EP3927717A1 - Construction de banques de brins guides et procédés d'utilisation associés - Google Patents

Construction de banques de brins guides et procédés d'utilisation associés

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Publication number
EP3927717A1
EP3927717A1 EP20759378.1A EP20759378A EP3927717A1 EP 3927717 A1 EP3927717 A1 EP 3927717A1 EP 20759378 A EP20759378 A EP 20759378A EP 3927717 A1 EP3927717 A1 EP 3927717A1
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EP
European Patent Office
Prior art keywords
polynucleotide
sequence
seq
dna
restriction enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP20759378.1A
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German (de)
English (en)
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EP3927717A4 (fr
Inventor
Joshua Yates
Jonathon Hill
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Pioneer Biolabs LLC
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Pioneer Biolabs LLC
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Publication of EP3927717A1 publication Critical patent/EP3927717A1/fr
Publication of EP3927717A4 publication Critical patent/EP3927717A4/fr
Withdrawn legal-status Critical Current

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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • C12N2330/31—Libraries, arrays

Definitions

  • the disclosure generally relates to compositions, polynucleotides, kits, methods, and systems for enzymatic construction of clustered regularly interspaced short palindromic repeats (CRISPR) guide strand libraries and methods of use for the same.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • a first polynucleotide which encodes an RNA bound by an enzyme which functions in the CRISPR system.
  • An exemplary first polynucleotide comprises a constant region encoding sequence for a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) recognized by a cas enzyme for example, having a non-palindromic recognition site recognized by a type II restriction enzyme oriented in said sequence such that a second operably linked polynucleotide is cleaved 17 to 27 base pairs from said recognition site, when said second polynucleotide is present.
  • sgRNA CRISPR single guide RNA
  • crRNA CRISPR targeting RNA
  • the type II restriction enzyme has methylase activity and cleavage activity and the recognition site comprises a nucleotide which is methylated by said type II restriction enzyme upon cleavage, methylation of said nucleotide altering said recognition site such that the type II enzyme no longer binds said site.
  • the constant region encoding sequence is operably linked to the second polynucleotide which encodes a variable or targeting region which hybridizes to a sequence of interest.
  • the non-palindromic recognition site when transcribed is capable of being incorporated within a stem-loop structure of the CRISPR sgRNA or CRISPR crRNA without disrupting Cas9 binding at the constant region in said polynucleotide.
  • the first polynucleotide is a plurality of first polynucleotides; at least a portion of the plurality of first polynucleotides is operably linked to a second polynucleotides to form a plurality of linked second polynucleotides; wherein the plurality of linked second polynucleotides are digested with the type II restriction enzyme having methylase activity to form a plurality of third polynucleotides encoding a plurality of CRISPR sgRNAs or CRISPR crRNAs, wherein at least one of the plurality of CRISPR sgRNAs or CRISPR crRNAs has a variable region different from the other CRISPR sgRNAs or CRISPR crRNAs.
  • Type II restriction enzyme sites useful in the compositions and methods of the invention include, without limitation, one or more of NmeAIII, Mmel, CstMI, EcoP15I, ApyPI, AquII, AquIII, AquIV, Cdpl, CstMI, DraRI, DrdIV, EsaSSI, Maql, NhaXI, NlaCI, PlaDI, PspOMII, PspPRI, Reel, RpaB5I, SdeAI, SpoDI, and Bsb.
  • the polynucleotides described above comprise one or more adapter sequences.
  • the polynucleotides can comprise a single stranded overhang of between 1 and 100 nucleotides on one or both ends in certain embodiments.
  • the constant region sequence contains a portion of the stem loop sequence and said second constant region polynucleotide encodes the remaining portion of a functional stem loop structure, wherein operable linkage via an adapter reforms a functional stem loop structure bound by a Cas enzyme.
  • the constant region sequence encodes elongated stem loops or additional sequences at a 5’ end, a 3’ end or both, while maintaining a CRISPR enzyme binding site.
  • An exemplary method comprises providing a polynucleotide sample; digesting said polynucleotide sample at protospacer adjacent motifs (PAM) to form target region containing fragments with a first restriction enzyme (RE) in the presence of a ligase and a first adapter sequence.
  • the first adapter sequence comprises a constant region having a CRISPR enzyme binding site, and the ligase operably links the first adapter sequence to one or both ends of the fragments, thereby forming an intermediate product which lacks a binding site recognized by said first RE, said intermediate product comprising at least one binding site for a second Type II RE.
  • the intermediate product is then digested with the second Type II RE in the presence of a ligase and a second adapter sequence, wherein the Type II RE having methylation and nuclease activity which methylates said product at said recognition site and cleaves said targeting region between 17 to 27 base pairs away from said recognition site to form methylated fragments comprising a CRISPR constant region and targeting region, the presence of said methyl group preventing further digestion of said fragments by the second Type II RE, wherein the second adapter optionally comprises one or more of a cloning sequence, an adapter sequence, and a vector backbone.
  • the first digestion and ligation are carried out in two steps.
  • the second digestion and ligation are carried out in two steps.
  • the second two steps are combined.
  • the polynucleotide sample to be digested at PAM motifs can be obtained from any source, e.g., from one or more of an organism of interest, an organism at a selected stage of development, a tissue of interest, a cell at a selected stage of differentiation, a cell at a particular stage of the cell cycle, a tissue or cell having a selected pathology.
  • the polynucleotide sample can comprise a nucleic acids obtained from any source of interest, including without limitation, cDNA synthesized from RNA, genomic DNA, mitochondrial DNA, human DNA, animal DNA, plant DNA, fungal DNA, archaeal DNA, or bacterial DNA.
  • the polynucleotides can be isolated using any method useful in the art for this purpose, including, but not limited to precipitation, hybridization, antibody isolation, and co-precipitation.
  • the first polynucleotide is a plurality of first polynucleotides; at least a portion of the plurality of first polynucleotides is operably linked to DNA to form a plurality of second linked polynucleotides; and the plurality of second linked
  • polynucleotides are digested with the type II restriction enzyme having methylase activity to form a plurality of third polynucleotides encoding a plurality of CRISPR sgRNAs or CRISPR crRNAs, wherein at least one of the plurality of CRISPR sgRNAs or CRISPR crRNAs is methylated and has a variable region different from the other CRISPR sgRNAs or CRISPR crRNAs.
  • the starting sample e.g., a cDNA sample
  • the adapters lack a 5’ phosphate.
  • the adapters contain at least six consecutive phosphorothioates at the 5’ end to render then resistant to nuclease digestion.
  • the nucleic acid to be obtained can be digested with enzymes, which include, but are not limited to, Hpall, Mspl, ScrFI, Bfal, and Pack
  • enzymes which include, but are not limited to, Hpall, Mspl, ScrFI, Bfal, and Pack
  • the ligated CRISPR sgRNA or CRISPR crRNA product comprises at least one nick.
  • the second adapter comprises a promoter sequence, e.g., a T7 RNA polymerase promoter sequence.
  • the ligated product does not maintain a G-U hydrogen bond at position 1 of the scaffold in the constant region sequence.
  • the inventive method can also comprise purifying the ligated CRISPR sgRNA or CRISPR crRNA product.
  • the first adapter sequence comprises a 5’ single stranded overhang, in certain cases to facilitate purification.
  • the ligated CRISPR sgRNA or CRISPR crRNA product is purified using a solid support operably linked to a capture
  • the solid support is a magnetic bead and the purification step includes magnetic separation.
  • the separated beads are suspended in a buffer comprising Bst 3.0 polymerase and nucleotide triphosphates (NTPs) at about 45°C for about 15 minutes, thereby repairing and extending the nicked CRISPR sgRNA or CRISPR crRNA product, said extension causing displacement of repaired CRISPR sgRNA or CRISPR crRNA product from the bead.
  • the method can also comprise transcribing the sgRNA template libraries in the presence of DNase I.
  • Another embodiment of the method comprises PCR amplification of said ligated CRISPR sgRNA or CRISPR crRNA product. In certain aspects of the foregoing method, the digestion and ligation steps are performed essentially simultaneously.
  • An exemplary method comprises digesting a polynucleotide sample from a source of interest at protospacer adjacent motifs (PAM) with a first restriction enzyme (RE) to form target region containing fragments in the presence of a ligase and a first adapter sequence, said first adapter sequence comprising a constant region having a CRISPR enzyme binding site, said ligase operably linking said first adapter sequence to one or both ends of said fragments, thereby forming an intermediate product which lacks a binding site recognized by said first RE, said intermediate product comprising at least one binding site for a second Type II RE.
  • PAM protospacer adjacent motifs
  • RE restriction enzyme
  • the intermediate product is then digested with the second Type II RE in the presence of a ligase and a second adapter sequence, said Type II RE having methylation and nuclease activity which methylates said product at said recognition site and cleaves said targeting region between 17 to 27 base pairs away from said recognition site to form one or more methylated fragments.
  • the resulting methylated polynucleotide fragments comprising operably linked CRISPR constant region and targeting region sequences, the presence of said methyl group preventing further digestion of said polynucleotide fragments by the second Type II RE, said second adapter optionally comprising one or more of a cloning sequence, one or more adapter sequences, and a vector backbone.
  • the method further comprises immobilizing said polynucleotides to a solid support by immobilizing one or more first single stranded polynucleotide(s) to a solid support, said first single stranded polynucleotide having 5’ and 3’ ends and having binding affinity for at least a portion of a second polynucleotide(s), said 5' end of the first single stranded polynucleotide protruding from the solid support.
  • the immobilized one or more first polynucleotide(s) are then contacted with one or more second polynucleotide(s) of sufficient complementarity such that a polynucleotide duplex forms, thereby immobilizing the one or more second polynucleotide(s) on the solid support.
  • the resulting polynucleotide duplexes are then contacted with a polymerase having strand displacement activity in the presence of dNTPs, the second polynucleotide serving as a template for extension by said polymerase, wherein the first polynucleotide is displaced by the extension of the second
  • polynucleotide and said extension preventing rehybridization between the first and second polynucleotides, said second polynucleotide encoding a CRISPR sgRNA or CRISPR crRNA.
  • An exemplary method comprises immobilizing one or more first single stranded polynucleotide(s) to a solid support, the first single stranded polynucleotide having 5’ and 3’ ends and having binding affinity for at least a portion of a second polynucleotide(s), said 5' end of the first single stranded polynucleotide protruding from the solid support; contacting the one or more first polynucleotide(s) with one or more second polynucleotide(s) of sufficient complementarity such that a polynucleotide duplex forms, thereby immobilizing the one or more second polynucleotide(s) on the solid support; and contacting the polynucleotide duplex(s) so formed with a polymerase having strand
  • the support is a bead or a column. In other embodiments, the support is a magnetic bead.
  • the attached polynucleotide is a plurality of polynucleotides, preferably, a plurality of polynucleotides with different sequences.
  • the method can further comprise purifying the second polynucleotide from a mixture.
  • the length of the hybridizing sequences is adjusted to increase or decrease the temperature at which hybridization will occur.
  • polynucleotides of interest are purified at different temperatures.
  • the polynucleotides are purified via a process selected from the group consisting of magnetic separation, precipitation, hybridization, antibody isolation, and co-precipitation.
  • one or more second polynucleotides encode for one or more CRISPR/Cas9 guide RNAs.
  • certain intermediate or ligated products comprise at least five
  • phosphorothioate linked polynucleotides at the 5’ end, and any unwanted polynucleotides present in the reaction are degraded by contacting the reaction with first exonuclease and second exonucleases which cleave double stranded and single stranded DNA respectively, said phosphorothioate containing polynucleotides being resistant to exonuclease cleavage.
  • the aforementioned phosphorothioates are incorporated by ligating adapters or via PCR.
  • kits for production of an sgRNA guide strand library comprising: a first polynucleotide which encodes an RNA bound by a Cas enzyme, comprising a constant region encoding sequence for a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) having a non-palindromic recognition site recognized by a type II restriction enzyme oriented in said sequence such that a second operably linked polynucleotide is cleaved 17 to 27 base pairs from said recognition site, when operably linked, said type II restriction enzyme having methylase activity and cleavage activity, said recognition site comprising a nucleotide which is methylated by said type II restriction enzyme upon cleavage, methylation of said nucleotide altering said recognition site such that said type II enzyme no longer binds said site.
  • sgRNA CRISPR single guide RNA
  • crRNA CRISPR targeting RNA
  • the kit can comprise second polynucleotides encoding a variable or targeting sequence.
  • the kit can also contain one or more ligases for operably linking the constant region and targeting polynucleotides or one or more adapters.
  • the kit may also contain one or more Type II restriction enzymes.
  • the kit includes a solid support.
  • the kit can comprise a strand displacing polymerase.
  • the kit may also include one or more adapter sequences encoding a promoter and/or cloning site and/or
  • the kit can further comprise buffers suitable for simultaneous digestion and ligation of a polynucleotide; and optionally, reagents suitable for PCR amplification.
  • the kit can also contain reagents suitable for normalization of input nucleic acid sample.
  • FIGURE 1 A DNA substrate is cleaved by a first type II restriction endonuclease (RE) containing a PAM motif in its recognition sequence and ligation of a first adapter containing the CRISPR guide RNA scaffold sequence is carried out in a single reaction.
  • a modification to the scaffold sequence prevents the first adapter from being cleaved from the DNA substrate after being ligated.
  • the adapter ligation is therefore irreversible and forces the reaction to proceed toward the formation of the intermediate product.
  • the first adapter was synthesized without a phosphate group on the end to be ligated. This prevents the first adapter from ligating to other first adapters, which could result in the final library having truncated products. Because the first adapter is not phosphorylated, the intermediate ligation product will contain a nick.
  • FIGURE 2 Digestion of the intermediate product (from the first reaction) by a second type II RE and ligation of a second adapter containing a promoter sequence is carried out in a single reaction.
  • the addition of a methyl moiety to the RE binding site in the first adapter by the second RE prevents cleavage of the second adapter after it has been ligated.
  • the second RE can still bind to the recognition sequence and cut across the nick.
  • the second adapter was also synthesized without a phosphate group on the end to be ligated. This prevents the second adapter from ligating to other second adapters, increasing the final yield of the library.
  • an engineered version of Mmel was used that would recognize a sequence which maintained this
  • the asterisk (*) indicates that the reaction is not strictly reversible because the methyl group is incorporated during digestion. Excess adapter drives the reaction to the final product.
  • the CRISPR Scaffold adapter is synthesized with a long, 5’, single- stranded overhang that is capable of hybridizing to a single stranded oligo immobilized on a solid support, such as a magnetic.
  • a solid support such as a magnetic.
  • a strand displacing polymerase can be added to permanently remove the fragments from the solid support. This is due to the directionality of the oligo attached to the beads and extension direction of the polymerase.
  • the captured oligos are used as a template by the polymerase and extension by the polymerase prevents re -hybridization. Double stranded DNA can then be preferentially isolated using standard silica-based separation techniques. Immobilization of the library to the solid support can occur at any point in the library construction process.
  • FIGURE 4. Phosphorothioate linkages (shown in black) are synthesized into the adapters on the outside ends of the 5' strand. After the library has been constructed, there could be many byproducts, but only the final product has both of the 5' ends protected from degradation.
  • PCR primers could also be used to incorporate the linkages into a DNA product that could then be subjected to the endonucleases.
  • a 3' to 5' endonuclease could also be used if protecting groups are incorporated into the outside ends of the 3' strand.
  • FIGURES 5A to 5D provide an example embodiment of the method wherein the first adaptor only contains a portion of the sgRNA or crRNA sequence.
  • FIGURE 5A shows a restriction digestion map of a representative template for an sgRNA modified to contain an Mmel site (SeqlD 454). Type II enzymes that cut once in the region are shown.
  • FIGURE 5B provides a first adapter created to contain an Mmel site, an overhang for ligation with a second DNA fragment, and the portion of the sgRNA template sequence up to the Setl restriction site.
  • FIGURE 5C shows a final product generated using the first adapter in FIGURE 5B and a second adapter containing a T7 promoter and a second restriction site (BamHI).
