachusetts Institute Of Technology Intracellular Delivery
US8865406B2 (en) 2012-12-12 2014-10-21 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
US8889356B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US20140356414A1 (en) 2013-06-03 2014-12-04 University Of Southern California Targeted Crosslinked Multilamellar Liposomes
US8906616B2 (en) 2012-12-12 2014-12-09 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
US20150040268A1 (en) 2013-04-25 2015-02-05 Morflora Israel Ltd Methods and compositions for the delivery of nucleic acids to seeds
US20150038767A1 (en) 2007-01-16 2015-02-05 Radiadyne, Llc Endorectal balloon with gas lumen and stopper
US20150047074A1 (en) 2013-08-09 2015-02-12 Massachusetts Institute Of Technology Nanobionic engineering of organelles and photosynthetic organisms
US20150059010A1 (en) 2013-08-22 2015-02-26 Pioneer Hi-Bred International Inc Genome modification using guide polynucleotide/cas endonuclease systems and methods of use
US8993233B2 (en) 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US20150208663A1 (en) 2013-10-15 2015-07-30 Board Of Trustees Of The University Of Arkansas Method of delivery of bio-active agents to plant cells by using nano-sized materials as carriers
WO2015131101A1 (fr) 2014-02-27 2015-09-03 Monsanto Technology Llc Compositions et procédés de modification génomique dirigée
US20150344912A1 (en) 2012-10-23 2015-12-03 Toolgen Incorporated Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US9222100B2 (en) 2010-08-24 2015-12-29 Monsanto Technology Llc Methods and DNA constructs for autoregulating transgene silencing
WO2016007347A1 (fr) 2014-07-11 2016-01-14 E. I. Du Pont De Nemours And Company Compositions et procédés de production de plantes résistantes à un herbicide, le glyphosate
US20160138008A1 (en) 2012-05-25 2016-05-19 The Regents Of The University Of California Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
WO2016166340A1 (fr) 2015-04-16 2016-10-20 Wageningen Universiteit Édition du génome médiée par une nucléase
US9490490B2 (en) 2013-05-30 2016-11-08 Bloom Energy Corporation Measurement device and method for determining fuel cell stack size variations
WO2016205711A1 (fr) * 2015-06-18 2016-12-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes
WO2017189308A1 (fr) * 2016-04-19 2017-11-02 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes associés
US9896696B2 (en) 2016-02-15 2018-02-20 Benson Hill Biosystems, Inc. Compositions and methods for modifying genomes
WO2018140899A1 (fr) 2017-01-28 2018-08-02 Inari Agriculture, Inc. Nouvelles cellules végétales, plantes et semences
WO2018208755A1 (fr) * 2017-05-09 2018-11-15 The Regents Of The University Of California Compositions et procédés d'étiquetage de protéines cibles à proximité d'une séquence de nucléotides d'intérêt
US10227110B1 (en) 2016-04-29 2019-03-12 Bombardier Recreational Products Inc. Retractable anchor fixtures
WO2020240523A1 (fr) * 2019-05-31 2020-12-03 The Governing Council Of The University Of Toronto Procédés et compositions pour l'édition de gènes multiplex
WO2021154866A1 (fr) * 2020-01-27 2021-08-05 Sherlock Biosciences Dosages de détection améliorés
WO2022159402A1 (fr) * 2021-01-19 2022-07-28 The Board Of Trustees Of The Leland Stanford Junior University Composition et procédé d'ingénierie génomique à multiplexage élevé à l'aide de réseaux crispr synthétiques

