WO2016205623A1 - Méthodes et compositions pour l'édition de génome dans des bactéries à l'aide de systèmes cas9-crispr - Google Patents

Méthodes et compositions pour l'édition de génome dans des bactéries à l'aide de systèmes cas9-crispr Download PDF

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WO2016205623A1
WO2016205623A1 PCT/US2016/038046 US2016038046W WO2016205623A1 WO 2016205623 A1 WO2016205623 A1 WO 2016205623A1 US 2016038046 W US2016038046 W US 2016038046W WO 2016205623 A1 WO2016205623 A1 WO 2016205623A1
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bacterium
genome
target dna
nucleic acid
region
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Chase BEISEL
Rodolphe Barrangou
Kurt M. SELLE
Michelle Luo
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North Carolina State University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the invention relates to the methods and compositions for genome editing in bacteria using CRISPR-Cas9 technologies as well as non-homologous end joining.
  • CRISPR-Cas9 has proven to be a powerful tool for genome editing (Hsu et al. Cell 157, 1262-1278 (2014)).
  • the CRISPR RNA guides the Cas9 endoribonuclease to complementary sequences flanked by a protospacer-adjacent motif, resulting in Cas9- mediated DNA cleavage.
  • the targeting RNA can be generated either from the native CRISPR array and the tracrRNA, or from a CRISPR RNA:tracrRNA hybrid called a single-guide RNA (sgRNA). In all forms of life, irreparable double-stranded breaks are lethal.
  • NHEJ non-homologous end-joining
  • HDR homology-directed repair
  • Indels random insertions and deletions
  • HDR homologous DNA is used as a template for the precise repair of the cleaved DNA; non-native sequences can be introduced as long as the ends of the DNA match the target location.
  • Both pathways have been exploited for multiplexed gene disruptions through the introduction of indels and precise editing through the use of a repair template with user-defined sequences in the middle. Because these pathways are highly efficient, DNA cleavage by CRISPR-Cas9 actively drives editing.
  • CRISPR-Cas9 targeting the genome with CRISPR-Cas9 is known to be lethal. It is likely that CRISPR has to be lethal because CRISPR naturally targets plasmids and bacteriophages and any repair of the targeted DNA may allow the invader to persist. By extension, targeting the genome would also not be repaired, resulting in lethality.
  • Each CRISPR-Cas system likely evolved means of blocking repair, whether by tightly binding both cleaved strands (Type II/Cas9) or by degrading the DNA through the action of a 3 " -to- 5' exonuclease (Type I). This effect has direct implications for genome editing with CRISPR-Cas9 in bacteria.
  • Cas9 kills any cells that did not undergo editing, thereby selecting for pre-existing mutants harboring the alteration.
  • genome editing in its current form relies on high transformation efficiencies and high rates of recombination. Otherwise, no bacterial cells would be recovered.
  • the present invention overcomes previous shortcomings in the art by providing compositions and methods for genome editing in bacteria using CRISPR-Cas9 technologies that circumvents the lethality of genome targeting.
  • a recombinant nucleic acid construct comprising a synthetic
  • CRISPR array a polynucleotide encoding an ATP-dependent DNA ligase D (LigD) and a polynucleotide encoding a DNA-end-binding protein Ku, optionally a polynucleotide encoding a recombinase.
  • LigD ATP-dependent DNA ligase D
  • Ku DNA-end-binding protein Ku
  • a recombinant nucleic acid construct comprising a synthetic CRISPR array, a tracr nucleic acid, a polynucleotide encoding an ATP-dependent DNA ligase D (LigD), a polynucleotide encoding a DNA-end-binding protein Ku, and a polynucleotide encoding a Cas9 nuclease, optionally wherein the CRISPR array and the tracr nucleic acid are fused to form a single guide nucleic acid.
  • LigD ATP-dependent DNA ligase D
  • Ku DNA-end-binding protein Ku
  • Cas9 nuclease optionally wherein the CRISPR array and the tracr nucleic acid are fused to form a single guide nucleic acid.
  • a further aspect of the invention provides a method of editing a target DNA in the genome of a bacterium, comprising introducing into a bacterium a synthetic CRISPR array, a polynucleotide encoding an ATP-dependent DNA ligase D (LigD) and a polynucleotide encoding a DNA-end-binding protein Ku (e.g., a recombinant nucleic acid construct of the invention), wherein the CRISPR array comprises at least one spacer having substantial complementarity to a target DNA in the genome of the bacterium, thereby editing the target DNA in the genome of the bacterium.
  • a synthetic CRISPR array comprising introducing into a bacterium a synthetic CRISPR array, a polynucleotide encoding an ATP-dependent DNA ligase D (LigD) and a polynucleotide encoding a DNA-end-binding protein Ku (e.g., a re
  • a method of editing a target DNA in the genome of a bacterium comprising introducing into a bacterium a synthetic CRISPR array, a tracr nucleic acid, a polynucleotide encoding an ATP-dependent DNA ligase D (LigD), a polynucleotide encoding a DNA-end-binding protein Ku, a polynucleotide encoding a Cas9 nuclease, and optionally a polynucleotide encoding a recombinase (e.g., a recombinant nucleic acid construct of the invention), optionally wherein the CRISPR array and the tracr nucleic acid can be fused to form a single guide nucleic acid, wherein the CRISPR array comprises at least one spacer having substantial complementarity to a target DNA in the genome of the bacterium, thereby editing the target DNA in the genome of the bacterium.
  • the CRISPR array
  • a method of editing a target DNA in the genome of a bacterium comprising introducing into a bacterium a synthetic CRISPR array and a polynucleotide encoding a Cas9 nuclease, wherein the Cas9 nuclease comprises a modified protospacer adjacent motif (PAM)-interacting domain (PIM) and the Cas9 having the modified PIM releases cleaved DNA more readily than a Cas9 nuclease without said modification, thereby editing a target DNA in the genome of the bacterium.
  • PAM protospacer adjacent motif
  • Another aspect of the invention provides a method of editing a target DNA in the genome of a bacterium, comprising introducing into a bacterium a synthetic CRISPR array, wherein the synthetic CRISPR array comprises at least one spacer having substantial complementarity to a target DNA in the genome of the bacterium, and the target DNA is adjacent to a sub-optimal protospacer adjacent motif (PAM), thereby editing the target DNA in the genome of the bacterium.
  • PAM sub-optimal protospacer adjacent motif
  • a method of editing a target DNA in the genome of a bacterium comprising introducing into a bacterium a synthetic CRISPR array, wherein the CRISPR array comprises at least one spacer having complementarity to a target DNA in the genome of the bacterium and said at least one spacer of the CRISPR array comprises a 5' region and a 3' region and the 5' region of said at least one spacer comprises about 50%
  • complementarity to about 99% complementarity to the target DNA in the genome of the bacterium and the 3' region of said at least one spacer comprises about 75% complementarity to about 99% complementarity to the target DNA in the genome of the bacterium, thereby editing the target DNA in the genome of the bacterium.
  • a further aspect of the invention provides a method of editing a target DNA in the genome of a bacterium, comprising introducing into a bacterium a synthetic CRISPR array, wherein the CRISPR array comprises at least one spacer having substantial complementarity to a target DNA in the genome of the bacterium, and the at least one spacer of the CRISPR array comprises a 5' region (non-seed) and a 3' region (seed) and the 5' region of the at least one spacer comprises about 10 consecutive nucleotides having about one to about five nucleotides that are non-complementary to the target DNA (i.e., non-complementary to the 3' region of the protospacer (having a 5' region and a 3' region)) in the genome of the bacterium and the 3' region of the at least one spacer comprises 100% complementary to the target DNA (i.e., 100% complementary to the 5' region of the protospacer) in the genome of the bacterium, thereby editing the target DNA in
  • expression cassettes and cells comprising the recombinant nucleic acid constructs, CRISPR arrays, and/or polynucleotides of the invention.
  • Fig. 1 provides the general experimental design for determining the effect of Ku and LigD on the ability of the transformed bacteria to repair damage done with CRISPR Cas9 (Example 1).