  • FIGURE 5C shows that the final product can be digested using BamHI and Setl and ligated into a plasmid containing the remainder of the sgRNA template and a cloning site digested with the above enzymes or an isoschizomer thereof, thus creating a complete sgRNA template.
  • Ellipses indicate that the two ends are connected.
  • FIGURE 6A shows the hairpin structure of a wildtype crRNA (SEQ ID NO: 423) for CRISPR Cas9.
  • FIGURE 6B shows polynucleotides of various embodiments encoding for an sgRNA or crRNA recognized by a CRIPSR Cas9 protein and having non-palindromic recognition site for Mmel, NmeAIII, Hpall or Mspl, ScrFI, and Bfal.
  • S. pyogenes crRNA sequence was modified to insert binding sites for restriction enzymes while maintaining secondary structure.
  • WT crRNA indicates the previously published CRISPR sgRNA sequence for Cas9 binding. Areas surrounded by dotted lines indicate the hairpin region. Binding sites are shown surrounded by a solid line.
  • SEQ ID NOs: 424 to 439 are shown in FIGURE 6B in descending order.
  • FIGURE 7A shows the hairpin structure of a wildtype crRNA (SEQ ID NO: 440) for CRISPR Cpfl.
  • FIGURE 7B shows polynucleotides of various embodiments encoding for an sgRNA or crRNA recognized by a CRIPSR Cpf 1 protein and having non-palindromic recognition site for Mmel and NmeAIII. F. novicida crRNA sequence was modified to insert an Mmel binding site while maintaining secondary structure.
  • WT crRNA indicates the previously published CRISPR sgRNA sequence for Cpfl binding. Areas surrounded by dotted lines indicate the hairpin region. Cpfl-based systems lack a tracrRNA. Mmel and NmeAIII binding sites are shown surrounded by a solid line. Underlined letters indicate bases that do not match the original sequence. O.H. indicates the presence of an overhang added to the crRNA.
  • SEQ ID NOs: 441 to 453 are shown in FIGURE 7B in descending order.
  • a rapid and efficient enzymatic method to generate sgRNA libraries is described herein, which is applicable to a large variety of DNA substrates.
  • the methods disclosed significantly reduce the cost of custom library generation while maintaining fidelity in the guide strands produced.
  • Input DNA is obtained using one of several different methods, and optionally normalized.
  • a series of digestion and ligation steps are performed to create DNA templates that are transcribed into sgRNAs.
  • By altering the sequence of the first stem loop of the sgRNA and by taking advantage of the methyltransferase activity of type IIS restriction enzymes we were able to develop a method that combines restriction digestion and ligation steps into single reactions.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • the following are non-limiting examples of polynucleotides: single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • polynucleotide and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • exogenous nucleic acid can refer to a nucleic acid that is not normally or naturally found in or produced by a given bacterium, organism, or cell in nature.
  • endogenous nucleic acid can refer to a nucleic acid that is normally found in or produced by a given bacterium, organism, or cell in nature.
  • nucleic acid DNA or RNA
  • protein is the product of various combinations of cloning, restriction, or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems.
  • construct is understood to mean a recombinant nucleic acid, generally recombinant DNA, which has been generated for the purpose of the expression or propagation of a nucleotide sequence(s) of interest, or is to be used in the construction of other recombinant nucleotide sequences.
  • promoter or“promoter polynucleotide” is understood to mean a regulatory sequence/element or control sequence/element that is capable of binding/recruiting an RNA polymerase and initiating transcription of sequence downstream or in a 3’ direction from the promoter.
  • a promoter can be, for example, constitutively active, or always on, or inducible in which the promoter is active or inactive in the presence of an external stimulus.
  • Example of promoters include T7 promoters or U6 promoters.
  • An“adapter or adaptor”, or a“linker” for use in the compositions and methods described herein is a short, chemically synthesized, single-stranded or double-stranded oligonucleotide that can be ligated to the ends of other DNA or RNA molecules.
  • Double stranded adapters can be synthesized to have blunt ends to both terminals or to have sticky end at one end and blunt end at the other, or sticky ends at both ends.
  • a double stranded DNA adapter can be used to link the ends of two other DNA molecules (i.e., ends that do not have "sticky ends", that is complementary protruding single strands by themselves).
  • a conversion adapter is used to join a DNA insert cut with one restriction enzyme, say EcoRl, with a vector opened with another enzyme, Bam HI. This adapter can be used to convert the cohesive end produced by Bam HI to one produced by Eco R1 or vice versa.
  • EcoRl restriction enzyme
  • Bam HI another enzyme
  • One of its applications is ligating cDNA into a plasmid or other vectors instead of using Terminal Deoxynucleotide
  • operably linked can mean the positioning of components in a relationship which permits them to function in their intended manner.
  • a promoter can be linked to a polynucleotide sequence to induce transcription of the polynucleotide sequence.
  • sequence identity refers to a specified percentage of residues in two nucleic acid or amino acid sequences that are identical when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
  • comparison window refers to a segment of at least about 20 contiguous positions in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally.
  • the comparison window is from 15 to 30 contiguous positions in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally.
  • the comparison window is usually from about 50 to about 200 contiguous positions in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally.
  • Normalization is the process of ensuring equal representation of different polynucleotides. For example, the number of RNA copies per gene in a sample will depend on the expression level of that gene. These levels can vary over several orders of magnitude. However, it is often desirable to identify or include genes with very low expression level during data analysis or library generation. In order to improve representation of low expression genes, a number of methods can be used to flatten the distribution of polynucleotides in the sample to ensure all polynucleotide species are represented at roughly equal levels.
  • complementarity refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 4, 5, and 6 out of 6 being 66.67%, 83.33%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • substantially complementary refers to a degree of complementarity that is at least 40%, 50%, 60%, 62.5%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%, or percentages in between over a region of 4, 5, 6, 7, and 8 nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • a "selected phenotype” refers to any phenotype, e.g., any observable characteristic or functional effect that can be measured in an assay such as changes in cell growth, proliferation, morphology, enzyme function, signal transduction, expression patterns, downstream expression patterns, reporter gene activation, hormone release, growth factor release, neurotransmitter release, ligand binding, apoptosis, and product formation.
  • assays include, e.g., transformation assays, e.g., changes in proliferation, anchorage dependence, growth factor dependence, foci formation, growth in soft agar, tumor proliferation in nude mice, and tumor vascularization in nude mice;
  • apoptosis assays e.g., DNA laddering and cell death, expression of genes involved in apoptosis; signal transduction assays, e.g., changes in intracellular calcium, cAMP, cGMP, IP3, changes in hormone and neurotransmitter release; receptor assays, e.g., estrogen receptor and cell growth; growth factor assays, e.g., EPO, hypoxia and erythrocyte colony forming units assays; enzyme product assays, e.g., FAD-2 induced oil desaturation; transcription assays, e.g., reporter gene assays; and protein production assays, e.g., VEGF ELISAs.
  • a candidate gene is "associated with" a selected phenotype if modulation of gene expression of the candidate gene causes a change in the selected phenotype
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast.
  • a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template” or “editing polynucleotide” or “editing sequence”.
  • an exogenous template polynucleotide may be referred to as an editing template.
  • the recombination is homologous recombination.
  • a guide sequence is any polynucleotide sequence having sufficient
  • the degree of complementarity between a guide sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g.
  • polynucleotide or polynucleotides encoding for a constant region of a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) comprising a non-palindromic recognition site for a type II restriction enzyme, the non-palindromic recognition site being oriented in a manner recognized by the type II restriction enzyme for cutting a site that is 17 to 27 base pairs past an end of the constant region in an operably linked target region polynucleotide, if present.
  • the polynucleotides encode for a plurality of sgRNAs or crRNAs.
  • the polynucleotide of various embodiments is double-stranded with sense and antisense strands and the non-palindromic recognition site of various embodiments is oriented in a manner recognized by the type II restriction enzyme for cutting a site that is 17 to 27 base pairs upstream from a 5’ end of the sense strand or downstream from a 3’ end of the antisense strand.
  • the polynucleotide is double-stranded with sense and antisense strands and the non-palindromic recognition site of various embodiments is oriented in a manner recognized by the type II restriction enzyme for cutting a site that is 17 to 27 base pairs downstream from a 3’ end of the sense strand or upstream from a 5’ end of the antisense strand.
  • CRISPR protein forms a complex with a guide RNA and is capable of binding or modifying by, for example, cleaving, nicking, methylating, or demethylating a target nucleic acid or a polypeptide associated with the target nucleic acid.
  • a CRISPR protein forms a complex with a guide RNA and is capable of binding or modifying by, for example, cleaving, nicking, methylating, or demethylating a target nucleic acid or a polypeptide associated with the target nucleic acid.
  • CRISPR/Cas9 is described in PCT Patent Application Publication No. WO 2016/196805 and references referred in WO 2016/196805, which are also incorporated in its entirety by reference herein.
  • the Cas9 protein utilizes variable regions to bind specific sequences of DNA in a genome. Examples of Cas9 proteins are from Streptococcus pyogenes or Staphylococcus aureus.
  • the Cas9 protein utilizes guide RNAs to bind specific regions of a DNA sequence.
  • Cpf 1 is another protein, which uses a guide RNA in order to bind a specific sequence in genomic DNA.
  • Cpf 1 is from
  • CRISPR proteins such as Cas9 and Cpfl utilize variable regions to bind specific sequences of DNA in a genome.
  • CRISPR proteins such as Cpfl and Cas9 use a guide RNA.
  • the guide RNA provides target specificity to the complex by having a nucleotide sequence that is complementary to a sequence of a target nucleic acid.
  • a number of methods have been employed to create guide RNAs.
  • two RNA segments known as CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) have complementary portions allowing them to combine to form an RNA duplex known as a crRNA/tracrRNA complex.
  • the crRNA/tracrRNA complex has structures such as or similar to hairpin/stem-loop structures and are recognized by CRISPR proteins.
  • Another method is using single RNA segments (e.g. single guide RNA or sgRNA) that can form hairpin or stem- loop structures and are recognized by CRISPR proteins.
  • the guide RNA such as crRNA and sgRNA includes two segments: a variable region, which is also known as a targeting region; and a constant region, which is also known as a scaffold region to which the CRISPR protein binds.
  • region is understood to mean a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in a nucleic acid molecule.
  • A“region” can also mean a
  • the guide RNA includes hairpin regions, which are conserved regions which bind to the CRISPR proteins such as Cas9 and Cpfl. These hairpin regions are located in the constant region of the guide RNA.
  • a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein.
  • Cas proteins include Casl,
  • CaslB Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.
  • the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2.
  • the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9.
  • the CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae.
  • the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence.
  • the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • a vector encodes a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • D10A aspartate-to-alanine substitution
  • pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A.
  • nickases may be used for genome editing via homologous recombination.
  • a first example may apply the principles in a first series of steps and/or compositions.
  • a second example may apply the principles in a second series of steps and/or compositions. These two examples or portions thereof may be merged or combined in a variety of ways to accomplish the principles described. Additionally, techniques, methods, and compositions that are not specifically described may be applied and still be within the scope of the principles described herein.
  • systems, methods, and compositions for the rapid and efficient construction of CRISPR/Cas9 guide RNA libraries may include a methyl moiety composition.
  • a polynucleotide encoding for a constant region of a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) may include a methyl moiety that prevents digestion of the polynucleotide by a restriction endonuclease.
  • the methyl moiety may be added by an enzyme containing methyltransferase activity.
  • the polynucleotide may further include a non-palindromic recognition site for a Type IIS restriction enzyme binding site.
  • a polynucleotide encoding for a constant region of a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) may include a sequence that when ligated to digested fragments does not restore the restriction enzyme binding site sequence used to digest the fragments.
  • the modifications may maintain the stem loop structure of the CRISPR guide RNA molecule in order for the specificity and endonuclease activity of CRISPR RNA complexes to function.
  • the polynucleotide may include a non-palindromic recognition site for a type II restriction enzyme and the non-palindromic recognition site may be oriented in a manner recognized by the type II restriction enzyme.
  • a kit for generating CRISPR guide RNA (gRNA) libraries may result in a polynucleotide containing a methyl moiety and/or encoding for a constant region of a CRISPR sgRNA or CRISPR crRNA and having a sequence that does not restore a restriction enzyme binding site.
  • the kit may include one or more of the following: a type II restriction enzyme, a solid support that is configured to immobilize the polynucleotide on the solid support, a strand displacing polymerase, a promoter polynucleotide recognized by an RNA polymerase and buffers that allow/facilitate digestion and ligation to occur in the same reaction vessel.
  • kits for generating CRISPR guide RNA (gRNA) libraries may include at least one of the following: a type II restriction enzyme, a solid support, wherein the polynucleotide is capable of being immobilized on the solid supports or is immobilized on the solid support.
  • a first polynucleotide may include a sequence encoding for a constant region of one of a CRISPR single guide RNA (sgRNA) and CRISPR targeting RNA (crRNA), the sequence may include a non-palindromic recognition site recognized by a type II restriction enzyme, wherein the non-palindromic recognition site is configured such that a second polynucleotide operably linked to the first polynucleotide is cleaved 17 to 27 base pairs from the recognition site by the type II restriction enzyme, where the type II restriction enzyme is configured to add a methyl moiety to the recognition site such that methylation of the recognition site prevents subsequent cleavage of the second polynucleotide.
  • sgRNA CRISPR single guide RNA
  • crRNA CRISPR targeting RNA
  • a first polynucleotide may include a sequence encoding for a constant region of one of a CRISPR single guide RNA (sgRNA) and CRISPR targeting RNA (crRNA).
  • the sequence may include a non-palindromic recognition site configured to be recognized by a type II restriction enzyme, wherein the non-palindromic recognition site is oriented such that a second polynucleotide operably linked to an end adjacent to the non-palindromic recognition site, the first polynucleotide configured to be cleaved 17 to 27 base pairs from the recognition site by the type II restriction enzyme.
  • the type II restriction enzyme may add a methyl moiety to the recognition site such that methylation of the recognition site prevents subsequent cleavage of the second polynucleotide.
  • a first polynucleotide may include a sequence encoding for a constant region of one of a CRISPR single guide RNA (sgRNA) and CRISPR targeting RNA (crRNA), and an end configured to operably link a second polynucleotide.
  • the sequence may include a non-palindromic recognition site recognized by a type II restriction enzyme, wherein the non-palindromic recognition site is configured to position and orient the type II restriction enzyme to cleave the second polynucleotide 17 to 27 base pairs from the recognition site.
  • the type II restriction enzyme is configured to add a methyl moiety to the recognition site such that the methyl moiety prevents subsequent cleavage of the second polynucleotide.
  • the non-palindromic recognition site is positioned and oriented such that the type II restriction enzyme is configured to cleave an operably linked second polynucleotide 17 to 27 base pairs from the recognition site.
  • a polynucleotide may include a sequence encoding for at least part of a protein binding segment of an RNA component of a CRISPR complex.
  • the polynucleotide may include a non-palindromic recognition sequence for a type II restriction enzyme configured to cleave at least 17 nucleotides outside of the non-palindromic recognition sequence, the non-palindromic recognition sequence being oriented such that the type II restriction enzyme is configured such it cannot cleave within the sequence encoding for at least part of a protein binding segment of an RNA component of a CRISPR complex.
  • a polynucleotide encoding at least part of a protein binding segment of an RNA component of a CRISPR complex may include: a non-palindromic recognition sequence for a type II restriction enzyme capable of cleaving at least 17 nucleotides outside of the non- palindromic recognition sequence, the recognition sequence being oriented such that, in the presence of the restriction enzyme, the DNA cleavage domain of the restriction enzyme is positioned outside of the sequence encoding for at least part of a protein binding segment of an RNA component of a CRISPR complex.
  • a method for generating double stranded inputs for enzymatic CRISPR library generation may include at least one of: selection of a DNA source and purification of polynucleotides from the source by physical or chemical separation.