Patent Citations (67)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5858742A (en) 1983-01-17 1999-01-12 Monsanto Company Chimeric genes for transforming plant cells using viral promoters
US6090627A (en) 1983-04-15 2000-07-18 Mycogen Plant Science, Inc. Octopine T-DNA structural genes
US5728925A (en) 1984-12-28 1998-03-17 Plant Genetic Systems, N.V. Chimaeric gene coding for a transit peptide and a heterologous polypeptide
US5188642A (en) 1985-08-07 1993-02-23 Monsanto Company Glyphosate-resistant plants
US5322938A (en) 1987-01-13 1994-06-21 Monsanto Company DNA sequence for enhancing the efficiency of transcription
US6015891A (en) 1988-09-09 2000-01-18 Mycogen Plant Science, Inc. Synthetic insecticidal crystal protein gene having a modified frequency of codon usage
US5641876A (en) 1990-01-05 1997-06-24 Cornell Research Foundation, Inc. Rice actin gene and promoter
WO1993003769A1 (fr) 1991-08-20 1993-03-04 THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Transfert induit par adenovirus de genes vers la voie gastro-intestinale
WO1993009239A1 (fr) 1991-11-08 1993-05-13 Research Corporation Technologies, Inc. Vecteurs basaux du virus 2 adeno-apparente
WO1993019191A1 (fr) 1992-03-16 1993-09-30 Centre National De La Recherche Scientifique Adenovirus recombinants defectifs exprimant des cytokines pour traitement antitumoral
WO1994012649A2 (fr) 1992-12-03 1994-06-09 Genzyme Corporation Therapie genique de la fibrose kystique
WO1994028938A1 (fr) 1993-06-07 1994-12-22 The Regents Of The University Of Michigan Vecteurs d'adenovirus pour therapie genique
WO1995000655A1 (fr) 1993-06-24 1995-01-05 Mc Master University Vecteurs a base d'adenovirus destines a la therapie genique
WO1995011984A2 (fr) 1993-10-25 1995-05-04 Canji, Inc. Vecteur recombinant d'adenovirus et procedes d'utilisation
US20020192813A1 (en) 1999-08-18 2002-12-19 Timothy W. Conner Plant expression vectors
US7151204B2 (en) 2001-01-09 2006-12-19 Monsanto Technology Llc Maize chloroplast aldolase promoter compositions and methods for use thereof
US20150038767A1 (en) 2007-01-16 2015-02-05 Radiadyne, Llc Endorectal balloon with gas lumen and stopper
US8420888B2 (en) 2007-02-26 2013-04-16 Monsanto Technology Llc Chloroplast transit peptides for efficient targeting of DMO and uses thereof
US20100311168A1 (en) 2009-04-07 2010-12-09 Dow Agrosciences Llc Nanoparticle mediated delivery of sequence specific nucleases
US20110093982A1 (en) 2009-10-16 2011-04-21 Dow Agrosciences Llc Use of dendrimer nanotechnology for delivery of biomolecules into plant cells
US20110247100A1 (en) 2010-03-31 2011-10-06 Dow Agrosciences Llc Plant peptide gamma-zein for delivery of biomolecules into plant cells
US20120023619A1 (en) 2010-07-07 2012-01-26 Dow Agrosciences Llc Linear dna molecule delivery using pegylated quantum dots for stable trasformation in plants
US9222100B2 (en) 2010-08-24 2015-12-29 Monsanto Technology Llc Methods and DNA constructs for autoregulating transgene silencing
US20120244569A1 (en) 2011-03-23 2012-09-27 Dow Agrosciences Llc Quantum dot carrier peptide conjugates suitable for imaging and delivery applications in plants
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US20140287509A1 (en) 2011-10-17 2014-09-25 Massachusetts Institute Of Technology Intracellular Delivery