  • Fig. 2 shows an exemplary spacer sequence (SEQ I NO:l) that was used in Example
  • Fig. 3 provides the killing efficiency of the bacteria transformed with Ku and LigD and Cas9 as compared to bacteria transformed only with Cas9 (Example 1)
  • phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y.
  • phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”
  • chimeric refers to a nucleic acid molecule or a polypeptide in which at least two components are derived from different sources (e.g., different organisms, different coding regions).
  • Complement as used herein can mean 100% complementarity or identity with the comparator nucleotide sequence or it can mean less than 100% complementarity (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like, complementarity).
  • Complement may also be used in terms of a “complement” to or “complementing" a mutation.
  • complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • sequence ⁇ -G-T
  • sequence binds to the complementary sequence "T-C-A.”
  • Complementarity between two single-stranded molecules may be "partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • contact refers to placing the components of a desired reaction together under conditions suitable for carrying out the desired reaction (e.g., integration, transformation, screening, selecting, killing, identifying, amplifying, and the like).
  • conditions suitable for carrying out the desired reaction e.g., integration, transformation, screening, selecting, killing, identifying, amplifying, and the like.
  • the methods and conditions for carrying out such reactions are well known in the art (See, e.g., Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109:E2579-E2586; M.R. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • Cas9 nuclease refers to a large group of endonucleases that catalyze the double stranded DNA cleavage in the CRISPR Cas system. These polypeptides are well known in the art and many of their structures (sequences) are characterized (See, e.g., WO2013/176772; WO/2013/188638).
  • the domains for catalyzing the cleavage of the double stranded DNA are the RuvC domain and the HNH domain.
  • the RuvC domain is responsible for nicking the (-) strand and the HNH domain is responsible for nicking the (+) strand (See, e.g., Gasiunas et al. PNAS 109(36):E2579-E2586 (September 4, 2012)).
  • a Cas9 nuclease useful with this invention can be a Cas9 nuclease that comprises one or more of the modifications as described herein, thereby resulting in a Cas9 nuclease having one or more modified activities relative to a wild-type Cas9 nuclease or a Cas9 nuclease not so modified.
  • a Cas9 nuclease useful with this invention can be a wild-type Cas9 nuclease that is introduced into and expressed or over-expressed in the natural host bacterium.
  • a Cas9 nuclease useful with this invention can be a wild-type Cas9 nuclease that is heterologous to the host bacterium into which it is introduced.
  • a Cas9 nuclease useful with the invention is a modified Cas9 nuclease that releases the cleaved target DNA (cleaved by the Cas9) more readily than a native Cas9 or a Cas9 that is not modified to more readily release cleaved target DNA as described herein.
  • a Cas9 nuclease modified to more readily release cleaved DNA comprises a modified protospacer adjacent motif (PAM)-interacting domain (PIM).
  • PAM protospacer adjacent motif
  • Modifications would reduce the affinity between Cas9 and the PAM without disrupting cleavage activity.
  • the corresponding mutations to Cas9 can be identified by saturation mutagenesis of sites within the PIM implicated in binding the PAM (e.g. XX) as well as regions important for the folding and structural integrity of the PIM (e.g. XX).
  • a Cas9 nuclease useful with the invention is a modified Cas9 nuclease that rather than cleaving the double stranded target DNA provides site-specific nicking of either the (-) or the (+) strand.
  • a Cas9 nuclease can comprise a mutation in the RiivC domain, thereby providing a Cas9 that is able to nick the (+) strand of the target DNA, or a mutation in the UNH domain, thereby providing a Cas9 that is able to nick the (-) strand of the target DNA.
  • a Cas9 nuclease can be introduced as a polypeptide.
  • a Cas9 nuclease can be introduced in a nucleic acid construct.
  • a “fragment” or “portion” of a nucleotide sequence will be understood to mean a nucleotide sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 70%, 71%, 72%», 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%), 93%o, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence.
  • a nucleic acid fragment or portion according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • a fragment of a polynucleotide can be a functional fragment that encodes a polypeptide that retains its function (e.g., a fragment of a Cas9 polypeptide retains one or more of the activities of a native Cas9 nuclease including, but not limited to, HNH nuclease activity, RuvC nuclease activity, DNA, RNA and/or PAM recognition and binding activities).
  • the invention may comprise a functional fragment of a Cas9 nuclease that is encoded by a fragment of a Cas9 polynucleotide.
  • the term "gene” refers to a nucleic acid molecule capable of being used to produce mRNA, tRNA, rRNA, miRNA, anti-microRNA, regulatory RNA, and the like.
  • Genes may or may not be capable of being used to produce a functional protein or gene product. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and/or 5' and 3 ! untranslated regions).
  • a gene may be "isolated” by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid.
  • genome includes an organism's chromosomal/nuclear genome as well as any mitochondrial, and/or plasmid genome.
  • a hairpin sequence is a nucleotide sequence comprising hairpins (e.g., that forms one or more hairpin structures).
  • a hairpin e.g., stem-loop, fold-back
  • a hairpin refers to a nucleic acid molecule having a secondary structure that includes a region of nucleotides that form a single strand that are further flanked on either side by a double stranded-region.
  • Such structures are well known in the art.
  • the double stranded region can comprise some mismatches in base pairing or can be perfectly complementary.
  • a repeat nucleotide sequence comprises, consists essentially of, consists of a hairpin sequence that is located within said repeat nucleotide sequence (i.e., at least one nucleotide (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) of the repeat nucleotide sequence is present on either side of the hairpin that is within said repeat nucleotide sequence).
  • a hairpin sequence of a nucleic acid construct can be located at the 3 'end of a tracr nucleic acid.
  • a “heterologous” or a “recombinant” nucleotide sequence is a nucleotide sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequence.
  • homologues include homologous sequences from the same and other species and orthologous sequences from the same and other species.
  • homologue refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins.
  • compositions and methods of the invention further comprise homologues to the nucleotide sequences and polypeptide sequences of this invention.
  • Orthologous refers to homologous nucleotide sequences and/ or amino acid sequences in different species that arose from a common ancestral gene during speciation.
  • a homologue of a nucleotide sequence of this invention has a substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) to said nucleotide sequence of the invention.
  • a homologue of a repeat, a tracr nucleic acid, a Cas9 polypeptide useful with this invention can be about 70% homologous or more to any known repeat, tracr nucleic acid, or Cas9 polypeptide.
  • hybridization refers to the binding of two complementary nucleotide sequences or substantially complementary sequences in which some mismatched base pairs are present.
  • the conditions for hybridization are well known in the art and vary based on the length of the nucleotide sequences and the degree of complementarity between the nucleotide sequences. In some embodiments, the conditions of hybridization can be high stringency, or they can be medium stringency or low stringency depending on the amount of complementarity and the length of the sequences to be hybridized.
  • the conditions that constitute low, medium and high stringency for purposes of hybridization between nucleotide sequences are well known in the art (See, e.g., Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109:E2579-E2586; M.R. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • enhanced “enhanced,” “enhancing,” and “enhancement” (and grammatical variations thereof) describe an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.
  • increased transcription of a target DNA can mean an increase in the transcription of the target gene of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.
  • an enhancement of genome editing in bacteria can mean an increase in the number/frequency of recoverable bacterial cells having edited/modified genomes by at least about 25%, 50%, 75%, 100%), 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more (or any range or value therein) as compared to the number/frequency recovered using traditional CRISPR-Cas9 methodologies for genome editing (e.g., Cas9 nuclease, CRISPR array, tracrRNA, sgRNA or Lambda Red).
  • CRISPR-Cas9 methodologies for genome editing e.g., Cas9 nuclease, CRISPR array, tracrRNA, sgRNA or Lambda Red.
  • a “native” or “wild type” nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence.
  • a wild type mRNA is a mRNA that is naturally occurring in or endogenous to the organism.
  • a “homologous” nucleic acid is a nucleotide sequence naturally associated with a host cell into which it is introduced.
  • an endogenous restriction enzyme means a restriction enzyme that is naturally occurring in (native to) the production host bacterium.
  • nucleic acid refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids.
  • dsRNA is produced synthetically, less common bases, such as inosine, 5- methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing.
  • nucleic acid constructs of the present disclosure can be DNA or RNA, but are preferably DNA.