  • the DNA source is selected by one or more of an organism of interest, an organism at a selected stage of development, a tissue of interest, a cell at a selected stage of differentiation, a cell at a particular stage of the cell cycle, a tissue or cell having a selected pathology.
  • the polynucleotides may be one or more of the following: cDNA created from RNA, genomic DNA, mitochondrial DNA, or other appropriate polynucleotides.
  • the polynucleotides may be selected by at least one of: precipitation isolation, hybridization isolation, antibody isolation, and other co -precipitation isolation.
  • the polynucleotides or one or more segments thereof may be amplified by PCR or other appropriate technique(s).
  • the polynucleotides may be normalized as described above.
  • a method for generating DNA templates for production of a CRISPR/Cas9 guide RNA library may include providing a polynucleotide sample; digesting the polynucleotide sample at protospacer adjacent motifs (PAM) to form fragments with a first restriction enzyme (RE) in the presence of a ligase and first adapter, wherein the first adapter sequence may include a constant region containing a sequence CRISPR enzyme can bind to and/or cloning site sequence; attaching polynucleotide sequences to one or both ends of the fragments with a ligase, thereby forming an intermediate product which lacks binding sites recognized by the first RE; and digesting the second polynucleotide with the type II restriction enzyme to form a third polynucleotide encoding a CRISPR sgRNA or CRISPR crRNA, wherein the type II restriction enzyme cuts the DNA at a site that is 17 to 27 base pairs from the end of the first polynucle
  • the method may also include the step of ligating a polynucleotide to an end of the third polynucleotide.
  • the ligated polynucleotide may include at least one of a promoter and a cloning adapter.
  • the first adapter and second adapter may or may not contain a 5’ phosphate group.
  • a method for generating DNA templates for production of a CRISPR/Cas9 guide RNA library may include one or more of the following steps: providing a polynucleotide sample; digesting the polynucleotide sample at protospacer adjacent motifs (PAM) to form fragments with a first restriction enzyme (RE); ligation to a first adapter, wherein the first adapter sequence may include a constant region that may be a sequence a CRISPR enzyme can bind to.
  • the method may further include connecting/linking polynucleotide sequences to one or both ends of the fragments with a ligase.
  • the method may include contacting the intermediate product with a Type II S RE having methylase activity, in the presence of a ligase, the ligase operably linking a second adapter sequence to digested, methylated fragments formed from digestion of the intermediate product, thereby forming a ligated product having a variable or targeting region, and a scaffold region, wherein the second adapter sequence may include a promoter element and/or cloning site sequence.
  • CRISPR/Cas9 guide RNA library may include: providing a polynucleotide sample; digesting the polynucleotide sample at protospacer adjacent motifs (PAM) to form fragments with a first restriction enzyme (RE) in the presence of a ligase and first adapter, wherein the first adapter sequence may include a constant region containing a sequence a CRISPR enzyme can bind to and/or cloning site sequence, polynucleotide sequences to one or both ends of the fragments with a ligase, thereby forming an intermediate product which lacks binding sites recognized by the first RE.
  • PAM protospacer adjacent motifs
  • RE restriction enzyme
  • the method may also include contacting the intermediate product with a Type II S RE, in the presence of a ligase, the ligase operably linking a second adapter sequence to the digested fragments formed from digestion of the intermediate product, thereby forming a ligated product having a variable or targeting region, and a scaffold region, wherein the second adapter sequence may include a promoter element and/or cloning site sequence.
  • the method may include re-digestion that is blocked by simultaneous DNA modification of the DNA upon digestion.
  • the modification in attachment of a methylation moiety.
  • a cloning site sequence may be present, and the ligated product may be cloned into a vector which may include an operable promoter and/or scaffold sequence.
  • a vector may include a scaffold sequence.
  • the first polynucleotide may be a plurality of first polynucleotides and at least a portion of the plurality of first polynucleotides are ligated with DNA to form a plurality of second polynucleotides.
  • the plurality of second polynucleotides may be digested with the type II restriction enzyme having methylase activity to form a plurality of third polynucleotides encoding a plurality of CRISPR sgRNAs or CRISPR crRNAs.
  • At least one of the plurality of CRISPR sgRNAs or CRISPR crRNAs may have a variable region different from the other CRISPR sgRNAs or CRISPR crRNAs.
  • the polynucleotide sample may be cDNA which is normalized to remove repeated transcripts from the cDNA, thereby increasing equal representation of transcripts in the library.
  • the input polynucleotide sample may be obtained from a source selected from the group including: an organism of interest, an organism at a selected stage of development, a tissue of interest, a cell at a selected stage of differentiation, a cell at a particular stage of the cell cycle, a tissue or cell having a selected pathology, or other appropriate tissue or organism.
  • the components and intermediate products may have a number of different characteristics.
  • the adapters may lack a 5’ phosphate and the adapters may contain at least six consecutive phophorothioates at the 5’ end.
  • the promoter may be a T7 RNA polymerase promoter.
  • the polynucleotide sequence described above may be digested with an enzyme selected from the group consisting of Hpall, Mspl, ScrFI, Bfal, and Pack
  • the ligated product of step may include at least one nick.
  • the method may include purifying the ligated product.
  • the ligated product may be purified using a capture oligonucleotide that may include a biotin at a 3’ end, which hybridizes to the scaffold portion of the ligated product operably linked to a solid support.
  • the solid support is a magnetic bead and the purification step includes magnetic separation.
  • the method may include suspending the separated beads in a buffer that may include Bst 3.0 polymerase and nucleotide triophosphates (NTPs) at about 45°C for about 15 minutes, thereby repairing and extending the nicked strand, the extension causing displacement of the repaired product from the bead.
  • NTPs nucleotide triophosphates
  • the method may also include transcribing the sgRNA template libraries in the presence of DNase I.
  • the method may further include elution of the sgRNA template from the beads followed by PCR amplification of the sgRNA template.
  • the first and second RE may be selected from the appropriate enzymes shown above or other appropriate enzymes. The digestion and ligation may be performed essentially simultaneously.
  • a functional gRNA template produced by the method described herein may include one or more of the following: operably linked sequences, a promoter, a protospacer, an adapter, a RE II site, and a modified scaffold sequence.
  • a genome wide library may include a plurality of unique CRISPR-Cas system guide sequences that are capable of targeting a plurality of target sequences in genomic loci, wherein the library is created using one of the methods described herein.
  • One method for eluting polynucleotides from solid supports may include at least one of the following steps: attaching single stranded polynucleotide(s) to a solid support such that the 5' end of the polynucleotide is protruding from the solid support and is capable of hybridizing with at least a portion of a second polynucleotide(s); contacting the attached polynucleotide with a second polynucleotide such that hybridization between the two polynucleotides can occur, forming a polynucleotide duplex, immobilizing the second polynucleotide; contacting the hybridized duplex with a polymerase exhibiting strand displacement activity in the presence of dNTPs, in which the second polynucleotide is used as a template for extension by the polymerase, wherein the first polynucleotide is displaced by the extension of the polymerase, and extension prevents
  • the second polynucleotide contains sequences encoding for CRISPR/Cas9 guide RNAs.
  • the support may be a bead which may or may not be magnetic or
  • the attached polynucleotide may be a plurality of polynucleotides which may or may not have different sequences.
  • the method above may include a step of using the support to purify the second polynucleotide from a mixture.
  • hybridization may occur at different temperatures and the length of the hybridizing sequence can be adjusted to increase or decrease the temperature at which hybridization will occur.
  • the method for eluting may also include a physical separation process.
  • Selectively degrading unwanted polynucleotide components of a mixture may provide enzymatic protection of a CRISPR guide RNA (gRNA) library.
  • a method for selectively degrading unwanted polynucleotide components of a mixture may include one or more of the following steps: incorporation of at least five nucleotides linked by phosphorothioates at the 5’ ends of the double stranded DNA fragment to be protected; contacting the double stranded DNA fragments with a first exonuclease that operates on double stranded DNA and is sensitive to phosphorothioate linkages and a second exonuclease that operates on single stranded DNA.
  • polynucleotides with no protection or with a single end are degraded.
  • phosphorothioates may be incorporated by ligating adapters and/or incorporated by PCR.
  • a method for manipulation of DNA substrates through enzymatic processes to produce nucleotide sequences may include one or more of the following steps: simultaneously digesting DNA by targeting a restriction enzyme to sites containing protospacer adjacent motif (PAM) sequences in the DNA to produce DNA fragments; ligating adaptors to ends of DNA fragments to produce an intermediate product containing the scaffold ligated with a DNA fragment of an arbitrary length; and simultaneously digesting and ligating the intermediate product to produce guide RNA templates.
  • PAM protospacer adjacent motif
  • Simultaneously digesting DNA and ligating adaptors to ends of the DNA fragments may include digesting the DNA in presence of a ligase and a first adaptor.
  • the digesting may include a type IIS restriction enzyme to digest the intermediate product to create a guide RNA that may include an 18 to 25 base pair protospacer connected to the engineered polynucleotide.
  • the type IIS restriction enzyme blocks re-digestion of the ligation product.
  • the type IIS restriction blocks its own function after digestion by chemically modifying its own binding site. Chemically modifying the binding site may include attaching a methyl group.
  • the ligation may include connection of the digestion product to an upstream adapter to produce a guide RNA template containing an upstream adapter, a proto spacer and the engineered polynucleotide.
  • the upstream adapter may include at least one of a promoter, a cloning site, or other DNA integration site. In some examples, the upstream adapter does not contain a 5’ phosphate on the ligated end and the ligated product contains at least one nick.
  • the method may further include purifying the guide RNA templates.
  • This purification may include attaching polynucleotides in a sequence dependent hybridization to a bead, washing to remove reagents and fragments that are not attached to the beads, and eluting the guide RNA templates from the bead.
  • the bead may be a magnetic or paramagnetic bead.
  • the purification may include elution and nick repair of the guide RNA templates. In some examples, the elution and nick repair occur in the same reaction and may both be performed by a single enzyme. This single enzyme may include a strand displacing polymerase to simultaneously elute and repair nicks in the attached polynucleotides.
  • the strand displacing polymerase may use the previous captured polynucleotide as a template to displace and fill in a sequence, thereby permanently displacing the polynucleotide from the beads.
  • a hybridized segment of the previously captured polynucleotide may be made double stranded by the polymerase, thereby preventing rehybridization to the
  • the method may also include selecting a DNA source, which may include choosing a species of organism, selecting at least one of: a developmental stage of the organism, a tissue maturation stage, a state of cell differentiation and a stage of a cell cycle, selecting environmental conditions the organism is subject to, and selecting a tissue or cell type from the organism to be the DNA source.
  • DNA or RNA may be extracted from the selected DNA source, where the extracting may include at least one of: a chemical separation of cellular components and a physical separation of cellular components.
  • the extracting may isolate a nucleic acid species of interest.
  • the chemical separation of DNA or RNA may include amplification of the nucleic acid species.
  • the chemical separation may include enzymatic amplification of at least one region of the nucleic acid species.
  • This enzymatic amplification may include polymerase chain reaction (PCR).
  • the physical separation of DNA or RNA may include isolation of DNA or RNA by at least one of: precipitation isolation, hybridization isolation, antibody isolation, and other co -precipitation isolation.
  • the extracting may include extracting RNA from the selected DNA source and then converting the RNA into DNA.
  • the extracting may include normalizing the DNA by enzymatic or chemical methods applied to the DNA, where the step of normalizing may include balancing quantities of various DNA components in the mixture to ensure more equal representation in an output library.
  • a method for generating DNA templates for production of a CRISPR/Cas9 guide RNA library may include digesting DNA into digested products in the presence of a ligase and an adaptor, wherein the digested products are chemically impeded/driven/prevented from
  • the digesting and ligating may include digesting the polynucleotide sample at protospacer adjacent motifs (PAM) to form fragments with a first restriction enzyme (RE) in the presence of a ligase and first adapter, wherein the first adapter sequence may include a constant region containing a sequence CRISPR enzyme can bind to and/or cloning site sequence, polynucleotide sequences to one or both ends of the fragments with a ligase, thereby forming an intermediate product which lacks binding sites recognized by the first RE.
  • PAM protospacer adjacent motifs
  • RE restriction enzyme
  • the first adapter sequence may include a constant region containing a sequence CRISPR enzyme can bind to and/or cloning site sequence, polynucleotide sequences to one or both ends of the fragments with a ligase, thereby forming an intermediate product which lacks binding sites recognized by the first RE.
  • the method may include digesting the second polynucleotide with the type II restriction enzyme to form a third polynucleotide encoding a CRISPR sgRNA or CRISPR crRNA, wherein the type II restriction enzyme cuts the DNA at a site that is 17 to 27 base pairs from the end of the first polynucleotide.
  • a method for manipulation of DNA substrates through enzymatic processes to produce nucleotide sequences includes at least one of the following steps:
  • selecting a DNA source includes one or more of the following:
  • extracting may include:
  • chemical separation of DNA or RNA may include amplification of the nucleic acid species, wherein chemical separation may include enzymatic amplification of at least one region of the nucleic acid species, wherein the enzymatic amplification may include a polymerase chain reaction (PCR); i. wherein physical separation of DNA or RNA includes isolation of DNA or RNA by at least one of: precipitation isolation, hybridization isolation, antibody isolation, and other co precipitation isolation;
  • PCR polymerase chain reaction
  • extracting may include extracting RNA from the selected DNA source and then converting the RNA into DNA;
  • extracting may include normalizing the DNA using enzymatic or chemical methods applied to the DNA, wherein normalizing may include balancing quantities of various DNA components in the mixture to ensure more equal representation in an output library;
  • ligating may include introducing an engineered polynucleotide sequence that can associate with a CAS9 molecule (scaffold) such that the engineered polynucleotide sequence provides at least one of the following:
  • ii. contains a type IIS binding site such that enzyme will cut between 18 and 25 base pairs upstream of the adapter
  • iv. allows ligation of the digested DNA fragments by having a blunt end or containing an overhang compatible with the ends produced by enzymatic digestion;
  • the engineered polynucleotide sequence does not maintain the G-U hydrogen bond of the G at position 1 of the scaffold sequence, wherein the G is replaced by another base to meet conditions described above;
  • the engineered polynucleotide sequence may include a single gRNA scaffold or crRNA sequence or an adapter sequence for cloning into a vector containing either a single gRNA scaffold or crRNA sequence;
  • digesting may include a type IIS restriction enzyme to digest the intermediate product to create a guide RNA may include an 18 to 25 base pair protospacer connected to the engineered polynucleotide;
  • the type IIS restriction enzyme blocks redigestion of the ligation product; ii. wherein the type IIS restriction blocks its own function after digestion by chemically modifying its own binding site; wherein the chemical modifying its own binding site may include attaching a methyl group;
  • ligation may include connection of the digestion product to an upstream adapter to produce a guide RNA template containing an upstream adapter, a protospacer and the engineered polynucleotide; wherein the upstream adapter may include at least one of a promoter, a cloning site, or other DNA integration site;
  • upstream adapter does not contain a 5’ phosphate on the ligated end; ii. wherein the ligated product contains at least one nick;
  • purifying may include attaching polynucleotides in a sequence dependent hybridization to a bead, washing to remove reagents and fragments that are not attached to the beads, and eluting the guide RNA templates from the bead;
  • bead may include a magnetic or paramagnetic bead
  • purifying may include elution and nick repair of the guide RNA templates
  • the single enzyme may include a strand displacing polymerase to simultaneously elute and repair nicks in the attached polynucleotides;
  • the strand displacing polymerase uses the previous captured polynucleotide as a template to displace and fill in a sequence, thereby permanently displacing the polynucleotide from the beads;
  • a DNA substrate is cleaved by a first type II restriction endonuclease (RE) containing a PAM motif in its recognition sequence and ligation of a first adapter containing the CRISPR guide RNA scaffold sequence is carried out in a single reaction.
  • a modification to the scaffold sequence prevents the first adapter from being cleaved from the DNA substrate after being ligated.
  • the adapter ligation is therefore irreversible and forces the reaction to proceed toward the formation of the intermediate product.