US20130145488A1 (en) 2011-12-06 2013-06-06 Iowa State University Research Foundation, Inc. Mesoporous silica nanoparticles suitable for co-delivery
US20130185823A1 (en) 2012-01-16 2013-07-18 Academia Sinica Mesoporous silica nanoparticle-mediated delivery of dna into arabidopsis root
US20160138008A1 (en) 2012-05-25 2016-05-19 The Regents Of The University Of California Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US20140096284A1 (en) 2012-10-01 2014-04-03 Iowa State University Research Foundation, Inc. Method for the delivery of molecules lyophilized onto microparticles to plant tissues
US20150344912A1 (en) 2012-10-23 2015-12-03 Toolgen Incorporated Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US8871445B2 (en) 2012-12-12 2014-10-28 The Broad Institute Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8906616B2 (en) 2012-12-12 2014-12-09 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
US8795965B2 (en) 2012-12-12 2014-08-05 The Broad Institute, Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8865406B2 (en) 2012-12-12 2014-10-21 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
US8889418B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
US8932814B2 (en) 2012-12-12 2015-01-13 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US8945839B2 (en) 2012-12-12 2015-02-03 The Broad Institute Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8771945B1 (en) 2012-12-12 2014-07-08 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8895308B1 (en) 2012-12-12 2014-11-25 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
US8889356B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US8999641B2 (en) 2012-12-12 2015-04-07 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8993233B2 (en) 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US20150040268A1 (en) 2013-04-25 2015-02-05 Morflora Israel Ltd Methods and compositions for the delivery of nucleic acids to seeds
US9490490B2 (en) 2013-05-30 2016-11-08 Bloom Energy Corporation Measurement device and method for determining fuel cell stack size variations
US20140356414A1 (en) 2013-06-03 2014-12-04 University Of Southern California Targeted Crosslinked Multilamellar Liposomes
US20150047074A1 (en) 2013-08-09 2015-02-12 Massachusetts Institute Of Technology Nanobionic engineering of organelles and photosynthetic organisms
US20150059010A1 (en) 2013-08-22 2015-02-26 Pioneer Hi-Bred International Inc Genome modification using guide polynucleotide/cas endonuclease systems and methods of use
US20150082478A1 (en) 2013-08-22 2015-03-19 E I Du Pont De Nemours And Company Plant genome modification using guide rna/cas endonuclease systems and methods of use
US20150208663A1 (en) 2013-10-15 2015-07-30 Board Of Trustees Of The University Of Arkansas Method of delivery of bio-active agents to plant cells by using nano-sized materials as carriers
WO2015131101A1 (fr) 2014-02-27 2015-09-03 Monsanto Technology Llc Compositions et procédés de modification génomique dirigée
US20170166912A1 (en) 2014-02-27 2017-06-15 Monsanto Technology Llc Compositions and methods for site directed genomic modification
WO2016007347A1 (fr) 2014-07-11 2016-01-14 E. I. Du Pont De Nemours And Company Compositions et procédés de production de plantes résistantes à un herbicide, le glyphosate