  • the nucleic acid constructs of this invention may be described and used in the form of DNA, depending on the intended use, they may also be described and used in the form of RNA.
  • a "synthetic" nucleic acid or polynucleotide refers to a nucleic acid or polynucleotide that is not found in nature but is constructed by the hand of man and as a consequence is not a product of nature.
  • polynucleotide refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a DNA fragment or portion, genomic DNA, synthetic ⁇ e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded or double stranded.
  • nucleic acid molecules are also used interchangeably herein to refer to a heteropolymer of nucleotides.
  • nucleic acid molecules and/or polynucleotides provided herein are presented herein in the 5' to 3' direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR ⁇ 1.821 - 1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25.
  • percent sequence identity refers to the percentage of identical nucleotides in a linear polynucleotide of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test ("subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned.
  • percent identity can refer to the percentage of identical amino acids in an amino acid sequence.
  • a "protospacer sequence” refers to the target double stranded DNA and specifically to the portion of the target DNA (e.g., or target region in the genome) that is fully or substantially complementary (and hybridizes) to the spacer sequence of the CRISPR spacer-repeat sequences, CRISPR repeat-spacer-repeat sequences, and/or CRISPR arrays.
  • a “sub-optimal protospacer sequence” refers to a target DNA to which a spacer is designed, wherein the spacer comprises greater than 50% complementarity and less than 100% complementarity to said protospacer sequence.
  • the reduced complementarity can come from, for example, truncating the spacer sequence at the 5' end by up to about 5 nucleotides, introducing up to 5 mismatches within the non-seed region, or introducing up to 3 mismatches within the seed region.
  • a "sub-optimal PAM sequence” refers to a PAM sequence that allows DNA cleavage but at a rate that is below an optimal PAM. For instance, the optimal PAM for the
  • Streptococcus pyogenes Cas9 is NGG, whereas the sub-optimal PAM for this same Cas9 is NAG.
  • Sub-optimal PAMs are commonly identified when applying high-throughput techniques for PAM elucidation.
  • “suppress,” and “decrease” describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%), 98%. 99%, or 100%» as compared to a control.
  • the reduction results in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even less than about 5%) detectable activity or amount.
  • a mutation in a Cas9 nuclease can reduce the nuclease activity of the Cas9 nuclease by at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or as compared to a control (e.g., wild-type Cas9 nuclease).
  • a “repeat sequence” as used herein, refers to, for example, any repeat sequence of a wild-type CRISPR locus or a repeat sequence of a synthetic CRISPR array that are separated by "spacer sequences" (e.g., a repeat-spacer-repeat sequence).
  • a repeat sequence useful with this invention can be any known or later identified repeat sequence of a CRISPR locus or it can be a synthetic repeat designed to function in a CRISPR Type II system.
  • a repeat sequence can be identical to or substantially identical to a repeat sequence from a wild- type CRISPR Type II loci.
  • a repeat sequence can comprise a portion of a wild type repeat sequence (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or more contiguous nucleotides of a wild type repeat sequence).
  • a repeat sequence comprises, consists essentially of, or consists of at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides, or any range therein).
  • a repeat sequence comprises, consists essentially of, or consists of at least about one to about 150 nucleotides.
  • a repeat sequence comprises, consists essentially of, or consists of at least about one nucleotide to about 100 nucleotides, or any range or value therein.
  • a repeat sequence can comprise, consist essentially of, or consist of about 3 nucleotides to about 100 nucleotides, about 10 nucleotides to about 100 nucleotides, about 15 nucleotides to about 100 nucleotides, about 20 to about 50 nucleotides, about 20 to about 40 nucleotides, about 20 to about 30 nucleotides, about 30 to about 40 nucleotides, about 25 to about 40 nucleotides, about 25 to about 45 nucleotides, and/or about 25 to about 50 nucleotides, or any range or value therein.
  • a repeat sequence can comprise, consist essentially of, or consist of about 25 nucleotides to about 38 nucleotides, or any range or value therein. In still further embodiments, a repeat sequence can comprise, consist essentially of, or consist of about 29 nucleotides. In yet further embodiments, the repeat sequence can comprise, consist essentially of, or consist of a hairpin only having at least about 20 to 30 nucleotides in length. In still other embodiments, a repeat sequence comprises, consists essentially of, or consists of at least about at least three nucleotides.
  • a repeat sequence linked to the 5' end of a spacer sequence can be about three nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, 10 nucleotides or more) and have at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the same region (e.g., 5' end) of a wild type repeat nucleotide sequence.
  • the portion of a repeat sequence linked to the 3' end of a spacer sequence can have 10 or more nucleotides having at least about 50% or more identity to a wild type repeat nucleotide sequence.
  • a repeat sequence can comprise, consist essentially of, or consist of a hairpin only having at least about 20 to 30 nucleotides in length.
  • a “CRISPR array” as used herein means a nucleic acid molecule that comprises two or more repeat sequences, or a portion of each of said repeat sequences, and at least one spacer sequence, wherein one of the two or more repeat sequences, or said portion thereof, is linked to the 5' end of the spacer sequence and the other of the two repeat sequences, or portion thereof, is linked to the 3' end of the spacer sequence.
  • the combination of repeat sequences and spacer sequences is synthetic, made by man and not found in nature.
  • a "CRISPR array” refers to a nucleic acid construct that comprises from 5' to 3' at least one spacer-repeat sequence (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more spacer-repeat sequences, and any range or value therein), wherein the 3' end of the 3' most spacer-repeat of the array is linked to a repeat sequence, thereby all spacers in said array are flanked on both the 5' end and the 3' end by a repeat sequence.
  • spacer-repeat sequence e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more spacer-repeat sequences, and any range or value therein
  • a CRISPR array of the invention can be of any length and comprise any number of spacer sequences alternating with repeat sequences, as described above.
  • a CRISPR array can comprise, consist essentially of, or consist of 1 to about 100 spacer sequences, each linked on its 5' end and its 3' end to a repeat sequence (e.g., repeat-spacer- repeat-spacer-repeat-spacer-repeat-spacer-repeat, and so on, so that each CRISPR array begins and ends with a repeat).
  • a recombinant CRISPR array of the invention can comprise, consist essentially of, or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more, spacer sequences each linked on its 5' end and its 3'
  • a CRISPR array may comprise two or more CRISPR spacer- repeat nucleic acids comprising a repeat sequence of a first CRISPR spacer-repeat nucleic acid of said two or more CRISPR spacer-repeat nucleic acids linked at the 3 ' end to a spacer sequence of a second CRISPR spacer-repeat nucleic acid of said two or more CRISPR spacer- repeat nucleic acids.
  • Additional CRISPR spacer-repeat nucleic acids can be similarly linked to one another (e.g., a third CRISPR spacer-repeat nucleic acid, a fourth CRISPR spacer-repeat nucleic acid, a fifth CRISPR spacer-repeat nucleic acid, a sixth CRISPR spacer-repeat nucleic acid, a seventh CRISPR spacer-repeat nucleic acid, an eighth CRISPR spacer-repeat nucleic acid, a ninth CRISPR spacer-repeat nucleic acid, a tenth CRISPR spacer-repeat nucleic acid, and so on).
  • a third CRISPR spacer-repeat nucleic acid e.g., a third CRISPR spacer-repeat nucleic acid, a fourth CRISPR spacer-repeat nucleic acid, a fifth CRISPR spacer-repeat nucleic acid, a sixth CRISPR spacer-repe
  • the repeat sequence of a first CRISPR spacer-repeat nucleic acid can be operably linked directly to the spacer of a second CRISPR spacer-repeat nucleic acid (i.e., no linking nucleotides) or via at least one linking nucleotide.
  • a repeat sequence of a first CRISPR spacer-repeat nucleic acid can be linked to a spacer of a second CRISPR spacer-repeat nucleic acid via at least about one to about 100 linking
  • nucleotides e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nucleotides or any range or value therein)).
  • sequence identity refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g. , nucleotides or amino acids. "Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey
  • spacer sequence is a nucleotide sequence that is complementary to a target DNA (i.e., target region in the genome or the "protospacer sequence,” which is adjacent to a protospacer adjacent motif (PAM) sequence).