  • the first adapter was synthesized without a phosphate group on the end to be ligated. This prevents the first adapter from ligating to other first adapters, which could result in the final library having truncated products.
  • Figure 2 depicts the digestion of the intermediate product (from the first reaction) by a second type II RE and ligation of a second adapter containing a promoter sequence is carried out in a single reaction.
  • the addition of a methyl moiety to the RE binding site in the first adapter by the second RE prevents cleavage of the second adapter after it has been ligated.
  • the second RE can still bind to the recognition sequence and cut across the nick.
  • the second adapter was also synthesized without a phosphate group on the end to be ligated.
  • the CRISPR Scaffold adapter is synthesized with a long, single-stranded, 5' overhang capable of hybridizing to a single stranded oligo immobilized on a solid support, such as a magnetic or agarose bead as shown in Figure 3.
  • Immobilization of the library on a solid support by hybridization with a complementary capture oligo, with the 5' end protruding allows the library to be purified from buffers, protein, and nucleic acid by physical separation.
  • a strand displacing polymerase can be added to permanently remove the fragments from the solid support. This is due to the directionality of the oligo attached to the beads and extension direction of the polymerase.
  • the captured oligos are used as a template by the polymerase and extension by the polymerase prevents re-hybridization. Double stranded DNA can then be preferentially isolated using standard silica-based separation techniques. Immobilization of the library to the solid support can occur at any point in the library construction process.
  • phosphorothioate linkages are synthesized into the adapters on the outside ends of the 5' strand as shown in Figure 4.
  • phosphorothioate linkages shown in black
  • PCR primers could also be used to incorporate the linkages into a DNA product that could then be subjected to the endonucleases.
  • a 3' to 5' endonuclease could also be used if protecting groups are incorporated into the outside ends of the 3' strand.
  • a constant region for sgRNA with nucleotide sequence of SEQ ID NO: 3 is recognized by CRISPR Cas9 protein.
  • the sgRNA with nucleotide sequence of SEQ ID NO: 3 can be transcribed from a double stranded polynucleotide having sense strand with nucleotide sequence of SEQ ID NO: 1 and an antisense with nucleotide sequence of SEQ ID NO: 2.
  • a constant region for sgRNA with nucleotide sequence of SEQ ID NO: 151 is recognized by CRISPR Cpfl protein.
  • the sgRNA with nucleotide sequence of SEQ ID NO: 151 can be transcribed from a double stranded polynucleotide having sense strand with nucleotide sequence of SEQ ID NO: 149 and an antisense with nucleotide sequence of SEQ ID NO: 1.
  • the CRISPR sgRNA(s) or cRNA(s) encoded by the polynucleotide or polynucleotides of various embodiments has at least one hairpin/stem-loop structure.
  • the CRISPR sgRNA(s) or crRNA(s) encoded by the polynucleotide or polynucleotides has one hairpin/stem-loop structure that are recognized by a CRISPR protein such as Cpfl.
  • the CRISPR sgRNA(s) or crRNA(s) encoded by the polynucleotide or polynucleotides has a plurality of hairpin/stem-loop structures that are recognized by a CRISPR protein such as Cas9.
  • recognition site has a homology or percent identity similar to an endogenous sequence of a CRISPR guide RNA or the hairpin regions such that the hairpin regions transcribed from the polynucleotide form and are recognized by a CRISPR protein.
  • the CRISPR sgRNA(s) or crRNA(s) encoded by the polynucleotide or polynucleotides of various embodiments are recognized by any CRISPR protein.
  • the CRISPR protein of various embodiments can include, for example, Class 1 or Class 2 CRISPR systems.
  • the CRISPR protein of various embodiments can include, for example, Type I, Type II, Type III, Type IV, or Type V CRISPR systems.
  • polynucleotides encoding for a constant region of a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) having the following sequence: 5’-CRlNl-RSN2-CR2N3-3’
  • sgRNA CRISPR single guide RNA
  • crRNA CRISPR targeting RNA
  • CR1 is a first constant region with a nucleotide(s) or modified nucleotide(s) including Adenine (A or a), Guanine (G or g), Cytosine (C or c), Thymine (T or t), and/or Uracil (U or u);
  • N1 is 0, 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, 105, 110, 115, 120 or more nucleotides;
  • RS is a non-palindromic recognition site for a type II restriction enzyme with a nucleotide(s) or modified nucleotide(s) including Adenine (A or a), Guanine (G or g), Cytosine (C or c), Thymine (T or t), or Uracil (U or u);
  • N2 is 4, 5, 6, 7, or 8;
  • CR2 is a second constant region with a nucleotide(s) or modified nucleotide(s) including Adenine (A or a), Guanine (G or g), Cytosine (C or c), Thymine (T or t), or Uracil (U or u); and
  • N3 is 0, 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, 105, 110, 115, 120 or more nucleotides;
  • RS is oriented in a manner recognized by the type II restriction enzyme for cutting a site that is 17 to 27 base pairs past a 5’ or 3’ end of the polynucleotide, in an operably linked targeting region sequence, if present.
  • polynucleotides encoding for a constant region of a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) having the at least one of the following sequences:
  • VR is a variable region (e.g. targeting region) with a nucleotide(s) or modified nucleotide(s) including Adenine (A or a), Guanine (G or g), Cytosine (C or c), Thymine (T or t), or Uracil (U or u);
  • Adenine A or a
  • Guanine G or g
  • Cytosine C or c
  • Thymine T or t
  • Uracil U or u
  • N4 is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides; and CR1, Nl, RS, and N2 are as described above. In various embodiments, N1 or N3 is range between any two number of nucleotides listed for N1 and N3 above.
  • N2 is range between any two number of nucleotides listed for N2 above.
  • N4 is range between any two number of nucleotides listed for N4 above.
  • the non-palindromic recognition site of the polynucleotide of various embodiments has a sequence recognized by a type IIS restriction enzyme.
  • the non-palindromic recognition site in a manner recognized by the type II restriction enzyme for cutting a site that is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 base pairs past an end of the polynucleotide, in an operably linked targeting region sequence, if present.
  • the cutting site is a range between any two base pair lengths past an end of the polynucleotide.
  • the non-palindromic recognition site is oriented in a manner recognized by the type II restriction enzyme for cutting a site that 17 to 27 base pairs past an end of the polynucleotide.
  • the non-palindromic recognition site is oriented in a manner recognized by the type II restriction enzyme for cutting a site that 18 to 24 base pairs past an end of the polynucleotide.
  • the type IIS restriction enzyme of various embodiments can include, for example, NmeAIII, Mmel, CstMI, EcoP15I, ApyPI, AquII, AquIII, AquIV, Cdpl, CstMI, DraRI, DrdIV, EsaSSI, Maql, NhaXI, NlaCI, PlaDI, PspOMII, PspPRI, Reel, RpaB5I, SdeAI, SpoDI, Bsbl, or combinations thereof.
  • the recognition sites for listed restriction enzymes are listed below (where the recognition site for each is followed by the cleavage distance):
  • the polynucleotide further includes a region having a sequence substantially complementary the sequence of non-palindromic recognition site.
  • the complementary region of various embodiments can be spaced either upstream of downstream from the non- palindromic recognition site.
  • the complementary sequence of sgRNA is capable of forming bonds with the non-palindromic recognition site such that hairpins or stem- loop structures form.
  • the CR1 and/or CR2 nucleotide can have a second restriction site or be prepared to have ends compatible with DNA digested with restriction enzymes that cut at protospacer adjacent motif (PAM) sites.
  • Restriction enzymes that cut at PAM sites include, for example: Hpall, Mspl, ScrFI, Bfal, and Pacl. The recognition sites for the listed restriction enzymes are listed below.
  • the polynucleotide or polynucleotides encoding for a constant region of a CRISPR sgRNA or crRNA excluding or including the non-palindromic recognition site or the non-palindromic recognition site and the complimentary region has at least 80%, 85%, 90%, 95%, 99%, or 100% identity to at least one of SEQ ID NO: 4; SEQ ID NO: 9; SEQ ID NO: 14; SEQ ID NO: 19; SEQ ID NO: 24; SEQ ID NO: 29; SEQ ID NO: 34; SEQ ID NO: 39; SEQ ID NO: 44; SEQ ID NO: 49; SEQ ID NO: 54; SEQ ID NO: 59; SEQ ID NO: 64; SEQ ID NO: 69; SEQ ID NO: 74; SEQ ID NO: 79; SEQ ID NO: 84; SEQ ID NO: 89; SEQ ID NO: 94; SEQ ID NO: 99; SEQ ID NO: 104; SEQ ID NO:
  • the polynucleotide or polynucleotides encoding for a constant region of a CRISPR sgRNA or crRNA comprise or are SEQ ID NO: 4; SEQ ID NO: 9; SEQ ID NO: 14; SEQ ID NO: 19; SEQ ID NO: 24; SEQ ID NO: 29; SEQ ID NO: 34; SEQ ID NO: 39; SEQ ID NO: 44; SEQ ID NO: 49; SEQ ID NO: 54; SEQ ID NO: 59; SEQ ID NO: 64; SEQ ID NO: 69; SEQ ID NO: 74; SEQ ID NO: 79; SEQ ID NO: 84; SEQ ID NO: 89; SEQ ID NO: 94; SEQ ID NO: 99; SEQ ID NO: 104; SEQ ID NO: 109; SEQ ID NO: 114; SEQ ID NO: 119; SEQ ID NO: 124; SEQ ID NO: 129; SEQ ID NO: 134; SEQ ID NO: 139
  • the following examples highlight Mmel site TCCRAC at an end, 1 base pair from an end, or 2 base pairs from an end.
  • the variable region of the examples can be 18, 19, or 20 base pairs long.
  • SEQ ID NO: 4 to SEQ ID NO: 148 relate to Cas9 systems.
  • the following examples highlights Mmel site TCCRAC at an end, 1 base pair from an end, or 2 base pairs from an end.
  • the variable region of the examples can be 18, 19, or 20 base pairs long.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 4 and antisense strand SEQ ID NO: 5.
  • the polynucleotide has sense strand SEQ ID NO: 6 and antisense strand SEQ ID NO: 7 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 8.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 9 and antisense strand SEQ ID NO: 10.
  • the polynucleotide has sense strand SEQ ID NO: 11 and antisense strand SEQ ID NO: 12 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 13.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 14 and antisense strand SEQ ID NO: 15.
  • the polynucleotide has sense strand SEQ ID NO: 16 and antisense strand SEQ ID NO: 17 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 18.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 19 and antisense strand SEQ ID NO: 20.
  • the polynucleotide can be digested with Hpall or Mspl to form a compatible end.
  • the polynucleotide has sense strand SEQ ID NO: 21 and antisense strand SEQ ID NO: 22 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 23.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 24 and antisense strand SEQ ID NO: 25.
  • the polynucleotide can be digested with ScrFI to form a compatible end.
  • the polynucleotide has sense strand SEQ ID NO: 26 and antisense strand SEQ ID NO: 27 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 28.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 29 and antisense strand SEQ ID NO: 30.
  • the polynucleotide can be digested with Bfal to form a compatible end.
  • the polynucleotide has sense strand SEQ ID NO: 31 and antisense strand SEQ ID NO: 32 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 33.
  • the following examples highlights NmeAIII site GCCGAG at an end, 1 base pair from an end, or 2 base pairs from an end.
  • the variable region of the examples can be 18, 19, or 20 base pairs long.
  • polynucleotide is double stranded having sense strand SEQ ID NO: 34 and antisense strand SEQ ID NO: 35.
  • the polynucleotide has sense strand SEQ ID NO: 36 and antisense strand SEQ ID NO: 37 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 38.
  • polynucleotide is double stranded having sense strand SEQ ID NO: 39 and antisense strand SEQ ID NO: 40.
  • DNA containing a variable region is ligated to the polynucleotide and subsequently digested with NmeAIII, the polynucleotide has sense strand SEQ ID NO: 41 and antisense strand SEQ ID NO: 42 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 43.
  • polynucleotide is double stranded having sense strand SEQ ID NO: 44 and antisense strand SEQ ID NO: 45.
  • DNA containing a variable region is ligated to the polynucleotide and subsequently digested with NmeAIII, the polynucleotide has sense strand SEQ ID NO: 46 and antisense strand SEQ ID NO: 47 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 48.
  • variable regions of the following examples can be 18 base pairs, 19 base pairs, or 20 base pairs long.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 49 and antisense strand SEQ ID NO: 50.
  • the polynucleotide has sense strand SEQ ID NO: 51 and antisense strand SEQ ID NO: 52 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 53.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 54 and antisense strand SEQ ID NO: 55.
  • the polynucleotide has sense strand SEQ ID NO: 56 and antisense strand SEQ ID NO: 57 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 58.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 59 and antisense strand SEQ ID NO: 60.
  • the polynucleotide has sense strand SEQ ID NO: 61 and antisense strand SEQ ID NO: 62 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 63.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 64 and antisense strand SEQ ID NO: 65.
  • the polynucleotide has sense strand SEQ ID NO: 66 and antisense strand SEQ ID NO: 67 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 68.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 69 and antisense strand SEQ ID NO: 70.
  • the polynucleotide has sense strand SEQ ID NO: 71 and antisense strand SEQ ID NO: 72 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 73.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 74 and antisense strand SEQ ID NO: 75.
  • the polynucleotide has sense strand SEQ ID NO: 76 and antisense strand SEQ ID NO: 77 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 78.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 79 and antisense strand SEQ ID NO: 80.
  • the polynucleotide has sense strand SEQ ID NO: 81 and antisense strand SEQ ID NO: 82 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 83.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 84 and antisense strand SEQ ID NO: 85.
  • the polynucleotide has sense strand SEQ ID NO: 86 and antisense strand SEQ ID NO: 87 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 88.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 89 and antisense strand SEQ ID NO: 90.
  • the polynucleotide has sense strand SEQ ID NO: 91 and anti sense strand SEQ ID NO: 92 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 93.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 94 and antisense strand SEQ ID NO: 95.
  • DNA containing a variable region is ligated to the polynucleotide and subsequently digested with Maql, the
  • polynucleotide has sense strand SEQ ID NO: 96 and antisense strand SEQ ID NO: 97 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 98.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 99 and antisense strand SEQ ID NO: 100.
  • the polynucleotide has sense strand SEQ ID NO: 101 and antisense strand SEQ ID NO: 102 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 103.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 104 and antisense strand SEQ ID NO: 105.
  • the polynucleotide has sense strand SEQ ID NO: 106 and antisense strand SEQ ID NO: 107 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 108.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 109 and antisense strand SEQ ID NO: 110.
  • the polynucleotide has sense strand SEQ ID NO: 111 and antisense strand SEQ ID NO: 112 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 113.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 114 and antisense strand SEQ ID NO: 115.
  • DNA containing a variable region is ligated to the polynucleotide and subsequently digested with PspOMII, the polynucleotide has sense strand SEQ ID NO: 116 and antisense strand SEQ ID NO:
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 119 and antisense strand SEQ ID NO: 120.
  • the polynucleotide has sense strand SEQ ID NO: 121 and antisense strand SEQ ID NO: 122 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 123.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 124 and antisense strand SEQ ID NO: 125.
  • the polynucleotide has sense strand SEQ ID NO: 126 and antisense strand SEQ ID NO: 127 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 128.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 129 and antisense strand SEQ ID NO: 130.
  • the polynucleotide has sense strand SEQ ID NO: 131 and antisense strand SEQ ID NO: 132 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 133.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 134 and antisense strand SEQ ID NO: 135.
  • the polynucleotide has sense strand SEQ ID NO: 136 and antisense strand SEQ ID NO: 137 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 138.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 139 and antisense strand SEQ ID NO: 140.
  • the polynucleotide has sense strand SEQ ID NO: 141 and antisense strand SEQ ID NO: 142 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 143.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 144 and antisense strand SEQ ID NO: 145.
  • the polynucleotide has sense strand SEQ ID NO: 146 and antisense strand SEQ ID NO: 147 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 148.