US20170306349A1 (en) 2014-07-11 2017-10-26 E I Du Pont De Nemours And Company Compositions and methods for producing plants resistant to glyphosate herbicide
US20180282713A1 (en) 2015-04-16 2018-10-04 Wageningen Universiteit Nuclease-mediated genome editing
WO2016166340A1 (fr) 2015-04-16 2016-10-20 Wageningen Universiteit Édition du génome médiée par une nucléase
US9790490B2 (en) 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
WO2016205711A1 (fr) * 2015-06-18 2016-12-22 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes
US9896696B2 (en) 2016-02-15 2018-02-20 Benson Hill Biosystems, Inc. Compositions and methods for modifying genomes
WO2017189308A1 (fr) * 2016-04-19 2017-11-02 The Broad Institute Inc. Nouvelles enzymes crispr et systèmes associés
US10227110B1 (en) 2016-04-29 2019-03-12 Bombardier Recreational Products Inc. Retractable anchor fixtures
WO2018140899A1 (fr) 2017-01-28 2018-08-02 Inari Agriculture, Inc. Nouvelles cellules végétales, plantes et semences
WO2018208755A1 (fr) * 2017-05-09 2018-11-15 The Regents Of The University Of California Compositions et procédés d'étiquetage de protéines cibles à proximité d'une séquence de nucléotides d'intérêt
WO2020240523A1 (fr) * 2019-05-31 2020-12-03 The Governing Council Of The University Of Toronto Procédés et compositions pour l'édition de gènes multiplex
WO2021154866A1 (fr) * 2020-01-27 2021-08-05 Sherlock Biosciences Dosages de détection améliorés
WO2022159402A1 (fr) * 2021-01-19 2022-07-28 The Board Of Trustees Of The Leland Stanford Junior University Composition et procédé d'ingénierie génomique à multiplexage élevé à l'aide de réseaux crispr synthétiques

Non-Patent Citations (55)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. KIM 12007
"Methods in Enzymology", vol. 266, 1996, ACADEMIC PRESS, INC., article "Computer Methods for Macromolecular Sequence Analysis"
ALI ET AL., HUM GENE THER, vol. 9, no. 81, 1998, pages 86
ALI ET AL., HUM MOL GENET, vol. 5, no. 591, 1996, pages 594
ALTSCHUL ET AL., MOL. BIOL., vol. 215, 1990, pages 403 - 410
BANDYOPADHYAY ANINDYA ET AL: "CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement", FRONTIERS IN PLANT SCIENCE, vol. 11, 2 November 2020 (2020-11-02), XP093004959, DOI: 10.3389/fpls.2020.584151 *
BEGEMANN ET AL., BIORXIV, 2017
BENNETT ET AL., INVEST OPTHALMOL VIS SCI, vol. 38, no. 2857, 1997, pages 2863
BORRAS ET AL., GENE THER, vol. 6, no. 515, 1999, pages 524
BROOTHAERTS ET AL., NATURE, vol. 433, 2005, pages 629 - 633
BURSTEIN ET AL., NATURE, 2016
CAS, no. 27306-78-1
CHOI ET AL., J. CONTROLLED RELEASE, vol. 235, 2016, pages 222 - 235
CONG ET AL., SCIENCE, vol. 339, 2013, pages 819 - 823
DI TOMASSO ET AL., NUCLEIC ACIDS RES., vol. 39, no. 3, 2011, pages 5284 - 5298
FLANNERY ET AL., PNAS, vol. 94, no. 6916, 1997, pages 10319
FLOTTE ET AL., PNAS, vol. 90, 1993, pages 10613 - 10617
GIRALDO ET AL., NATURE MATERIALS, vol. 13, 2014, pages 400 - 409
GOMEZ ET AL., PLANT SIGNAL BEHAV., vol. 5, 2010, pages 1517 - 1519
HENDEL ET AL., NATURE BIOTECHNOL., vol. 33, 2015, pages 985 - 991
JARVER, MOL. THERAPY-NUCLEIC ACIDS,, vol. l, no. e27, 2012, pages l - 17
JOMARY ET AL., GENE THER, vol. 4, no. 683, 1997, pages 690
JORES, NATURE COMMUNICATIONS, vol. 7, 2016, pages 12036 - 12051
KEIFTBATEY, RNA, vol. 10, no. 6, 2004, pages 988 - 995