  • PAM protospacer adjacent motif
  • the spacer sequence can have one, two, three, four, or five mismatches as compared to the target DNA, which mismatches can be contiguous or noncontiguous.
  • the spacer sequence can have 70% identity to a target DNA.
  • the spacer nucleotide sequence can have 80% identity to a target DNA. In still other embodiments, the spacer nucleotide sequence can have 85% identity, 90% identity, 95%, 96%, 97%, 98%. 99% identity, and the like, to a target nucleotide sequence of a target gene. In representative embodiments, the spacer sequence has 100% complementarity to the target DNA. In particular embodiments, a spacer sequence has complete identity or substantial identity over a region of a target nucleotide sequence that is at least about 8 nucleotides to about 150 nucleotides in length.
  • the 5' region of a spacer sequence can be identical to a target DNA while the 3' region of said spacer can be substantially identical to the said target DNA and therefore the overall complementarity of the spacer sequence to the target DNA is less than 100%.
  • the first 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and the like, nucleotides in the 3' region of a 20 nucleotide spacer sequence (seed region) can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA.
  • the first 7 to 12 nucleotides of the 3' end of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 50% complementary (e.g., 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to the target DNA.
  • 50% complementary e.g., 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%
  • he 3' end of the spacer sequence can be 75%-99% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence can be at least about 50% to about 99% complementary to the target DNA.
  • the first 7 to 10 nucleotides in the 3' end of the spacer sequence can be 75%-99% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are at least about 50% to about 99% complementary to the target DNA.
  • the first 7 to 10 nucleotides in the 3' end of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA.
  • the first 10 nucleotides (within the seed region) of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA.
  • the 5' region of a spacer sequence (e.g., the first 8 nucleotides at the 5' end, the first 10 nucleotides at the 5' end, the first 15 nucleotides at the 5' end, the first 20 nucleotides at the 5' end) can have about 75% identity or more (75% to about 100% identity) to a target DNA, while the remainder of the spacer sequence can have about 50% or more identity to the target DNA.
  • the first 8 nucleotides at the 5' end of a spacer sequence can be 100% identical to the target nucleotide sequence or it can have one or two mutations and therefore can be about 88%> identical or about 75% identical to a target DNA, respectively, while the remainder of the spacer nucleotide sequence can be at least about 50% or more identical to the target DNA.
  • a spacer sequence of this invention can be about 15 nucleotides to about 150 nucleotides in length.
  • a spacer nucleotide sequence of this invention can be about 15 nucleotides to about 100 nucleotides in length (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
  • a spacer nucleotide sequence can be a length of about 8 to about 150 nucleotides, about 8 to about 100 nucleotides, about 8 to about 50 nucleotides, about 8 to about 40 nucleotides, about 8 to about 30
  • nucleotides about 8 to about 25 nucleotides, about 8 to about 20 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40, about 10 to about 30, about 10 to about 25, about 10 to about 20, about 15 to about 50, at least about 8, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 1 10, at least about 120, at least about 130, at least about 140, at least about 150 nucleotides in length, or more, and any value or range therein.
  • a spacer sequence of a CR ISPR spacer-repeat nucleic acid of the invention comprises at least about 16 consecutive nucleotides of a target DNA or target nucleic acid, wherein at the 3' end of said spacer at least about 10 consecutive nucleotides of said at least about 16 consecutive nucleotides comprise at least about 90% complementarity to the target nucleic acid, wherein the target nucleic acid is adjacent to a protospacer adjacent motif (PAM) sequence in the genome of a bacterium of interest.
  • PAM protospacer adjacent motif
  • repair template refers to a natural or engineered nucleic acid (e.g., DNA) that can serve as a specific or non-specific nucleic acid molecule to
  • bridge/repair/replace damaged DNA e.g., dsDNA breaks, single-stranded nicks, oxidative damage, pyrimidine cross-links, etc
  • repair/replication machinery e.g., an endogenous polynucleotide encoding RecABCD, a heterologous polynucleotide encoding an ATP-dependent DNA ligase D (LigD), a heterologous polynucleotide encoding a DNA-end-binding protein Ku, and/or a heterologous polynucleotide encoding a recombinase.
  • a repair template useful with this invention can be dsDNA or ssDNA (with homology arms flanking the sequence to be mutated), which may be linear or circularized and can include but is not limited to DNA oligonucleotide(s), plasmid(s), or whole gDNA.
  • the phrase "substantially identical,” or “substantial identity” in the context of two nucleic acid molecules, nucleotide sequences or protein sequences refers to two or more sequences or subsequences that have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • sequence comparison algorithms or by visual inspection.
  • substantial identity can refer to two or more sequences or subsequences that have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95, 96, 96, 97, 98, or 99% identity.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA).
  • An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e. , the entire reference sequence or a smaller defined part of the reference sequence.
  • Percent sequence identity is represented as the identity fraction multiplied by 100.
  • the comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence.
  • percent identity may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlcngth
  • E expectation
  • BLOSUM62 scoring matrix see I Ienikoff &
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)).
  • One measure of similarity provided by the BL AST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001.
  • the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.001.
  • a target DNA refers to a region of an organism's genome that is fully complementary or substantially complementary (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a spacer sequence in a CRISPR array.
  • a target region may be about 10 to about 40 consecutive nucleotides in length located immediately adjacent to a PAM sequence (PAM sequence located immediately 3 ' of the target region) in the genome of the organism.
  • a target nucleotide sequence or target DNA is located adjacent to or flanked by a PAM (protospacer adjacent motif). While PAMs are often specific to the particular CRISPR-Cas system, a PAM sequence can be determined by those skilled in the art through established experimental and computational approaches. Thus, for example, experimental approaches include targeting a sequence flanked by all possible nucleotides sequences and identifying sequence members that do not undergo targeting, such as through in vitro cleavage of target DNA (Patanayak et al. 2013. Nat. Biotechnol. 31 :839-843) or the transformation of target plasmid DNA (Esvelt et al. 2013. Nat. Methods 10: 1 1 16-1121 ; Jiang et al. 2013. Nat. Biotechnol. 31 :233-239).
  • a computational approach can include performing BLAST searches of natural spacers to identify the original target DNA sequences in
  • a "trans-activating CRISPR (tracr) nucleic acid” or “tracr nucleic acid” as used herein refers to any tracr RNA (or its encoding DNA).
  • a tracr nucleic acid comprises from 5' to 3' a bulge, a nexus hairpin and terminal hairpins, and optionally, at the 5' end, an upper stem (See. Briner et al. (2014 ) Molecular Cell. 56(2):333-339).
  • a tracr nucleic acid functions in hybridizing to the repeat portion of mature or immature crRNAs. recruits Cas9 protein to the target site, and may facilitate the catalytic activity of Cas9 by inducting structural
  • tracrRNAs are specific to the CRISPR-Cas system and can be variable. Any tracr nucleic acid, known or later identified, can be used with this invention.
  • a tracr nucleic acid can be fused to a CRISPR array to form a single guide nucleic acid and therefore, in some embodiments, the tracr nucleic acid and CRISPR array can be introduced as a single guide.
  • any polynucleotide, nucleotide sequence and/or recombinant nucleic acid molecule of this invention e.g., a CRISPR array, a tracr, a heterologous polynucleotide encoding a Cas9 nuclease, a Cas9 nuclease, a heterologous polynucleotide encoding an ATP-dependent DNA ligase D (LigD), a heterologous polynucleotide encoding a DNA-end-binding protein Ku, and/or a heterologous polynucleotide encoding a recombinase can be codon optimized for expression in any species of interest.
  • a CRISPR array e.g., a CRISPR array, a tracr, a heterologous polynucleotide encoding a Cas9 nuclease, a Cas9 nuclease, a heterolog
  • Codon optimization is well known in the art and involves modification of a nucleotide sequence for codon usage bias using species-specific codon usage tables.
  • the codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the species of interest.
  • the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest.
  • the modifications of the nucleotide sequences are determined by comparing the species-specific codon usage table with the codons present in the native polynucleotide sequences.
  • nucleotide sequence and/or recombinant nucleic acid molecule of this invention can be codon optimized for expression in the particular organism/species of interest.