  • SEQ ID NO: 152 to SEQ ID NO: 406 relate to Cpfl systems.
  • Fig. 6A shows the hairpin structure of a wildtype cRNA for CRISPR Cpfl.
  • the following examples highlights Mmel site TCCRAC at an end, 1 base pair from an end, or 2 base pairs from an end.
  • the variable region of the examples can be 18, 19, or 20 base pairs long.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 152 and antisense strand SEQ ID NO: 153.
  • the polynucleotide has sense strand SEQ ID NO: 154 and antisense strand SEQ ID NO: 155 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 156.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 157 and antisense strand SEQ ID NO: 158.
  • the polynucleotide has sense strand SEQ ID NO: 159 and antisense strand SEQ ID NO: 160 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 161.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 162 and antisense strand SEQ ID NO: 163.
  • the polynucleotide can be prepared to have an AT overhang allowing the nucleotide to be compatible with DNA digested with Pacl.
  • the polynucleotide has sense strand SEQ ID NO: 164 and antisense strand SEQ ID NO: 165 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 166.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 167 and antisense strand SEQ ID NO: 168.
  • the polynucleotide can be prepared to have an AT overhang allowing the nucleotide to be compatible with DNA digested with Pacl.
  • the polynucleotide has sense strand SEQ ID NO: 169 and antisense strand SEQ ID NO: 170 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 171.
  • the following examples highlights NmeAIII site GCCGAG at an end, 1 base pair from an end, or 2 base pair from an end.
  • the variable region of the examples can be 18, 19, or 20 base pairs long.
  • polynucleotide is double stranded having sense strand SEQ ID NO: 172 and antisense strand SEQ ID NO: 173.
  • the polynucleotide has sense strand SEQ ID NO: 174 and antisense strand SEQ ID NO: 175 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 176.
  • polynucleotide is double stranded having sense strand SEQ ID NO: 177 and antisense strand SEQ ID NO: 178.
  • DNA containing a variable region is ligated to the polynucleotide and subsequently digested with NmeAIII, the polynucleotide has sense strand SEQ ID NO: 179 and antisense strand SEQ ID NO: 180 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 181.
  • polynucleotide is double stranded having sense strand SEQ ID NO: 182 and antisense strand SEQ ID NO: 183.
  • the polynucleotide can be prepared to have an AT overhang allowing the nucleotide to be compatible with DNA digested with Pad.
  • the polynucleotide has sense strand SEQ ID NO: 184 and antisense strand SEQ ID NO: 185 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 186.
  • polynucleotide is double stranded having sense strand SEQ ID NO: 187 and antisense strand SEQ ID NO: 188.
  • the polynucleotide can be prepared to have an AT overhang allowing the nucleotide to be compatible with DNA digested with Pad.
  • the polynucleotide has sense strand SEQ ID NO: 189 and antisense strand SEQ ID NO: 190 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 191.
  • variable regions of the following examples can be 19 base pairs or 20 base pairs long.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 192 and antisense strand SEQ ID NO: 193.
  • the polynucleotide has sense strand SEQ ID NO: 194 and antisense strand SEQ ID NO: 195 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 196.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 197 and antisense strand SEQ ID NO: 198.
  • the polynucleotide has sense strand SEQ ID NO: 199 and antisense strand SEQ ID NO: 200 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 201.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 202 and antisense strand SEQ ID NO: 203.
  • the polynucleotide has sense strand SEQ ID NO: 204 and antisense strand SEQ ID NO: 205 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 206.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 207 and antisense strand SEQ ID NO: 208.
  • the polynucleotide has sense strand SEQ ID NO: 209 and antisense strand SEQ ID NO: 210 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 211.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 212 and antisense strand SEQ ID NO: 213.
  • the polynucleotide has sense strand SEQ ID NO: 214 and antisense strand SEQ ID NO: 215 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 216.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 217 and antisense strand SEQ ID NO: 218.
  • the polynucleotide has sense strand SEQ ID NO: 219 and antisense strand SEQ ID NO: 220 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 221.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 222 and antisense strand SEQ ID NO: 223.
  • the polynucleotide has sense strand SEQ ID NO: 224 and antisense strand SEQ ID NO: 225 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 226.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 227 and antisense strand SEQ ID NO: 228.
  • the polynucleotide has sense strand SEQ ID NO: 229 and antisense strand SEQ ID NO: 230 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 231.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 232 and antisense strand SEQ ID NO: 233.
  • the polynucleotide has sense strand SEQ ID NO: 234 and antisense strand SEQ ID NO: 235 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 236.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 237 and antisense strand SEQ ID NO: 238.
  • the polynucleotide has sense strand SEQ ID NO: 239 and antisense strand SEQ ID NO: 240 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 241.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 242 and antisense strand SEQ ID NO: 243.
  • the polynucleotide has sense strand SEQ ID NO: 244 and antisense strand SEQ ID NO: 245 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 246.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 247 and antisense strand SEQ ID NO: 248.
  • the polynucleotide has sense strand SEQ ID NO: 249 and antisense strand SEQ ID NO: 250 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 251.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 252 and antisense strand SEQ ID NO: 253.
  • the polynucleotide has sense strand SEQ ID NO: 254 and antisense strand SEQ ID NO: 255 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 256.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 257 and antisense strand SEQ ID NO: 258.
  • DNA containing a variable region is ligated to the polynucleotide and subsequently digested with
  • the polynucleotide has sense strand SEQ ID NO: 259 and antisense strand SEQ ID NO: 260 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 261.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 262 and antisense strand SEQ ID NO: 263.
  • the polynucleotide has sense strand SEQ ID NO: 264 and antisense strand SEQ ID NO: 265 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 266.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 267 and antisense strand SEQ ID NO: 268.
  • the polynucleotide has sense strand SEQ ID NO: 269 and antisense strand SEQ ID NO: 270 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 271.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 272 and antisense strand SEQ ID NO: 273.
  • the polynucleotide has sense strand SEQ ID NO: 274 and antisense strand SEQ ID NO: 275 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 276.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 277 and antisense strand SEQ ID NO: 278.
  • the polynucleotide has sense strand SEQ ID NO: 279 and antisense strand SEQ ID NO: 280 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 281.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 282 and antisense strand SEQ ID NO: 283.
  • the polynucleotide has sense strand SEQ ID NO: 284 and antisense strand SEQ ID NO: 285 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 286.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 287 and antisense strand SEQ ID NO: 288.
  • the polynucleotide has sense strand SEQ ID NO: 289 and antisense strand SEQ ID NO: 290 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 291.
  • variable regions of the following examples can be 18 base pairs or 19 base pairs long.
  • the polynucleotide is double stranded having sense strand SEQ ID NO: 292 and antisense strand SEQ ID NO: 293.
  • the polynucleotide has sense strand SEQ ID NO: 294 and antisense strand SEQ ID NO: 295 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 296.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:297 and antisense strand SEQ ID NO: 298.
  • the polynucleotide has sense strand SEQ ID NO: 299 and antisense strand SEQ ID NO: 300 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 301.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:302 and antisense strand SEQ ID NO: 303.
  • the polynucleotide has sense strand SEQ ID NO: 304 and antisense strand SEQ ID NO: 305 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 306.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:307 and antisense strand SEQ ID NO: 308.
  • the polynucleotide has sense strand SEQ ID NO: 309 and antisense strand SEQ ID NO: 310 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 311.
  • the polynucleotide is double stranded having sense strand SEQ ID NOG 12 and antisense strand SEQ ID NO: 313.
  • the polynucleotide has sense strand SEQ ID NO: 314 and antisense strand SEQ ID NO: 315 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 316.
  • the polynucleotide is double stranded having sense strand SEQ ID NOG 17 and antisense strand SEQ ID NO: 318.
  • the polynucleotide has sense strand SEQ ID NO: 319 and antisense strand SEQ ID NO: 320 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 321.
  • the polynucleotide is double stranded having sense strand SEQ ID NOG22 and antisense strand SEQ ID NO: 323.
  • the polynucleotide has sense strand SEQ ID NO: 324 and antisense strand SEQ ID NO: 325 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 326.
  • the polynucleotide is double stranded having sense strand SEQ ID NOG27 and antisense strand SEQ ID NO: 328.
  • the polynucleotide has sense strand SEQ ID NO: 329 and antisense strand SEQ ID NO: 330 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 331.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:332 and antisense strand SEQ ID NO: 333.
  • the polynucleotide has sense strand SEQ ID NO: 334 and antisense strand SEQ ID NO: 335 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 336.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:337 and antisense strand SEQ ID NO: 338.
  • the polynucleotide has sense strand SEQ ID NO: 339 and antisense strand SEQ ID NO: 340 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 341.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:342 and antisense strand SEQ ID NO: 343.
  • the polynucleotide has sense strand SEQ ID NO: 344 and antisense strand SEQ ID NO: 345 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 346.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:347 and antisense strand SEQ ID NO: 348.
  • the polynucleotide has sense strand SEQ ID NO: 349 and antisense strand SEQ ID NO: 350 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 351.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:352 and antisense strand SEQ ID NO: 353.
  • the polynucleotide has sense strand SEQ ID NO: 354 and antisense strand SEQ ID NO: 355 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 356.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:357 and antisense strand SEQ ID NO: 358.
  • the polynucleotide has sense strand SEQ ID NO: 359 and antisense strand SEQ ID NO: 360 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 361.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:362 and antisense strand SEQ ID NO: 363.
  • the polynucleotide has sense strand SEQ ID NO: 364 and antisense strand SEQ ID NO: 365 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 366.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:367 and antisense strand SEQ ID NO: 368.
  • the polynucleotide has sense strand SEQ ID NO: 369 and antisense strand SEQ ID NO: 370 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 371.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:372 and antisense strand SEQ ID NO: 373.
  • the polynucleotide has sense strand SEQ ID NO: 374 and antisense strand SEQ ID NO: 375 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 376.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:377 and antisense strand SEQ ID NO: 378.
  • the polynucleotide has sense strand SEQ ID NO: 379 and antisense strand SEQ ID NO: 380 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 381.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:382 and antisense strand SEQ ID NO: 383.
  • the polynucleotide has sense strand SEQ ID NO: 384 and antisense strand SEQ ID NO: 385 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 386.
  • variable regions of the following examples can be 18 base pairs long.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:387 and antisense strand SEQ ID NO: 388.
  • the polynucleotide has sense strand SEQ ID NO: 389 and antisense strand SEQ ID NO: 390 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 391.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:392 and antisense strand SEQ ID NO: 393.
  • the polynucleotide has sense strand SEQ ID NO: 394 and antisense strand SEQ ID NO: 395 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 396.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:397 and antisense strand SEQ ID NO: 398.
  • the polynucleotide has sense strand SEQ ID NO: 399 and antisense strand SEQ ID NO: 400 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 401.
  • the polynucleotide is double stranded having sense strand SEQ ID NO:402 and antisense strand SEQ ID NO: 403.
  • DNA containing a variable region is ligated to the polynucleotide and subsequently digested with Bsbl, the
  • polynucleotide has sense strand SEQ ID NO: 404 and antisense strand SEQ ID NO: 405 that can be transcribed to an sgRNA or crRNA with sequence SEQ ID NO: 406.
  • SEQ ID NOS: 424-439 and 441 to 453 to are shown in Figures 6 and 7, respectively.
  • the sequences in the Tables below list a number additional sequences that are useful in the methods of the present invention. Each sequence satisfies the following criteria:
  • the Type IIS sequence is situated such that the enzyme cuts at least 17bp and not more than 25 bp upstream or downstream of the scaffold/constant region as cas9 has the variable region upstream, whereas in a Cpf 1 system, the variable region is downstream
  • Base pairing interactions in the stem loop are maintained without introducing a second Type II RS enzyme site (e.g., a second Mmel site).
  • overhangs can be included to aid ligation which could be generated using Mung Bean Nuclease (or similar enzyme) during digestion and ligation to blunt the products for ligation.
  • a representative embodiment entails use of the combination of ScrFI with Mmel (SEQ ID NOs: 454/455. This sequence contains the normal Mmel site where it will cut 18 bp upstream, most often, and generate a separate two-basepair N overhang.
  • Type IIS enzymes are useful in the methods of the invention (SEQ ID NOs: 480/481, 482/483, 484/485, 486/487, 488/489, 490/491, 492/493, 494/495, 496/497, 498/499, 500/501) 3.
  • engineered Type IIS nucleases that recognize different motifs are employed (SEQ ID NOs: 456/457, 458/459, 460/461, 462/463, 468/469, 472/473, 476/477)
  • sequence(s) listed and characterized below in Table III, or any combination thereof, may be used in the compositions, methods, or kits described herein.
  • any sequence(s) listed and characterized in Table IV, or any combinations thereof, may be used in the compositions, methods or kits described herein.
  • the polynucleotide(s) further include(s) a modification at least one modified sugar moiety, at least one modified internucleotide linkage, at least one modified nucleotide, or combinations thereof.
  • the modification of various embodiments can be located at or adjacent to the end of the polynucleotide.
  • the internucleotide linkage includes phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, or combinations thereof.
  • the modified nucleotide is selected from a peptide nucleic acid, a locked nucleic acid (LNA), or combination thereof.
  • the modified sugar moiety is selected from: a 2'-0-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-0-alkyl modified sugar moiety, a bicyclic sugar moiety, or combinations thereof.
  • the last two nucleotides from an end of the polynucleotide of various embodiments can have phosphate backbones that have been modified to include phosphorothioate.
  • phosphorothioate is resistant to nuclease degradation and allows for the
  • polynucleotide of various embodiments to be ligated with various types of DNA when during the ligation is occurring in the presence of nucleases.
  • the polynucleotide(s) is/are attached, affixed, or immobilized on a support such as a solid support.
  • the support of various embodiments can include two-dimensional surfaces such as microarray slides or three-dimensional surfaces such as beads or micro- spheres including polystyrene micro- spheres, magnetic microspheres, silica micro-spheres, or fluorescent micro- spheres.
  • expression cassettes, plasmid, or vectors including the polynucleotide encoding for the CRISPR sgRNA or CRISPR crRNA In other embodiments are disclosed expression cassettes, plasmid, or vectors including the polynucleotide encoding for the CRISPR sgRNA or CRISPR crRNA of various embodiments and a promoter polynucleotide operably linked to the polynucleotide of various embodiments, wherein the promoter polynucleotide is recognized by an RNA polymerase and is capable of directing the RNA polymerase to transcribe the CRISPR sgRNA or CRISPR crRNA from the polynucleotide of various embodiments.
  • the polynucleotide could be oriented within a plasmid including a topoisomerase as described in U.S. Patent No. 5,766,891, which is incorporated in its entirety by reference herein, or a cloning system such as a TOPO® Cloning System (Thermo Fisher Scientific).
  • a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein.
  • Reagents may be provided in any suitable container.
  • a kit may provide one or more reaction or storage buffers.
  • Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form).
  • a buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof.
  • the buffer is alkaline.
  • the buffer has a pH from about 7 to about 10.
  • the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element.
  • the kit comprises a homologous recombination template polynucleotide.
  • the buffer contains 50 mM Potassium Acetate; 20 mM Tris Acetate; 10 mM Magnesium Acetate; 100 pg/ml Bovine Serum Albumin; 1 mM ATP and 7.5% Polyethylene Glycol 6000.
  • Reaction mixes for an exemplary two step reaction process are set forth below.
  • CRISPR-Cas9 Most current applications of CRISPR-Cas9 take advantage of this simplicity to target single or small sets of genetic loci. Although this results in rapid and efficient targeting of single genes, CRISPR-Cas9 is not widely used for screening mutants to discover novel genes and pathways because of the expense of synthesizing complex libraries.
  • the advantage of the second is that it removes the need for blunting the digestion products.
  • the presently described guide strand libraries can be used in any species and requires only a small amount of enzymes.
  • the protocol can be adapted for all sources of DNA and can be completed in under four hours.