KIM, BIOCONJUGATE CHEM., vol. 22, 2011, pages 2558 - 2567
LEE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 110, no. 14, 2013, pages 5416 - 5421
LEPPEKSTOECKLIN, NUCLEIC ACIDS RES., vol. 452, no. 2, 2014, pages e13
LI ET AL., INVEST OPTHALMOL VIS SCI, vol. 35, no. 2543, 1994, pages 2549
LIANG ET AL., NATURE COMMUNICATIONS, vol. 8, 2017, pages 14261
LIDAVIDSON, PNAS, vol. 92, no. 7700, 1995, pages 7704
LIU ET AL., FRONTIERS IMMUNOL., vol. 10, pages 456
LU ET AL., J. AGRIC. FOOD CHEM., vol. 58, 2010, pages 2288 - 2294
MENDELSON ET AL., VIROL, vol. 166, 1988, pages 154 - 165
METH. MOL. BIOL.,, vol. 70, 1997, pages 173 - 187
MOL. BIOL., vol. 48, 1970, pages 443 - 453
O'BRIANLUMMIS: "11", BMC BIOTECHNOL., no. 66, 2011, pages 71
PARK ET AL., NATURE COMMUNICATIONS, 2018
PARROTT ET AL., NUCLEIC ACIDS RES., vol. 28, no. 2, 2000, pages 489 - 497
QUAX ET AL., MOLECULAR CELL, vol. 59, 2015, pages 149 - 161
RAN ET AL., NATURE PROTOCOLS, vol. 8, 2013, pages 2281 - 2308
ROLLING ET AL., HUM GENE THER, vol. 10, no. 641, 1999, pages 648
SAKAMOTO ET AL., H GENE THER, vol. 5, no. 1088, 1999, pages 1097
SAMULSKI ET AL., J. VIR., vol. 63, 1989, pages 3822 - 3828
SHEN ET AL., THERANOSTICS, vol. 2, 2012, pages 283 - 294
SHMAKOV ET AL., MOL. CELL, vol. 60, 2015, pages 385 - 397
SRISAWATENGELKE, METHODS, vol. 26, no. 2, 2002, pages 156 - 161
TAKAHASHI ET AL., J VIROL, vol. 73, no. 7812, 1999, pages 7816
UNNAMALAI ET AL., FEBS LETTERS, vol. 566, 2004, pages 307 - 310
VERMAECKSTEIN, ANNU. REV. BIOCHEM., vol. 67, 1998, pages 99 - 134
WANG ET AL., J. AM. CHEM. SOC. COMM., vol. 132, 2010, pages 9274 - 9276
WONG ET AL., NANO LETT., vol. 16, 2016, pages 1161 - 1172
ZETSCHE ET AL., CELL, vol. 163, 2015, pages 759 - 771
ZHANG ET AL., J. CONTROLLED RELEASE,, vol. 123, 2007, pages 1 - 10
ZHAO ET AL., NANOSCALE RES. LETT., vol. 11, 2016, pages 195 - 203
ZHENGBAUM, METHODS MOL. BIOL., vol. 434, 2008, pages 205 - 219

Similar Documents

Publication Publication Date Title
EP3320092B1 (fr) Compositions de crispr-cas9 manipulées et procédés d'utilisation
US11634722B2 (en) Plant gene editing systems, methods, and compositions
JP2022028812A (ja) 肝臓ターゲティングおよび治療のためのＣＲＩＳＰＲ－Ｃａｓ系、ベクターおよび組成物の送達および使用
CN110959039A (zh) 新型cas13b直向同源物crispr酶和系统
US20160362667A1 (en) CRISPR-Cas Compositions and Methods
CN104080462A (zh) 用于修饰预定的靶核酸序列的组合物和方法
US20230235349A1 (en) Novel maize cells and maize plants
US11976291B2 (en) Genetically enhanced maize plants
US20220333101A1 (en) Compositions, systems, and methods for genome editing
EP4299739A1 (fr) Compositions, systèmes et procédés d'édition de génomes
EP4299733A1 (fr) Compositions, systèmes et procédés pour l'édition de génomes
US20220372524A1 (en) Compositions, systems, and methods for genome editing
US11459551B1 (en) Compositions, systems, and methods for genome editing
WO2024005864A1 (fr) Compositions, systèmes et procédés d'édition génomique
WO2024005863A1 (fr) Compositions, systèmes et procédés d'édition génomique
JP2024501892A (ja) 新規の核酸誘導型ヌクレアーゼ
US11859219B1 (en) Methods of altering a target nucleotide sequence with an RNA-guided nuclease and a single guide RNA
US20230022635A1 (en) Delivery of biological molecules to plant cells
US20220195446A1 (en) Plant transformation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22760851

Country of ref document: EP

Kind code of ref document: A1