  • the recombinant nucleic acids molecules, nucleotide sequences and polypeptides of the invention are "isolated.”
  • An “isolated” nucleic acid molecule, an “isolated” nucleotide sequence or an “isolated” polypeptide is a nucleic acid molecule, nucleotide sequence or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • an isolated nucleic acid molecule, nucleotide sequence or polypeptide may exist in a purified form that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • the isolated nucleic acid molecule, the isolated nucleotide sequence and/or the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more pure.
  • an isolated nucleic acid molecule, polynucleotide or polypeptide may exist in a non-native environment such as, for example, a recombinant host cell.
  • a non-native environment such as, for example, a recombinant host cell.
  • isolated means that it is separated from the chromosome and/or cell in which it naturally occurs.
  • a polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs in and is then inserted into a genetic context, a chromosome and/or a cell in which it does not naturally occur (e.g., a different host cell, different regulatory sequences, and/or different position in the genome than as found in nature).
  • polynucleotides and their encoded polypeptides are "isolated" in that, by the hand of man, they exist apart from their native environment and therefore are not products of nature, however, in some embodiments, they can be introduced into and exist in a recombinant host cell.
  • polynucleotides comprising tracr nucleic acids and/or CRJSPR arrays or, and polynucleotides encoding a Cas9 polypeptide, an ATP-dependent DNA ligase D (LigD), a heterologous polynucleotide encoding a DNA-end-binding protein Ku, and/or a heterologous polynucleotide encoding a recombinase can be operatively associated with a variety of promoters, terminators and other regulatory elements for expression in various organisms or cells.
  • at least one promoter and/or terminator can be operably linked to a polynucleotide of the invention.
  • Any promoter useful with this invention can be used and includes, for example, promoters functional with the organism of interest including but not limited to constitutive, inducible, developmental ly regulated, and the like, as described herein.
  • a regulatory element as used herein can be endogenous or heterologous.
  • an endogenous regulatory element derived from the subject organism can be inserted into a genetic context in which it does not naturally occur (e.g., a different position in the genome than as found in nature), thereby producing a recombinant or non-native nucleic acid.
  • operably linked or “operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related.
  • operably linked or “operably associated” as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated.
  • a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence.
  • a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of said nucleotide sequence.
  • control sequences e.g., promoter
  • the control sequences need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof.
  • intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered "operably linked" to the nucleotide sequence.
  • a “promoter” is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (i. e. , a coding sequence) that is operably associated with the promoter.
  • the coding sequence may encode a polypeptide and/or a functional RNA.
  • a “promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase and directs the initiation of transcription.
  • promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence.
  • the promoter region may comprise other elements that act as regulators of gene expression. These include, but are not limited to, a - 35 element consensus sequence and a -10 consensus sequence (Simpson. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:3233-3237).
  • Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated promoters for use in the preparation of recombinant nucleic acid constructs, polynucleotides, expression cassettes and vectors comprising the polynucleotides and recombinant nucleic acid constructs of the invention. These various types of promoters are known in the art.
  • expression of a construct of the invention can be made constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated promoters using the recombinant nucleic acid constructs of the invention operatively linked to the appropriate promoter functional in an organism of interest.
  • repression can be made reversible using the recombinant nucleic acid constructs of the invention operatively linked to, for example, an inducible promoter functional in an organism of interest.
  • promoter will vary depending on the quantitative, temporal and spatial requirements for expression, and also depending on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge present in the art, the appropriate promoter can be selected for the particular host organism of interest. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms and such knowledge can be readily accessed and implemented in other systems as appropriate.
  • promoters include useful with this invention include promoters functional in bacteria.
  • a promoter useful with bacteria can include, but is not limited to, L-arabinose inducible (araBAD, PHAD) promoter, any lac promoter, L-rhamnose inducible (rhaP BAD ) promoter, T7 RNA polymerase promoter, trc promoter, tac promoter, lambda phage promoter (PL , PL-9G-50), anhydrotetracycline-inducible (tetA) promoter, trp, Ipp, phoA, recA, proU, cst-1, cad A, nor, Ipp-lac, cspA, ⁇ -lac operator, T3-lac operator, T4 gene 32, ' V5-lac operator, nprM- lac operator.
  • arabin inducible araBAD, PHAD
  • any lac promoter L-rhamnose inducible (rhaP BAD ) promoter
  • Vhb Protein A, corynebacterial-E. coli like promoters, thr, horn, diphtheria toxin promoter, sig A, sig B, nusG, SoxS, katb, a-amylase (Pamy).
  • Ptms, P43 (comprised of two overlapping RNA polymerase ⁇ factor recognition sites, ⁇ , ⁇ ), Ptms, P43, rplK-rplA, ferredoxin promoter, and/or xylose promoter.
  • inducible promoters can be used.
  • chemical-regulated promoters can be used to modulate the expression of a gene in an organism through the application of an exogenous chemical regulator. Regulation of the expression of nucleotide sequences of the invention via promoters that are chemically regulated enables the RNAs and/or the polypeptides of the invention to be synthesized only when, for example, an organism is treated with the inducing chemicals.
  • the promoter may be a chemical-inducible promoter, where application of a chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • a promoter can also include a light-inducible promoter, where application of specific wavelengths of light induce gene expression (Levskaya et al. 2005.
  • a nucleic acid construct of the invention can be an "expression cassette” or can be comprised within an expression cassette.
  • expression cassette means a recombinant nucleic acid construct comprising one or more polynucleotides of the invention, wherein said recombinant nucleic acid construct is operably associated with at least one control sequence (e.g., a promoter).
  • control sequence e.g., a promoter
  • An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • An expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • An expression cassette also can optionally include a transcriptional and/or translational termination region (i.e., termination region) that is functional in the selected host cell.
  • a transcriptional and/or translational termination region i.e., termination region
  • a variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation.
  • the termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host cell, or may be derived from another source (i.e.. foreign or heterologous to the promoter, to the nucleotide sequence of interest, to the host, or any combination thereof).
  • terminators can be operably linked to the recombinant nucleic acid molecule and CRISPR array of the invention.
  • An expression cassette also can include a nucleotide sequence encoding a selectable marker, which can be used to select a transformed host cell.
  • selectable marker means a nucleotide sequence that when expressed imparts a distinct phenotype to the host cell expressing the marker and thus allows such transformed cells to be distinguished from those that do not have the marker.
  • Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic and the like), or whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., fluorescence).
  • a selective agent e.g., an antibiotic and the like
  • screening e.g., fluorescence
  • polynucleotides described herein e.g., polynucleotides comprising a CRISPR array and/or a tracr nucleic acid, and
  • vectors refers to a composition for transferring, delivering or introducing a nucleic acid (or nucleic acids) into a cell.
  • a vector comprises a nucleic acid molecule comprising the nucleotide sequence(s) to be transferred, delivered or introduced.
  • Vectors for use in transformation of host organisms are well known in the art.
  • Non-limiting examples of general classes of vectors include but are not limited to a viral vector, a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a fosmid vector, a bacteriophage, an artificial chromosome, or an Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable.
  • a vector as defined herein can transform a prokaryotic host either by integration into the cellular genome or exist extrachromosomal ly (e.g. autonomous replicating plasmid with an origin of replication).
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, such as broad-host plasmids or shuttle vectors with multiple origins-of-replication.
  • the nucleic acid in the vector is under the control of. and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell.
  • the vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • the recombinant polynucleotides of this invention and/or expression cassettes comprising the recombinant polynucleotides of this invention can be comprised in vectors as described herein and as known in the art.
  • the terms "contacting,” “'introducing. “ “”delivering, “ and “”administering” can refer to a process by which the recombinant polynucleotides of the present invention are delivered to a cell, in order to edit a target DNA in the genome of a cell comprising said target DNA having substantial complementarity to at least one spacer of a CRISPR array.
  • introduction in the context of a polynucleotide of interest means presenting a polynucleotide of interest to a host organism or a cell of said organism (e.g., host cell such as a bacterial cell) in such a manner that the polynucleotide gains access to the interior of a cell and includes such terms as
  • these polynucleotides can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different expression constructs or transformation vectors. Accordingly, these polynucleotides can be introduced into cells in a single transformation event and/or in separate transformation events. Thus, in some aspects of the present invention one or more polynucleotides of this invention can be introduced into a cell of a host bacterium.