  • a kit for practicing the methods disclosed can comprise magnetic beads with attached oligos (e.g, streptavidin beads with biotinylated oligos), at least two different enzymes, optionally a mung bean nuclease, one or more enzyme digestion buffers, polyethylene glycol (PEG), ATP, and a T7 promoter (or other suitable promoter) oligo.
  • oligos e.g, streptavidin beads with biotinylated oligos
  • PEG polyethylene glycol
  • ATP adenote
  • T7 promoter or other suitable promoter
  • Lambda phage DNA commonly used as a control DNA substrate, was ordered from New England Biolabs.
  • the phage DNA was isolated from a dam- E. coli strain, which can be digested with restriction endonucleases that are sensitive to dam methylation.
  • the small, well-defined genome results in a library of 656 predicted sgRNAs when used as a substrate for library generation.
  • a 200 base pair fragment corresponding to the first exon of the tyrosinase gene in zebrafish was amplified by PCR. This fragment was selected due to the single Hpall cut site in the middle of the fragment.
  • the library results in 2 sgRNAs that target the coding region of the Tyr gene, which could be used in knockout experiments.
  • Oligos were synthesised and ordered from IDT and were either purified using standard desalting purification or PAGE purification for longer oligos. Complementary oligos were combined in Cutsmart buffer from New England Biolabs, and hybridized by heat denaturation at 95°C for two minutes and slowly cooled to room temperature using a PCR machine.
  • the scaffold adapter contained either the standard sgRNA sequence, or a sequence with an extended first stem loop that more closely resembles the native two-part crRNAdracrRNA system and has been shown to be more efficient in some cases.
  • the longer adapter also contained a cloning adapter sequence that could be digested for cloning into a plasmid using Golden Gate assembly.
  • the adapter also contained either the sequence for the standard Mmel enzyme or a rationally engineered variant that recognized G instead of C in the sixth position of the binding site.
  • the adapter was also designed to contain a two base pair sticky end that matches the enzyme first used to digest the starting DNA substrate.
  • the adapter also contained a long (-20 nt) single stranded overhang that could be used to capture the library from solution using magnetic beads.
  • the promoter adapter was designed to contain either the T7 RNA polymerase promoter sequence for direct transcription of the library or a cloning adapter sequence that could be digested for cloning into a plasmid using Golden Gate assembly.
  • the adapter was designed to contain a two base-pair, NN overhang compatible with the NN overhang created by Mmel digestion.
  • the first adapter contains a non-palindromic Type II restriction site within a sequence coding for only a portion of the CRISPR guide, with the remainder added via the second adapter or by cloning the final product into a vector containing the remaining portion of the sequence.
  • the Cas9 sgRNA sequence e.g., SEQ ID NO: 1 contains restriction sites for the Type II enzymes Alul, Setl, Msel, TspGWI, Banl, NlalV, Hinfl, Plel, and Mlyl.
  • An adapter containing a modified sgRNA sequence containing a non-palindromic Type II restriction site and the guide RNA sequence up to and including the restriction enzyme binding site of choice could be synthesized, used in our method, and then cloned into a vector containing the remaining sgRNA template using said restriction enzyme.
  • the resulting vector would then contain a complete sgRNA or crRNA constant region capable of coding for an RNA that binds Cas9.
  • the desired site can also be engineered into the constant region sequence if appropriate modifications are made to maintain the necessary stem loop structures.
  • other cloning methods such as Gibson, Golden Gate, blunt-ended cloning, or synthesized compatible overhangs that do not require the presence of a restriction site, can also be employed.
  • CRISPR sgRNA template libraries were created enzymatically using a novel method presented here.
  • Input DNA was first digested using the Hpall enzyme, which digests at the PAM containing motif CCGG, and simultaneously ligated in the same reaction to an sgRNA scaffold template modified to contain an Mmel site and a ligation overhang.
  • the ligation overhang is designed to be compatible with the CG overhang created by Hpall digestion without regenerating the site upon ligation to prevent redigestion of the ligation product.
  • the ligation product was then purified from the reaction using magnetic beads containing a capture oligo for the sgRNA scaffold template.
  • the beads were washed and resuspended in a second reaction containing Mmel to digest 18-19 bases upstream of the scaffold, creating a protospacer, and ligase to attach a U6 promoter.
  • the reaction was washed and the resulting sgRNA template eluted from the beads.
  • the library was amplified using 10 cycles of PCR.
  • Capture oligos were designed to be complementary to the long single stranded end of the scaffold adapter.
  • the oligos were designed to hybridize at 50°C so that they could hybridize to the scaffold adapter at room temperature. They were also designed with a biotin moiety on the 3' end, so that the 5' end was protruding from the beads.
  • the tube was placed in a magnetic rack for 1 minute or until the solution was clear. The supernatant was then removed and discarded from the tube. The beads were then resuspended in the solution that contained the final library template or an intermediate product in order to hybridize it to the capture oligo, thereby providing a method of physical separation using a magnetic field. The beads were then washed with Cutsmart buffer to remove reagents or products not attached to the beads. The beads were then suspended in a buffer that contained the strand displacing polymerase Bst 3.0 and the dNTPs required for extension and incubated at 45 °C for 15 minutes.
  • the polymerase has reduced activity, but the library is not likely to melt at the nicks still in the library at this stage. Because of the directionality of the oligo attached to the beads and extension direction of the polymerase, the single stranded end of the adapter can be used as a template and extension by the polymerase displaces the capture oligo and extension prevents re-hybridization. The polymerase will also repair the nicks in the library by extending the DNA strand at the nick and displacing the nicked strand.
  • sgRNA Transcription either the MEGAscriptTM T7 Transcription Kit (Thermo Fisher Scientific) or the HiScribeTM T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) was used to transcribe the sgRNA template libraries. In either case 210-300 ng of the template was used and incubated at 37°C for 2 hours. DNase I was then added and the reaction was then incubated for an additional 15 minutes at 37°C. The sgRNA was then purified using either the RNA Clean & Concentrator-5 Kit (Zymo Research) or by phenol-chloroform extraction.
  • Cas9 Nuclease from S. pyogenes and 10 x NEBuffer 3.1 were purchased from New England Biolabs. The transcribed sgRNA library was allowed to complex with Cas9 molecules in the buffer. The DNA substrate to be digested was then added to the solution. cDNA synthesis and normalization
  • the mRNA population constitutes approximately 1% of total RNA with the number of transcripts varying from several thousand to several tens of thousands.
  • the high abundance transcripts (several thousand mRNA copies per cell) of as few as 5-10 genes account for 20% of the cellular mRNA.
  • the intermediate abundance transcripts (several hundred copies per cell) of 500-2000 genes constitute about 40-60% of the cellular mRNA.
  • the remaining 20-40% of mRNA is represented by rare transcripts (from one to several dozen mRNA copies per cell). Such an enormous difference in abundance complicates large-scale transcriptome analysis, which results in recurrent sequencing of more abundant cDNAs.
  • cDNA normalization decreases the prevalence of high abundance transcripts and equalizes transcript concentrations in a cDNA sample, thereby dramatically increasing the efficiency of sequencing and rare gene discovery.
  • One approach for nucleic acid normalization exemplified in PANC1 Rat Beta Cells entails collection of cells by trypsinizing a 10 cm plate and placing the sample in Trizol solution. Total RNA was then extracted using the Zymo Directzol kit (Zymo Research) using standard procedures. cDNA of polyadenylated RNA species was then generated using the Mint-2 cDNA synthesis kit (Evrogen) and normalized by subtractive hybridization using the Trimmer-2 kit (Evrogen).
  • CRISPR libraries are rapidly becoming an important tool for gene discovery across a wide- range of fields and applications, including drug screening, crop improvement, molecular studies of embryonic development, and others.
  • Current methods for these experiments synthesize large pooled libraries of individually designed CRISPR sgRNAs. While synthesis is straight-forward, it is limited by several factors. First, although prices have decreased in recent years, synthesis of large pools remains expensive and requires a turnaround time of 3-4 weeks. In addition, synthesis reactions can introduce errors, lowering the percentage of guides with the correct sequence. Cost constraints also limit the coverage of the library to a few guides per gene and severely hinder the ability to make custom libraries targeted to a specific cell-type or tissue. Finally, these libraries require significant a priori knowledge of the genome as they rely
  • the resulting process involves the collection of DNA or RNA from a
  • the initial digestion and ligation steps are combined.
  • the scaffold adapter sequence is modified in the repeat: anti-repeat duplex, a preferred modification is effective to prevent digestion of the scaffold adapter.
  • the restriction enzyme binding site can be destroyed during the ligation if the double stranded region has a base pair change. Destroying the restriction enzyme binding site upon ligation of the adapter would prevent the Hpall restriction enzyme from cleaving off the adapter, and permit digestion and ligation in a single reaction, simplifying the procedure. It would also force the reaction to use all of the scaffold adapter, increasing the efficiency and yield of the reaction.
  • the enzyme In order to combine the first digestion and ligation step into a single step, the enzyme must work in the same buffer. To increase the ligation speed and efficiency, high concentrations of ATP and 7.5% polyethylene glycol (PEG) were used in the ligation steps as a macromolecular crowding reagent. The activity of some enzymes is enhanced if PEG is included in the reaction buffer, but the activity of others is inhibited.
  • PEG polyethylene glycol
  • Mspl which has the same binding site as Hpall
  • Mmel is a Type IIS restriction enzyme that simultaneously methylates its binding site while cutting the DNA approximately 20 bp upstream. In biotechnological applications, this methylation is purely an artifact of the digestion and can actually be detrimental as it potentially inhibits downstream uses of the DNA, such as blocking methylation sensitive enzymes. As Mmel itself is methylation sensitive, the change has the effect of blocking re-digestion of the same DNA by Mmel itself. This is generally not desirable, as it limits the effectiveness of the enzyme by decreasing the number of enzymes bound to the DNA and forming the dimers required for Mmel function.
  • the DNA can be digested with Hpall. If the DNA is re-ligated to itself or another digested DNA fragment, it will be digested again. However, if it is ligated to an adapter, it will not be digested because the binding site is destroyed. A similar process happens during the subsequent Mmel digestion and T7 ligation. Although the binding site for Mmel cannot be destroyed, when it cleaves, it methylates the A in the second position, preventing Mmel from cutting again at that site, and therefore will not be able to cut off the T7 adapter after it has been ligated.
  • each ligation reaction involves unique overhangs not compatible with any other product or the oligos are not phosphorylated.
  • the T7 adapter cannot ligate directly to the first adapter, even if present during the initial digestion. Therefore, the four steps are combinable, and all four steps can be carried out in a single reaction.
  • the nick generated is repaired using a polymerase.
  • the second adapter can also ligate to itself, decreasing the efficiency of the library construction process.
  • the second set of adapters were also synthesized without a phosphate group. In vitro digestion with the Mmel restriction enzyme demonstrated that it cuts across this nick with high efficiency. In certain instances, the ratio of the DNA input to the adapter was increased to 2:1, which reduces the formation of any aberrant shorter library products below detectable levels.
  • PEG polyethylene glycol
  • the library was constructed on the surface of strep tavidin coated magnetic beads.
  • Magnetic beads with a poly-T oligonucleotide can be used to capture mRNA in the preparation of cDNA.
  • Some methods for building out strands of DNA on magnetic beads have also been published (Pengpumkiat et al., 2016). Because magnetic beads have liquid-phase like reaction kinetics, they are suitable for construction of the library using the method reactions described herein.
  • Streptavidin magnetic beads (50 pL) bind about 100 pmol of biotinylated DNA. Different biotinylated oligos were synthesized and the binding capacity of the beads was tested in both
  • an alternative approach could entail increasing the concentration of Mmel and performing digestion prior to bead attachment.
  • the present method where the substrates were purified before cutting, allowed the buffer to be exchanged for a buffer optimized for the endonuclease activity of Mmel.
  • Magnetic beads have been used in biological reactions as a solid support to facilitate post reaction cleanup, buffer exchange, and enzyme removal. Oligonucleotides can be attached to solid supports using several different methods, but removal from the beads is often desired for
  • One method employs a restriction enzyme to cleave the DNA.
  • Another method employs a photo-cleavable biotin moiety attached to the scaffold adapter.
  • This biotin moiety has been synthesized previously and can be attached to
  • an oligonucleotide Upon irradiation with ultraviolet light, the biotin is cleaved from the oligo and leaves the oligo with a phosphorylated 5' end. We found that in a clear solution, the biotin is readily cleaved with the UV light. However, when attached to magnetic beads, only a small fraction of the biotin was actually cleaved, likely due to the opacity of the beads interfering with passage of light to the photocleavable moiety. Using agarose beads could have solved this problem, but the handling capabilities of the magnetic beads are very convenient and can easily be
  • An alternative approach entails displacement of the library from the beads using a short oligo.
  • DNA nanotechnology applications can use strand displacement reactions to construct 3 dimensional structures (Ijas et ak, 2018). Strand displacement reactions can also be used to build logical circuits and therefore carry out mathematical calculations (Jiang et al., 2019).
  • Toehold- mediated strand displacement entails use of a toehold (e.g., a short, single stranded overhang on a DNA duplex).
  • a third oligonucleotide is added that is complementary not only to the duplex, but the toehold as well, this oligo can displace the shorter oligo of the duplex.
  • the strand could be displaced by the Bst 3.0 polymerase used to repair nicks in the library, and thus we proceed with the Bst 3.0 based approach.
  • the strand displacing polymerase Bst 3.0 has high displacement activity and can extend DNA at nicks, displacing the strand as it fills in the template. Because there was a nick in the DNA where the capture oligo hybridized during the capture of the library ( Figure 3), the Bst polymerase should displace the capture oligo and fill in the gap at the same time, essentially eluting the library from the beads.
  • An adapter was designed with a 19 bp overhang with a melting temperature of approximately 50°C that could hybridize to the short biotinylated capture oligo attached to the beads.
  • the adapter can then be attached to the beads by simply incubating at room temperature with the capture beads. Incubating the beads with Bst 3.0 elongates the DNA at the nick and displaces the library from the beads. Because the polymerase extends the DNA to the end of the fragment, this reaction is not reversible, and the library remains detached from the beads.
  • other strand-displacing enzymes in addition to Bst3.0 e.g., Phi29, Bstl.O, Bst 2.0 and large Klenow fragment
  • Bst3.0 e.g., Phi29, Bstl.O, Bst 2.0 and large Klenow fragment
  • polymerase dependent strand displacement can be used to irreversibly elute oligonucleotides from solid supports, creating complete, double- stranded DNA in the process.
  • the adapters would still hybridize to the beads in the presence of PEG.
  • Our data reveal that DNA actually hybridizes better in PEG and experiments show that hybridization of the adapter to the beads takes place in 7.5% PEG when resuspended well. It is, therefore, possible to carry out ligations in PEG and subsequently hybridize the DNA to the beads.
  • the nicks must first be repaired.
  • T4 polynucleotide kinase has little activity on nicked DNA substrates, so the nicks could not simply be sealed by phosphorylation and ligation.
  • nicks were placed in positions that would allow nick repair using a strand displacing polymerase.
  • the final library was contacted with the DNA polymerase Bst 3.0 and dNTPs and the data demonstrated that it effectively repaired the nicks in the library.
  • the polymerase has full activity at 65°C and 75% activity at 45°C.
  • the overlap holding the library fragments at this point is only 18 bp long, we incubated the beads at 45°C to prevent the fragments from coming apart.
  • the strand displacing polymerase phi29 works at lower temperatures and could also be used to displace the library, although the displacement activity of this polymerase is not as high. In this approach, the single stranded fragments left after this process can then be removed using standard methods.
  • An alternative purification method is to selectively degrade any incomplete DNA products. This entails protecting both sides of the final library with adapters that included phosphorothioate linkages to protect from exonuclease degradation and then adding the appropriate nucleases. The presence of 6 consecutive phosphorothioates in place of phosphates at the 5' end of an
  • oligonucleotide can effectively block digestion by lambda exonuclease (Eckstein and Gish, 2018).
  • Lambda Exonuclease works on double stranded and single stranded DNA substrates in the 5’ to 3’ direction but is effectively blocked by 6 consecutive phosphorothioates on the 5’ end (shown by black bold letters in Figure 3).
  • Exonuclease I can only act on single stranded DNA substrates and digests in the 3’ to 5’ direction.
  • Adapters were synthesized with phosphorothioates such that digestion by exonucleases was prevented if and only if both adapters are attached at opposite ends of the product. Because the final library will be the only product with the protecting groups on both ends, it will be the only product that will not be degraded when both exonucleases are added to the solution ( Figure 4). This method can be used in place of the bead purification method.
  • a rapid, efficient method for enzymatically generating CRISPR sgRNA libraries by combining restriction and ligation steps in a single reaction vessel is disclosed. Combining these steps makes the process faster and more efficient. Mixing the enzymes into a single reaction eliminates the need for a heat inactivation step and a second incubation step, reducing the amount of time and number of sample manipulations required in the process. Combining these steps also increases the efficiency of the reaction. In both steps, the elimination of additional magnetic bead separation and wash steps limits the losses of reaction product that occur during these steps. Further advantages are achieved in the first step because the reagents are designed to drive the reaction to completion.
  • the ligation reaction two products can occur: the ligation with the scaffold, which is desirable, and ligation with another digestion product, which is not.
  • the undesirable ligation of two products from the first digestion are normally removed during later purification steps as an unwanted side-product. As these fragments are not included in the final library, more input DNA must be supplied to account for these losses.
  • Our inventive modification to the scaffold results in a ligation product without the restriction site, thereby preventing re-digestion of the desired product. Accordingly, the desired ligation products accumulate, while ligations between two DNA input fragments are re-digested by the restriction enzyme— driving the
  • CRISPR gRNA libraries generated using the process described herein can be used in highly customizable CRISPR library applications as they can be generated from a wide variety of DNA sources.
  • the first step in enzymatically generating an sgRNA library is to decide on a DNA substrate to use as a starting material, as the substrate that is acquired will determine what the library can be used for in downstream applications.
  • sources that can be used as a starting substrate. For example
  • PANC1 Rat Beta Cells were collected by trypsinizing a 10 cm plate and DNA extracted using the GeneCatcher gDNA (genomic DNA) Blood Kit (ThermoFisher).
  • PANC1 Rat Beta Cells were collected by trypsinizing a 10 cm plate and placing the sample in Trizol solution. Total RNA was then extracted using the Zymo Directzol kit (Zymo Research) using standard procedures. cDNA of polyadenylated RNA species was then generated using the Mint-2 cDNA synthesis kit (Evrogen) and normalized by subtractive hybridization using the Trimmer-2 kit (Evrogen).
  • Chromatin immunoprecipitation was performed as previously described (Schaffer et ah, 2013). Cells were first crosslinked with formaldehyde and then lysed and DNA collected.
  • the putative enhancer region driving insulin expression was amplified by PCR. As the region was 20 kb long, 10 PCR reactions were created, each producing a 2kb fragment. The PCR products were then purified using the Zymo Research DNA Clean and Concentrate kit and pooled for library generation.
  • Beta cell transcriptome sgRNA library was cloned into a modified lentiviral vector containing the Cas9 coding sequence under the control of the eflalpha core promoter.
  • a single lentivims will produce both Cas9 and the sgRNA.
  • Calculations based on previously published RNA-seq data showed that beta-cells express approximately 11,000 genes.
  • our library contains approximately 200,000 sgRNA sequences targeting each expressed gene an average of 16 times— providing a high coverage library targeted to beta-cell relevant genes.
  • Lentiviral constructs were then electroporated into DH5alpha cells and grown overnight before harvesting the plasmids using the Monarch Plasmid Miniprep kit (New England Biolabs). A total of 50 electroporations were conducted to ensure at least lOx coverage of the library.
  • the sgRNA templates in the resulting library were PCR amplified and sequenced on an
  • Digest source DNA e.g. genomic DNA, a PCR product, or double-stranded cDNA (30 min, concurrent with step 1)
  • Digest and ligate source DNA e.g. genomic DNA, a PCR product, or double-stranded
  • Ligate T7 promoter (10 minutes) 4.1. Add 50 m ⁇ T7 promoter mix, 1 m ⁇ Ligase and 1 m ⁇ ATP + PEG to beads. Rock for 5 minutes, place on magnet for 1 minute and discard supernatant.
  • Digest source DNA e.g., genomic DNA, a PCR product, or double-stranded cDNA (30 min)
  • Digest and ligate source DNA e.g. genomic DNA, a PCR product, or double-stranded
  • Digest and ligate source DNA e.g. genomic DNA, a PCR product, or double-stranded cDNA (30 min)
  • Mspl 1 m ⁇ Ligase
  • Mmel (G6) enzyme 1 ul 2.5 mM SAM
  • 22 m ⁇ T7 promoter mix 1 m ⁇ ATP + PEG.
  • the library produced as described above can be used in a variety of applications. For example, there is increasing interest in creating libraries containing thousands of different sgRNAs targeting large sets of genes (Shalem et ah, 2014). Several labs have been able to create sgRNA for a few model organisms (Shalem et ah, 2014, Doenchet ah, 2016). One such library was designed and chemically synthesized for use in Drosophila cells. This library was composed of 40,279 different sgRNAs and targeted 13,501 different genes (Bassett et ah, 2015).
  • the library was cloned into a plasmid containing the ubiquitous U6 promoter and a separate expression construct consisting of an actin promoter followed by the Cas9 mRNA sequence.
  • the plasmid library was then maintained in millions of bacterial colonies. After recovery of the plasmid library, it was transfected into a
  • Drosophila cell line which was then screened for cellular phenotypes.
  • the targeting regions of the sgRNAs in the plasmids were sequenced and mutated genes could then be inferred based on the sequence. Similar screens have been carried out in human cells (Wang et ah, 2014), as well as cancer cells (Hart et ah, 2014). Additionally, these sgRNA libraries can be used to study noncoding regions of the genome such as enhancer elements (Korkmaz et ah, 2016). These screens show that complex sgRNA libraries can be powerful tools in genomic research.
  • an sgRNA library targeting the genome along with Cas9 protein could be delivered to the germline of an organism. After the germline is mutated, a traditional mating scheme could be set up and resultant mutants screened.
  • Cas9 over ENU as a mutagen has a distinct advantage: a library could be constructed that would only target a subset of the genes, making the screen more focused on the genes of interest. A different approach would be to dilute the sgRNA library into small pools and PCR amplify the pools.
  • sgRNA libraries Fusing other proteins to Cas9 has led to innovative applications of sgRNA libraries.
  • One of these applications is the ability to paint a chromosomal locus with a fluorescent marker (Lane et al., 2015).
  • a bright version of GFP can be fused to a nuclease-deficient dCas9 and an sgRNA library was generated to target a small region of a chromosome.
  • the dCas9-GFP molecules are complexed with the sgRNA library and the library is injected into living cells.
  • the fluorescent Cas9 essentially tiles the chromosomal region and can be visualized under a fluorescent microscope. Cas9 has also been used to make changes to the epigenome.
  • Proteins that act as activators or repressors have been fused to Cas9 and have been shown to efficiently upregulate or downregulate transcription in human and yeast cells (Gilbert et al., 2013).
  • Cas9 has been fused to proteins that modify histones such as histone demethylases (Kearns et al., 2015). Large scale epigenetic changes could be made by using an sgRNA library in combination with these techniques.
  • Enzymatically generating customized gRNA libraries will enable these and many other CRISPR applications by allowing the researcher to focus the study on the genes of interest while broadening the use of CRISPR libraries into new species and new paradigms.
  • a first polynucleotide which encodes an RNA bound by a cas enzyme may include a constant region encoding sequence selected from a CRISPR single guide RNA (sgRNA) and a CRISPR targeting RNA (crRNA) having a non-palindromic recognition site recognized by a type II restriction enzyme oriented in the sequence and configured to cleave a second operably linked polynucleotide 17 to 27 base pairs from the recognition site, the type II restriction enzyme having methylase activity and cleavage activity, the recognition site may include a nucleotide which may be methylated by the type II restriction enzyme upon cleavage, methylation of the nucleotide altering the recognition site such that the type II restriction enzyme no longer binds the site.
  • sgRNA CRISPR single guide RNA
  • crRNA CRISPR targeting RNA
  • a first polynucleotide may be operably linked to the second polynucleotide which encodes a variable or targeting region which hybridizes to a sequence of interest.
  • the non- palindromic recognition site when transcribed may be capable of being incorporated within a stem- loop structure of the CRISPR sgRNA or CRISPR crRNA without disrupting Cas9 binding at the constant region in the polynucleotide.
  • a first polynucleotide which encodes an RNA bound by a cas enzyme may include a constant region encoding sequence for a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) having a non-palindromic recognition site recognized by a type II restriction enzyme oriented in the sequence such that a second operably linked polynucleotide may be cleaved 17 to 27 base pairs from the recognition site, when present, the type II restriction enzyme having methylase activity and cleavage activity, the recognition site may include a nucleotide which may be methylated by the type II restriction enzyme upon cleavage, methylation of the nucleotide altering the recognition site such that the type II enzyme no longer binds the site, wherein the first polynucleotide may be a plurality of first polynucleotides; at least a portion of the plurality of first polynucleotides being operably linked to a second polynucleot
  • the polynucleotide or the plurality of third polynucleotides may be operably linked to a promoter sequence(s).
  • the type II restriction enzyme binding site sequence may optionally be methylated.
  • the Type II restriction enzyme site may be selected from the group consisting of one or more of NmeAIII, Mmel, CstMI, EcoP15I, ApyPI, AquII, AquIII, AquIV, Cdpl, CstMI, DraRI, DrdIV, EsaSSI, Maql, NhaXI, NlaCI, PlaDI, PspOMII, PspPRI, Reel, RpaB5I,
  • the polynucleotides above may also include one or more sequence adapters suitable for cloning.
  • the polynucleotide above may be double stranded and/or may include a single stranded overhang of between 1 and 100 nucleotides on one or both ends.
  • the constant region may contain at least a portion of the stem loop sequence and the second constant region polynucleotide may encode the remaining portion of a functional stem loop structure, wherein the operable linkage reform a functional stem loop structure bound by cas enzyme.
  • the constant region comprises elongated stem loops or additional sequences at a 5’ end, a 3’ end or both, while maintaining a CRISPR enzyme binding site.
  • a method for generating DNA templates for production of a CRISPR/Cas guide strand library may include providing a polynucleotide sample, digesting the polynucleotide sample at protospacer adjacent motifs (PAM) to form target region containing fragments with a first restriction enzyme (RE) in the presence of a ligase and a first adapter sequence, the first adapter sequence may include a constant region having a CRISPR enzyme binding site, the ligase operably linking the first adapter sequence to one or both ends of the fragments, thereby forming an intermediate product which lacks a binding site recognized by the first RE, the intermediate product may include at least one binding site for a second Type II RE; digesting the intermediate product with the second Type II RE in the presence of a ligase and a second adapter sequence, the Type II RE having methylation and nuclease activity which methylates the product at the recognition site and cleaves the targeting region between 17 to 27 base pairs away from the recognition site to form
  • the first digestion and ligation may be carried out in two steps. Additionally or alternatively, the second digestion and ligation are carried out in two steps. In some examples, the first digestion and ligation and second digestion and ligation are carried out in a single reaction vessel.
  • the polynucleotide sample may be obtained from a source selected from one or more of an organism of interest, an organism at a selected stage of development, a tissue of interest, a cell at a selected stage of differentiation, a cell at a particular stage of the cell cycle, a tissue or cell having a selected pathology.
  • the polynucleotide sample may be selected from cDNA synthesized from RNA, genomic DNA, mitochondrial DNA, human DNA, animal DNA, and plant DNA.
  • the polynucleotide sample may contain polynucleotides isolated via a method selected from precipitation, hybridization, antibody isolation, and co-precipitation.
  • the second adapter may include a promoter element.
  • the cloning site sequence may be present and the ligated product may be cloned into a vector the vector optionally includes an operably linked promoter sequence. All the steps may be performed in a single reaction vessel.
  • the first polynucleotide may be a plurality of first polynucleotides. At least a portion of the plurality of first polynucleotides may be operably linked to DNA to form a plurality of second linked polynucleotides.
  • the plurality of second linked polynucleotides may be digested with the type II restriction enzyme having methylase activity to form a plurality of third polynucleotides encoding a plurality of CRISPR sgRNAs or CRISPR crRNAs. At least one of the plurality of CRISPR sgRNAs or CRISPR crRNAs may have a variable region different from the other CRISPR sgRNAs or CRISPR crRNAs.
  • the polynucleotide sample may be a cDNA sample which may be normalized to remove repeated transcripts from the cDNA sample, thereby increasing equal representation of transcripts in the library.
  • the adapters may lack a 5’ phosphate.
  • the adapters may contain at least six consecutive phosphorothioates at the 5’ end.
  • the polynucleotide sequence may be digested with an enzyme selected from the group consisting of Hpall, Mspl, ScrFI, Bfal, and Pack
  • the ligated CRISPR sgRNA or CRISPR crRNA product may include at least one nick.
  • the second adapter may include a promoter sequence.
  • the promoter may be T7 RNA polymerase promoter.
  • the ligated product does not maintain a G-U hydrogen bond at position 1 of the scaffold sequence.
  • the method may further comprise purifying the ligated CRISPR sgRNA or CRISPR crRNA product.
  • the first adapter sequence may include a 5’ single stranded overhang.
  • the ligated CRISPR sgRNA or CRISPR crRNA product may be purified using a solid support operably linked to a capture oligonucleotide at a 3’ end, where the oligonucleotide hybridizing to the overhang of the adapter sequence.
  • the solid support may be a magnetic bead and the purification step includes magnetic separation.
  • Suspending the separated beads in a buffer may include Bst 3.0 polymerase and nucleotide triophosphates (NTPs) at about 45°C for about 15 minutes, thereby repairing and extending the nicked CRISPR sgRNA or CRISPR crRNA product, the extension causing displacement of repaired CRISPR sgRNA or CRISPR crRNA product from the bead.
  • the method may include at least one of the following: transcribing the sgRNA template libraries in the presence of DNase I; PCR amplification of the ligated CRISPR sgRNA or CRISPR crRNA product; and the digestion and ligation steps may be performed essentially simultaneously.
  • a method for generating DNA templates for production of a CRISPR/Cas guide strand library may include: a) providing a polynucleotide sample; b) digesting the polynucleotide sample at protospacer adjacent motifs (PAM) to form a target region containing fragments with a first restriction enzyme (RE) in the presence of a ligase and a first adapter sequence, the first adapter sequence may include a constant region having a CRISPR enzyme binding site, the ligase operably linking the first adapter sequence to one or both ends of the fragments, thereby forming an intermediate product which lacks a binding site recognized by the first RE, the intermediate product may include at least one binding site for a second Type II RE; c) digesting the intermediate product with the second Type II RE in the presence of a ligase and a second adapter sequence, the Type II RE having methylation and nuclease activity which methylates the product at the recognition site and cleaves the targeting region between 17 to 27 base
  • the first polynucleotide may be displaced by the extension of the second polynucleotide, and the extension preventing rehybridization between the first and second polynucleotides, the second polynucleotide encoding a CRISPR sgRNA or CRISPR crRNA.
  • a method for eluting polynucleotides from solid supports may include a) by immobilizing one or more first single stranded polynucleotide(s) to a solid support, the first single stranded polynucleotide having 5’ and 3’ ends and having binding affinity for at least a portion of a second polynucleotide(s), the 5' end of the first single stranded polynucleotide protruding from the solid support; b) contacting the one or more first polynucleotide(s) with one or more second
  • the support may be a bead.
  • the bead may be a magnetic bead.
  • polynucleotide may be a plurality of polynucleotides and the second polynucleotide may be a plurality of polynucleotides with different sequences.
  • the method may further comprise purifying the second polynucleotide from a mixture.