  • transformation refers to the introduction of a heterologous polynucleotide into a cell. Such introduction into a cell may be stable or transient.
  • a host cell or host organism is stably transformed with a nucleic acid molecule of the invention.
  • a host cell or host organism is transiently transformed with a recombinant nucleic acid molecule of the invention.
  • polynucleotide is introduced into the cell and does not integrate into the genome of the cell.
  • “Stable transformation” or “stably transformed” as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations.
  • “Genome” as used herein also includes the nuclear and the plasmid genome, and therefore includes integration of the nucleic acid construct into, for example, the plasmid genome.
  • Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a
  • minichromosome or a plasmid minichromosome or a plasmid.
  • Transient transformation may be detected by. for example, an enzyme-linked
  • ELISA immunosorbent assay
  • Western blot which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism.
  • transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a bacterium).
  • Stable transformation of a cell can be detected by. for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into said cell.
  • Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PGR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and/or hybridization protocols well known in the art.
  • PGR polymerase chain reaction
  • Transformation can also be detected by direct sequencing and/or hybridization protocols well known in the art.
  • the polynucleotide sequences, nucleic acid constructs, expression cassettes, and/or vectors of the invention can be expressed transiently and/or they can be stably incorporated into the genome of the host organism.
  • a polynucleotide of the invention can be introduced into a cell by any method known to those of skill in the art.
  • Exemplary methods of transformation include transformation via electroporation of competent cells, passive uptake by competent cells, chemical transformation of competent cells, as well as any other electrical, chemical, physical (mechanical) and/or biological mechanism that results in the introduction of nucleic acid into a cell, including any combination thereof.
  • transformation of a cell comprises nuclear transformation. In some embodiments of the invention, transformation of a cell comprises plasmid transformation and conjugation.
  • a nucleotide sequence therefore can be introduced into a host organism or its cell in any number of ways that are well known in the art.
  • the methods of the invention do not depend on a particular method for introducing one or more nucleotide sequences into the organism, only that they gain access to the interior of the cell.
  • they can be assembled as part of a single nucleic acid construct, or as separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, the nucleotide sequences can be introduced into the cell of interest in a single transformation event, or in separate transformation events.
  • the inventors have developed new compositions useful for genome editing of prokaryotes using CRIPSR-Cas9 technologies.
  • the genome editing comprises a synthetic CRISPR array and polypeptides that can carry out nonhomologous end-joining, ATP-dependent DNA ligase D (LigD), a heterologous polynucleotide encoding a DNA-end-binding protein u, and/or a heterologous polynucleotide encoding a recombinase.
  • novel spacer design including but not limited to identification of PAM sequences whose use can improve genome editing in bacteria
  • novel Cas9 nucleases modified to provide improved genome editing in bacteria.
  • Such methods can be broadly used for editing of target DNA in bacteria, whether to edit the genome, plasmids, or bacteriophages with such being useful in a range of applications from basic research in bacterial genetics to strain development for biomanufacturing, biotechnology, agriculture, medicine, and food production.
  • the present invention is directed to compositions and methods for improving or enhancing genome editing in bacteria, wherein the compositions and methods are improved or enhanced as compared to traditional methods of genome editing in bacteria with CRISPR-Cas9 methodologies (e.g., Cas9 nuclease, CRISPR array, tracrRNA, sgRNA or Lambda Red); the improvement or enhancement in genome editing increases the frequency of recovering or number of recovered bacterial cells comprising the prescribed genetic alteration.
  • CRISPR-Cas9 methodologies e.g., Cas9 nuclease, CRISPR array, tracrRNA, sgRNA or Lambda Red
  • a recombinant nucleic acid construct comprising a synthetic CRISPR array, a polynucleotide encoding an ATP-dependent DNA ligase D (LigD), a heterologous polynucleotide encoding a DNA-end-binding protein Ku, and/or a heterologous polynucleotide encoding a recombinase.
  • a synthetic CRISPR array comprising a synthetic CRISPR array, a polynucleotide encoding an ATP-dependent DNA ligase D (LigD), a heterologous polynucleotide encoding a DNA-end-binding protein Ku, and/or a heterologous polynucleotide encoding a recombinase.
  • the recombinant nucleic acid construct further comprises a tracr nucleic acid, optionally wherein the CRISPR array and the tracr nucleic acid may be fused together to form a single guide nucleic acid, which, in some embodiments, may be operably linked to a promoter.
  • the recombinant nucleic acid construct further comprises a polynucleotide encoding a Cas9 nuclease.
  • the CRISPR array, the polynucleotide encoding a LigD, the polynucleotide encoding Ku, the polynucleotide encoding a Cas9 nuclease and/or the tracr nucleic acid may be operably linked to one or more promoters, wherein said one or more promoters may be heterologous to the polynucleotides comprised in the nucleic acid constructs or may be heterologous to the organism into which a nucleic acid construct or polypeptide may be introduced (e.g., bacterium).
  • a recombinant nucleic acid construct comprising a synthetic CRISPR array, a tracr nucleic acid, a polynucleotide encoding an ATP-dependent DNA ligase D (LigD), a polynucleotide encoding a DNA-end-binding protein Ku, and a polynucleotide encoding a Cas9 nuclease, optionally wherein the CRISPR array and the tracr nucleic acid may be fused to form a single guide nucleic acid.
  • LigD ATP-dependent DNA ligase D
  • Ku DNA-end-binding protein Ku
  • Cas9 nuclease optionally wherein the CRISPR array and the tracr nucleic acid may be fused to form a single guide nucleic acid.
  • the CRISPR array, the tracr nucleic acid, the polynucleotide encoding LigD, the polynucleotide encoding Ku, the polynucleotide encoding Cas9 nuclease, and/or the single guide nucleic acid may be operably linked to one or more promoters, wherein said one or more promoters may be heterologous to the polynucleotides comprised in the nucleic acid constructs or to the organism into which a nucleic acid construct or polypeptide may be introduced (e.g., bacterium).
  • the recombinant nucleic acid construct may also comprise a heterologous polynucleotide encoding a recombinase.
  • LigD and Ku Any known or later identified LigD and Ku may be useful with this invention.
  • Exemplars- LigD and Ku polypeptides include those from Pseudomonas aeruginosa,
  • Bacillus halodurans, and/or Bordatella pertussis Bacillus halodurans, and/or Bordatella pertussis.
  • Recombinases useful with this invention include but are not limited to RecT from
  • Lactobacillus reuteri the beta protein from the E. coli Lambda phage, and/or the gamma, beta, and/or exo proteins from E. coli Lambda phage
  • Cas9 nuclease, the single guide nucleic acid and/or the polynucleotide encoding the recombinase can be comprised in a single nucleic acid construct or can be comprised in more than one nucleic construct, which can be operably linked to one or more promoters, wherein said one or more promoters may be heterologous to the polynucleotides comprised in said nucleic acid constructs or to the organism into which a nucleic acid construct or polypeptide may be introduced (e.g., bacterium).
  • the CRISPR arrays, tracr nucleic acids, polynucleotides, nucleic acid constructs of the invention can be use for editing of a target DNA in the genome of a bacterium. Accordingly, in some embodiments, the CRISPR arrays, tracr nucleic acids, polynucleotides, nucleic acid constructs of the invention may be introduced into a bacterium that comprises an endogenous Cas9 nuclease and/or a CRISPR Type II system. In other
  • the CRISPR arrays, tracr nucleic acids, polynucleotides, nucleic acid constructs of the invention may be introduced into a bacterium that does not comprise an endogenous Cas9 nuclease and/or a CRISPR Type II system.
  • a method of editing a target DNA in the genome of a bacterium comprising introducing into a bacterium a synthetic CRISPR array, a polynucleotide encoding an ATP-dependent DNA ligase D (LigD) and a polynucleotide encoding a DNA-end-binding protein Ku, optionally a polynucleotide encoding a recombinase, (e.g., a recombinant nucleic acid construct of the invention), wherein the CRISPR array comprises at least one spacer having substantial complementarity to a target DNA in the genome of the bacterium, thereby editing the target DNA in the genome of the bacterium.
  • the CRISPR array comprises at least one spacer having substantial complementarity to a target DNA in the genome of the bacterium, thereby editing the target DNA in the genome of the bacterium.
  • the synthetic CRISPR array, the polynucleotide encoding LigD and the polynucleotide encoding Ku, optionally a polynucleotide encoding a recombinase may be comprised in one or more recombinant nucleic acid constructs.
  • the method further comprises introducing into the bacterium a heterologous polynucleotide encoding a Cas9 nuclease, optionally on the same or different nucleic acid construct as the synthetic CRISPR array, the polynucleotide encoding LigD and/or the polynucleotide encoding Ku, optionally a polynucleotide encoding a recombinase.
  • the method comprises introducing into the bacterium a tracr nucleic acid, optionally wherein the CRISPR array and the tracr nucleic acid may be fused to form a single guide nucleic acid.
  • the method of editing a target DNA in the genome of a bacterium further comprises introducing into the bacterium a repair template, thereby providing a template for repair or replacement of the target DNA in the genome of the bacterium and/or for producing a mutation (e.g., a point mutation, a substitution mutation, a missense mutation, a nonsense mutation, or a silent mutation ) in the target DNA in the genome of the bacterium.
  • a mutation e.g., a point mutation, a substitution mutation, a missense mutation, a nonsense mutation, or a silent mutation
  • a method of editing a target DNA in the genome of a bacterium comprising introducing into a bacterium a synthetic CRISPR array, a tracr nucleic acid, a polynucleotide encoding an A TP-dependent DNA ligase D (LigD), a polynucleotide encoding a DNA-end-binding protein Ku, and a polynucleotide encoding a Cas9 nuclease, optionally a polynucleotide encoding a recombinase (e.g., a recombinant nucleic acid construct of the invention), optionally wherein the CRISPR array and the tracr nucleic acid can be fused to form a single guide nucleic acid, wherein the CRISPR array comprises at least one spacer having substantial complementarity to a target DNA in the genome of the bacterium, thereby editing the target DNA in the genome of the bacterium
  • the polynucleotide encoding a Cas9 nuclease, the polynucleotide encoding a recombinase, and/or single guide nucleic acid may each be comprised in one or more recombinant nucleic acid constructs.
  • the method of editing a target DNA in the genome of a bacterium further comprises introducing into the bacterium a repair template, thereby providing a template for repair or replacement of the target DNA in the genome of the bacterium and/or for producing a mutation (e.g.. a point mutation, a substitution mutation, a missense mutation, a nonsense mutation, or a silent mutation) in the target DNA in the genome f the bacterium.
  • a mutation e.g. a point mutation, a substitution mutation, a missense mutation, a nonsense mutation, or a silent mutation
  • any of the methods of editing of a target DNA in the genome of the bacterium can comprise site-specific nicking.
  • the methods of editing of a target DNA in the genome of the bacterium can comprise site-specific nicking.
  • polynucleotide encoding a Cas9 nuclease that is introduced into a bacterium comprises a point mutation in an RuvC active site motif, thereby nicking of the (+) strand of the target DNA in the genome of the bacterium.
  • the polynucleotide encoding a Cas9 nuclease that is introduced into a bacterium comprises a point mutation in an in an HNH active site motif, thereby nicking a (-) strand of the target DNA in the genome of the bacterium.
  • the Cas9 nuclease that is introduced can comprise a modified protospacer adjacent motif (PAM)-interacting domain (PIM), wherein the Cas9 having the modified PIM releases cleaved DNA more readily than a Cas9 nuclease without said modification.
  • PIM protospacer adjacent motif
  • the target DNA may be selected to provide enhanced genome editing in a bacterium.
  • the methods of editing a target DNA in the genome of a bacterium can comprise a target DNA that is located adjacent to a sub-optimal protospacer adjacent motif (PAM) in the genome of the bacterium. Having the target DNA located to a suboptimal PAM may result in the Cas9 nuclease more readily releasing the cleaved DNA, and allowing the repair pathways (endogenous and/or heterologous) as described herein to repair the break (e.g., LigD, Ku, recombinasc. repair template). Mutations to either region are known to reduce but not eliminate Cas9 binding and cleavage.
  • PAM sub-optimal protospacer adjacent motif
  • the method of editing a target DNA in the genome of a bacteria comprises designing spacers that improve the efficiency of genome editing in bacteria using CRISPR-Cas9 technologies.
  • any of the methods of editing a target DNA in the genome of a bacteria can further comprise introducing a CRISPR array, wherein at least one spacer of the CRISPR array comprises a 5' region and a 3" region and the 5' region of said at least one spacer comprises about 50% complementarity to about 99% complementarity to the target DNA in the genome of the bacterium and the 3 " region of said at least one spacer comprises about 75% complementarity to about 99% complementarity to the target DNA in the genome of the bacterium.
  • any of the method of editing a target DNA in the genome of a bacteria can further comprise introducing a CRISPR array, wherein at least one spacer of the CRISPR array comprises a 5 * region (non-seed) and a 3' region (seed) and the 5' region of the at least one spacer comprises about 10 consecutive nucleotides having about one to about five nucleotides that are non-complementary to the target DNA (i.e., non- complementary to the 3' region of the proto spacer (having a 5' region and a 3' region)) in the genome of the bacterium and the 3' region of the at least one spacer comprises 100%
  • a method of editing a target DNA in the genome of a bacterium comprising introducing into a bacterium a synthetic CRISPR array and a
  • the method further comprises introducing into the bacterium a tracr nucleic acid, optionally wherein the tracr nucleic acid and the CRISPR array are fused to form a single guide nucleic acid.
  • a tracr nucleic acid optionally wherein the tracr nucleic acid and the CRISPR array are fused to form a single guide nucleic acid.
  • the method further comprises introducing into the bacterium a polynucleotide encoding a Cas9 nuclease, optionally wherein the polynucleotide encoding a Cas9 nuclease further comprises a point mutation in an RuvC active site motif, thereby nicking a (+) strand of the target DNA in the genome of the bacterium or a point mutation in an HNH active site motif, thereby nicking a (-) strand of the target DNA in the genome of the bacterium.
  • the synthetic CRISPR array comprises at least one spacer having substantial complementarity to a target DNA in the genome of the bacterium and the target DNA is adjacent to a sub-optimal protospacer adjacent motif (PAM) in the genome of the bacterium.
  • PAM sub-optimal protospacer adjacent motif
  • a method of editing a target DNA in the genome of a bacterium comprising introducing into a bacterium a synthetic CRISPR array, wherein the synthetic CRISPR array comprises at least one spacer having substantial complementarity to a target DNA in the genome of the bacterium, and the target DNA is adjacent to a sub-optimal protospacer adjacent motif (PAM), thereby editing the target DNA in the genome of the bacterium.
  • the method further comprises introducing into the bacterium a tracr nucleic acid, optionally wherein the tracr nucleic acid and the CRISPR array are fused to form a single guide nucleic acid.
  • the method further comprises introducing into the bacterium a polynucleotide encoding a Cas9 nuclease, optionally wherein the polynucleotide encoding a Cas9 nuclease comprises a modified protospacer adjacent moti (PAM)-interacting domain (PIM), and/or comprises (1) a point mutation in an RuvC active site motif, thereby nicking a (+) strand of the target DNA in the genome of the bacterium or (2) a point mutation in an HNH active site motif, thereby nicking a (-) strand of the target DNA in the genome of the bacterium.
  • PAM protospacer adjacent moti
  • a method of editing a target DNA in the genome of a bacterium comprising introducing into a bacterium a synthetic CRISPR array, wherein the CRISPR array comprises at least one spacer having complementarity to a target DNA in the genome of the bacterium and said at least one spacer of the CRISPR array comprises a 5' region and a 3' region and the 5' region of said at least one spacer comprises about 50%
  • the method further comprised introducing into the bacterium a tracr nucleic acid, optionally wherein the tracr nucleic acid and the CRISPR array are fused to form a single guide nucleic acid.
  • the method further comprises introducing into the bacterium a polynucleotide encoding a Cas9 nuclease, optionally wherein the polynucleotide encoding a Cas9 nuclease comprises a modified protospacer adjacent motif (PAM)-interacting domain (PIM), and/or comprises (1) a point mutation in an RuvC active site motif, thereby nicking a (+) strand of the target DNA in the genome of the bacterium or (2) a point mutation in an HNH active site motif, thereby nicking a (-) strand of the target DNA in the genome of the bacterium.
  • the target DNA can be adjacent to a sub-optimal protospacer adjacent motif (PAM) in the genome of the bacterium.
  • PAM sub-optimal protospacer adjacent motif
  • a method of editing a target DNA in the genome of a bacterium comprising introducing into a bacterium a synthetic CRISPR array, wherein the CRISPR array comprises at least one spacer having substantial complementarity to a target DNA in the genome of the bacterium, and the at least one spacer of the CRISPR array comprises a 5' region (non-seed) and a 3' region (seed) and the 5' region of the at least one spacer comprises about 10 consecutive nucleotides having about one to about five nucleotides that are non- complementary to the target DNA (i.e..
  • the method further comprised introducing into the bacterium a tracr nucleic acid, optionally wherein the tracr nucleic acid and the CRISPR array are fused to form a single guide nucleic acid.
  • the method further comprises introducing into the bacterium a polynucleotide encoding a Cas9 nuclease, optionally wherein the polynucleotide encoding a Cas9 nuclease comprises a modified protospacer adjacent motif (PAM)-interacting domain (PIM), and/or comprises (1) a point mutation in an RuvC active site motif, thereby nicking a (+) strand of the target DNA in the genome of the bacterium or (2) a point mutation in an HNH active site motif, thereby nicking a (-) strand of the target DNA in the genome of the bacterium.
  • the target DNA can be adjacent to a sub-optimal protospacer adjacent motif (PAM) in the genome of the bacterium.
  • PAM sub-optimal protospacer adjacent motif
  • Example 1 Reduced lethality and indel formation into bacteria lacking an endogenous Cas9.
  • Polynucleotides encoding the ku and ligD genes from Pseudomonas aeruginosa or those from Mycobacterium tuberculosis are introduced into a strain lacking an endogenous CRISPR- Cas9 (e.g. Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum) by either recombineering the genes into the genome or by expressing the genes from a plasmid.
  • the number of viable colonies is evaluated following transformation of a plasmid that expresses a Cas9 (e.g.
  • NHEJ and Cas9 in E. coli E. coli and a number of other bacteria do not naturally possess the NHEJ repair pathway. Instead, to repair dsDNA breaks, they rely on the HDR pathway.
  • NHEJ pathway To introduce the NHEJ pathway, previous researchers introduced the ku and ligD genes from Mycobacterium tuberculosis into the E. coli genome (Malyarchuk et al. DNA Repair (Amst). Oct 1 :6(10): 1413-24 (2007)). These strains are E. coli pJH35 (WT) and pJH32 (+Ku/LigD).
  • WT E. coli pJH35
  • pJH32 (+Ku/LigD
  • a single-guide RNA was designed to target the xyl A gene in E. coli encoding a gene involved in D-xylose catabolism.
  • the Cas9 plasmid was transformed into the strains followed by the sgRNA plasmid (See, Fig. 1).
  • the spacer used in this experiment is provided in Fig. 2.
  • Transformation efficiency between the WT and the HEJ-competent strains was determined and the results are shown in Fig. 3. This was done by measuring the transformation efficiency relative to a non-targeting control.
  • the NHEJ-competent strain had a greater number of escapes than the WT strain meaning that the NHEJ-competent strain was able to repair the DNA damaged by Cas9.
  • the NHEJ-competent strain grew much slower than the WT, presumably due to the mild cytotoxicity of expressing the two repair proteins.
  • Example 2 Homology directed repair in E. coli.
  • a DNA repair template in the form of an oligonucleotide are introduced along with polynucleotides encoding ku and ligD genes that mutate the target sequence.
  • the repair template is designed to prevent subsequent attack by Cas9 following HDR, such as by introducing disruptive mutations in the PAM or seed region or deleting a portion of the target sequence.
  • a recombinase that promotes recombination of an oligonucleotide e.g. RecT from Lactobacillus reuteri, the beta protein from the E. coli Lambda phage
  • a double-stranded DNA e.g. the gamma, beta, and exo proteins from E. coli Lambda phage
  • the colonies containing the mutation prescribed by the oligonucleotide are identified.
  • Example 3 Indel formation and HDR in bacteria encoding an endogenous CRISPR-Cas9.
  • This example relies on bacteria with an endogenous CRISPR-Cas9 that is actively expressed (e.g. Streptoccocus thermophilus, Streptococcus pyogenes, Lactobacillus buchneri,
  • Polynucleotides encoding ku and ligD genes are encoded in a selectable plasmid for the species (e.g. the LMD-9 plasmid for Streptococcus thermophilus).
  • a genome-targeting CRISPR array is encoded on the same plasmid or a different plasmid. with or without an oligonucleotide or double-stranded DNA repair template is then transformed into the S. thermophilus LMD-9.
  • the number of viable colonies is assessed in the presence or absence of Jcu/ligD and sequence the target site for viable colonies. In the presence of ku/ligD will lead to high efficiencies of indel formation in the absence of the repair template, and high efficiencies of HDR in the presence of the repair template.
  • Example 4 Enhanced HDR when relying on a sub-optimal PAM.
  • This example applies to bacteria with an endogenous CRISPR-Cas9 (e.g. Streptococcus thermophilus, Lactobacillus buchneri, Campylobacter jejuni) or lacking an endogenous CRISPR-Cas9 (e.g. Escherichia coli, Bacillus subtilis).
  • a CRISPR RNA or arra is designed to target a desired sequence in the genome flanked by a non-optimal PAM.
  • the NAG PAM for the Streptococcus pyogenes Cas9 the NNGGAAT PAM for the Streptococcus thermophilus CRISPR 1. Cas9 and are typically determined by established high-throughput PAM screens that reveal weakly-active PAMs.
  • the CRISPR RNA or array is encoded on the same plasmid as the double-stranded DNA repair template. The bacterium is already
  • the plasmid with the CRISPR RNA/array and repair template is transformed, the number colony-forming units containing the transformed plasmid is measured, and the target site is sequenced. Genome- targeting, with and without the repair template, leads to a similar, small drop in the
  • transformation efficiency compared to a non-targeting control.
  • the transformation efficiencies are similar because the target DNA undergoes efficient repair because of the sub-optimal Cas9.
  • targeting a sequence with an optimal PAM e.g. NGG for the Streptococcus pyogenes Cas9, the NNAGAAT PAM for the Streptococcus thermophilus CRISPRl Cas9 results in a greatly reduced transformation efficiency compared to a target with a sub-optimal PAM.
  • the presence of the repair template yields the desired mutation in a fraction of the transformed colonies.
  • Example 5 Enhanced HDR when relying on partial targeting or a nicking Cas9.
  • Example 4 This example is similar to Example 4, only the DNA target is partially complementary to the spacer (e.g. at least one mutation in the seed region).
  • a nicking Cas9 is used in which a point mutation is introduced into the HNH endonuclease domain or the RuvC endonuclease domain as known in the art.
  • this mutation is introduced through homologous recombination.
  • the target is flanked by an optimal PAM (e.g. NGG for the Streptococcus pyogenes Cas9, the NNAGAAT PAM for the Streptococcus thermophilus CRISPRl Cas9).
  • the target sequence ismore amenable to HDR. Accordingly, the constructs described in this example when used as described in Example 4, a higher the transformation efficiency of the CRISPR RN A/array plasmid is achieved than with the equivalent target that is perfectly complementary to the target or relies on a wild-type Cas9. Furthermore, the presence of the repair template yields the desired mutation in a fraction of the transformed colonies.

Abstract

L'invention concerne des méthodes et des compositions pour l'édition de génome dans des bactéries au moyen de technologies CRISPR-Cas9 ainsi que des technologies de ligature d'extrémités non homologues.
PCT/US2016/038046 2015-06-17 2016-06-17 Méthodes et compositions pour l'édition de génome dans des bactéries à l'aide de systèmes cas9-crispr WO2016205623A1 (fr)

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