  • the length of the hybridizing sequences may be adjusted to increase or decrease the temperature at which hybridization will occur.
  • the polynucleotides may be purified at different temperatures and/or the polynucleotides are separated via a process selected from the group consisting of magnetic separation, precipitation, hybridization, antibody isolation, and co-precipitation.
  • One or more second polynucleotides may encode for one or more
  • the intermediate or ligated product may include at least five phosphorothioate linked polynucleotides at the 5’ end.
  • the method may also include degrading any unwanted polynucleotides present in the reaction by contacting the reaction with first exonuclease and second exonucleases which cleave double stranded and single stranded DNA respectively, the phosphorothioate containing polynucleotide being resistant to exonuclease cleavage.
  • the polynucleotides lacking phosphorothioate linkages may be degraded.
  • the polynucleotides with a single phosphorylated end may be degraded.
  • the phosphorothioates may be incorporated by ligating adapters and/or incorporated by PCR.
  • a kit for production of an sgRNA guide strand library may include: a first polynucleotide which encodes an RNA bound by a cas enzyme, may include a constant region encoding sequence for a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) having a non- palindromic recognition site recognized by a type II restriction enzyme oriented in the sequence such that a second operably linked polynucleotide may be cleaved 17 to 27 base pairs from the recognition site, when present, the type II restriction enzyme having methylase activity and cleavage activity, the recognition site may include a nucleotide which may be methylated by the type II restriction enzyme upon cleavage, methylation of the nucleotide altering the recognition site such that the type II enzyme no longer binds the site.
  • sgRNA CRISPR single guide RNA
  • crRNA CRISPR targeting RNA
  • the kit may also include one or more of: a second polynucleotide encoding a variable or targeting sequence, one or more ligases for operably linking the constant region and targeting polynucleotides, one or more Type II restriction enzymes, a solid support, a strand displacing polymerase, one or more adapter sequences encoding a promoter and/or cloning site, buffers suitable for simultaneous digestion and ligation of a polynucleotide and optionally reagents suitable for PCR amplification, reagents suitable for normalization of input nucleic acid sample.
  • a buffer may include one or more of, or all of: 50 mM Potassium Acetate, 20 mM Tris Acetate, 10 mM Magnesium Acetate, 100 ug/ml Bovine Serum Albumin, 1 mM ATP and 7.5% Polyethylene Glycol 6000.
  • a polynucleotide encoding for a constant region of a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA) may include a methyl moiety that prevents digestion of the polynucleotide by a restriction endonuclease.
  • the methyl moiety may be added by an enzyme containing methyltransferase activity.
  • the polynucleotide may further include: a non-palindromic recognition site for a Type IIS restriction enzyme binding site and a sequence that when ligated to DNA fragments digested with a second restriction enzyme does not restore the recognition sequence for the second restriction enzyme.
  • the modifications maintain the stem loop structure of the CRISPR guide RNA molecule in order for the specificity and endonuclease activity of CRISPR RNP complexes to function.
  • the polynucleotide may include a non-palindromic recognition site for a type II restriction enzyme, the non-palindromic recognition site being oriented in a manner recognized by the type II restriction enzyme to cleave upstream of the sequence encoding for a constant region of a CRISPR single guide RNA (sgRNA) or CRISPR targeting RNA (crRNA).
  • sgRNA CRISPR single guide RNA
  • crRNA CRISPR targeting RNA
  • kits for generating CRISPR guide RNA (gRNA) libraries may include/result in a polynucleotide containing a methyl moiety and a sequence encoding for a constant region of a CRISPR sgRNA or CRISPR crRNA and having a sequence that does not restore a restriction enzyme binding site.
  • the kit may further include one or more of the following: the type II restriction enzyme, a solid support, wherein the polynucleotide may be capable of being immobilized on the solid supports or is immobilized on the solid support, a strand displacing polymerase, a promoter polynucleotide recognized by an RNA polymerase, and buffers that allow digestion and ligation to occur in the same reaction vessel.
  • a method for generating double stranded DNA inputs for enzymatic CRISPR library generation may include one or more of the following steps: a) selection of a DNA source; and b) purification of polynucleotides from the source by physical or chemical separation.
  • the DNA source may be selected from one or more of: an organism of interest, an organism at a selected stage of development, a tissue of interest, a cell at a selected stage of differentiation, a cell at a particular stage of the cell cycle, a tissue or cell having a selected pathology.
  • the polynucleotides may be cDNA created from RNA, and/or the polynucleotides may be genomic DNA or mitochondrial DNA.
  • the polynucleotides may be selected by at least one of: precipitation isolation, hybridization isolation, antibody isolation, and other co -precipitation isolation.
  • the polynucleotides or one or more segments thereof may be amplified by PCR and/or normalized.
  • a method for generating DNA templates for production of a CRISPR/Cas9 guide RNA library may include: a) providing a polynucleotide sample; b) digesting the polynucleotide sample at protospacer adjacent motifs (PAM) to form fragments with a first restriction enzyme (RE) in the presence of a ligase and first adapter, wherein the first adapter sequence may include a constant region containing a sequence CRISPR enzyme can bind to and/or cloning site sequence,
  • the method may further include ligating a polynucleotide to an end of the third polynucleotide.
  • the ligated polynucleotide may include a promoter and/or a cloning adapter.
  • the first adaptor and/or second adapter may or may not contain a 5’ phosphate group.
  • a method for generating DNA templates for production of a CRISPR/Cas9 guide RNA library may include: a) providing a polynucleotide sample; b) digesting the polynucleotide sample at protospacer adjacent motifs (PAM) to form fragments with a first restriction enzyme (RE); c) ligation to a first adapter, wherein the first adapter sequence comprises a constant region containing a sequence CRISPR enzyme can bind to and/or cloning site sequence, polynucleotide sequences to one or both ends of the fragments with a ligase; d) contacting the intermediate product with a Type II S RE having methylase activity, in the presence of a ligase, the ligase operably linking a second adapter sequence to digested, methylated fragments formed from digestion of the intermediate product, thereby forming a ligated product having a variable or targeting region, and a scaffold region, wherein the second adapter sequence comprises at least
  • a method for generating DNA templates for production of a CRISPR/Cas9 guide RNA library may include one or more of the following steps: a) providing a polynucleotide sample; b) digesting the polynucleotide sample at protospacer adjacent motifs (PAM) to form fragments with a first restriction enzyme (RE) in the presence of a ligase and first adapter, wherein the first adapter sequence comprises a constant region containing at least one of: a sequence CRISPR enzyme can bind to and cloning site sequence, joining polynucleotide sequences to one or both ends of the fragments with a ligase, thereby forming an intermediate product which lacks binding sites recognized by the first RE; c) contacting the intermediate product with a Type II S RE, in the presence of a ligase, the ligase operably linking a second adapter sequence to the digested fragments formed from digestion of the intermediate product, thereby forming a ligated product having a
  • a cloning site sequence may be present and the ligated product may be cloned into a vector, the vector may optionally include an operable promoter and/or scaffold sequence.
  • the promoter may be present and the ligated product may be cloned into a vector that may include at least part of a scaffold sequence.
  • steps a-c may be performed in a single reaction vessel.
  • the first polynucleotide may include a plurality of first polynucleotides; at least a portion of the plurality of first polynucleotides may be ligated with DNA to form a plurality of second polynucleotides; and the plurality of second polynucleotides may be digested with the type II restriction enzyme that may include methylase activity to form a plurality of third polynucleotides encoding a plurality of CRISPR sgRNAs or CRISPR crRNAs.
  • the plurality of CRISPR sgRNAs or CRISPR crRNAs may have a variable region different from the other CRISPR sgRNAs or CRISPR crRNAs.
  • the method described above may include polynucleotide sample(s) in which cDNA may be normalized to remove abundant transcripts from the cDNA, thereby increasing equal representation of transcripts in the library.
  • the method above may include an input polynucleotide sample that may be obtained from a source selected from the group consisting of: an organism of interest, an organism at a selected stage of development, a tissue of interest, a cell at a selected stage of differentiation, a cell at a particular stage of the cell cycle, and a tissue or cell having a selected pathology.
  • the method may also include adapters have or do not have a 5’ phosphate. For example, the adapters contain at least six consecutive phophorothioates at the 5’ end.
  • the promoter may include a T7 RNA polymerase promoter.
  • the polynucleotide sequence of step a) may be digested with variety of appropriate enzymes, including an enzyme selected from the group consisting of Hpall, Mspl, ScrFI, Bfal, and Pacl.
  • the ligated product of step c) may include at least one nick and/or may not maintain a G-U hydrogen bond at position 1 of the scaffold sequence.
  • the method may further include purifying the ligated product.
  • the ligated product may be purified using a capture oligonucleotide that may include a biotin at a 3’ end, which hybridizes to the scaffold portion of the ligated product operably linked to a solid support.
  • the solid support may be a magnetic bead and the purification step may include magnetic separation.
  • the method may further or alternatively include suspending the separated beads in a reaction that includes Bst 3.0 polymerase and nucleotide triophosphates (NTPs) at about 45°C for about 15 minutes, thereby repairing and extending the nicked strand. This extension may cause displacement of the repaired product from the bead.
  • the method may include transcribing the sgRNA template libraries in the presence of DNase I and/or elution of the sgRNA template from the beads followed by PCR amplification of the sgRNA template.
  • the first first RE may be one of Mspl, Hpall, ScrFI, BsaJI.
  • the digestion and ligation of step b) may be performed essentially simultaneously, and/or the digestion and ligation of step c) may be performed essentially
  • a functional gRNA template may be produced by the method(s) described above, the template may include at least one or more operably linked sequences: a promoter, a protospacer, an adapter, a type II RE recognition sequence, and a modified scaffold sequence.
  • a genome wide library may include a plurality of unique CRISPR-Cas system guide sequences that are capable of targeting a plurality of target sequences in genomic loci, wherein the library may be created using one or more of the methods described above.
  • An illustrative method of eluting polynucleotides from solid supports may include one or more of the following steps, including: a) attaching a single stranded polynucleotide to a solid support such that the 5' end of the polynucleotide may be protruding from the solid support and may be capable of hybridizing with at least a portion of a second polynucleotide; b) contacting the attached single stranded polynucleotide with the second polynucleotide such that hybridization between the two polynucleotides forms a polynucleotide duplex, thereby immobilizing the second polynucleotide; c) contacting the polynucleotide duplex with a polymerase exhibiting strand displacement activity in the presence of dNTPs, in which the second polynucleotide may be used as a template for extension by the polymerase, wherein the first polynucleotide may
  • the support may include one or more beads, which may or may not have magnetic or paramagnetic properties.
  • the attached single stranded polynucleotide may include a plurality of polynucleotides.
  • the second polynucleotide may include a plurality of polynucleotides with different sequences.
  • the method may also include the use of the support to purify the second polynucleotide from a mixture.
  • a length of length of the hybridizing sequence can be adjusted to increase or decrease a temperature at which hybridization occurs. In some examples, hybridization occurs at room temperature. For example, the hybridization may occur at 0-50°C.
  • the method may include a physical separation process for purification of oligonucleotides.
  • the second polynucleotide contains a sequence encoding for CRISPR/Cas9 guide RNAs.
  • a method for selectively degrading unwanted polynucleotide components of a mixture may include: a) incorporation of at least five nucleotides linked by phosphorothioates at 5’ ends of a double stranded DNA fragment to be protected; b) contacting the double stranded DNA fragments with a first exonuclease that operates on double stranded DNA and may be sensitive to
  • phosphorothioate linkages and a second exonuclease that operates on single stranded DNA may be degraded.
  • polynucleotides with a single end may be degraded.
  • phosphorothioates may be incorporated by ligating adapters using any appropriate method including by PCR.
  • An illustrative method for manipulation of DNA substrates through enzymatic processes to produce nucleotide sequences may include: simultaneously digesting DNA by targeting a restriction enzyme to sites containing protospacer adjacent motif (PAM) sequences in the DNA to produce DNA fragments and ligating adaptors to ends of DNA fragments to produce an intermediate product containing the scaffold ligated with a DNA fragment of an arbitrary length; and simultaneously digesting and ligating the intermediate product to produce guide RNA templates.
  • PAM protospacer adjacent motif
  • simultaneously digesting DNA and ligating adaptors to ends of the DNA fragments may include digesting the DNA in presence of a ligase and a first adaptor.
  • the digesting may include a type IIS restriction enzyme to digest the intermediate product to create a guide RNA including an 18 to 25 base pair protospacer connected to and engineered polynucleotide.
  • the type IIS restriction enzyme blocks redigestion of the ligation product.
  • the type IIS restriction blocks its own function after digestion by chemically modifying its own binding site.
  • the chemical modification of the binding site may include attaching a methyl group.
  • Ligation may include a connection of the digestion product to an upstream adapter to produce a guide RNA template containing an upstream adapter, a proto spacer and the engineered
  • the upstream adapter may include at least one of a promoter, a cloning site, or other DNA integration site. Additionally or alternatively, the upstream adapter does not contain a 5’ phosphate on the ligated end. In one example, the ligated product contains at least one nick.
  • the method may include purifying the guide RNA templates. The step of purifying may include attaching polynucleotides in a sequence dependent hybridization to a bead, washing to remove reagents and fragments that are not attached to the beads, and eluting the guide RNA templates from the bead. Additionally or alternatively, purifying may include elution and nick repair of the guide RNA templates.
  • the elution and nick repair may occur in a same reaction.
  • the bead may be a magnetic or paramagnetic bead.
  • the elution and nick repair may be both performed by a single enzyme.
  • the single enzyme may include a strand displacing polymerase to
  • the strand displacing polymerase may use the previous captured polynucleotide as a template to displace and fill in a sequence, thereby permanently displacing the polynucleotide from the beads.
  • a hybridized segment of the previously captured polynucleotide may be made double stranded by the polymerase thereby preventing rehybridization of the polynucleotide to the beads.
  • the method may include selecting a DNA source, wherein selecting may include choosing a species of organism, selecting at least one of: a developmental stage of the organism, a tissue maturation stage, a state of cell differentiation and a stage of a cell cycle, selecting environmental conditions the organism may be subject to, and selecting a tissue or cell type from the organism to be the DNA source.
  • the method may additionally or alternatively include extracting DNA or RNA from the selected DNA source, wherein extracting comprises at least one of: a chemical separation of cellular components and a physical separation of cellular components, wherein the extracting isolates a nucleic acid species of interest.
  • the chemical separation of DNA or RNA may include amplification of the nucleic acid species.
  • the chemical separation may include enzymatic amplification of at least one region of the nucleic acid species.
  • the enzymatic amplification may include polymerase chain reaction (PCR).
  • the physical separation of DNA or RNA may include isolation of DNA or RNA by at least one of: precipitation isolation, hybridization isolation, antibody isolation, and other co-precipitation isolation.
  • the step of extracting may include extracting RNA from the selected DNA source and then converting the RNA into DNA.
  • the extracting may include normalizing the DNA by enzymatic or chemical methods applied to the DNA, wherein normalizing comprises balancing quantities of various DNA
  • a method for manipulation of DNA substrates through enzymatic processes to produce nucleotide sequences may include one or more of the following steps, including selecting a DNA source by: choosing a species of organism and selecting at least one of a developmental stage of the organism, a tissue maturation stage, a state of cell differentiation and a stage of a cell cycle, selecting environmental conditions the organism may be subject to, and selecting a tissue or cell type from the organism to be the DNA source.
  • the method may also include extracting DNA or RNA from the selected DNA source, wherein extracting comprises at least one of: a chemical separation of cellular components and a physical separation of cellular components, wherein the extracting isolates a nucleic acid species of interest; wherein chemical separation of DNA or RNA comprises amplification of the nucleic acid species, wherein chemical separation comprises enzymatic amplification of at least one region of the nucleic acid species, wherein the enzymatic amplification comprises polymerase chain reaction (PCR), wherein physical separation of DNA or RNA comprises isolation of DNA or RNA by at least one of: precipitation isolation, hybridization isolation, antibody isolation, and other co-precipitation isolation; wherein extracting comprises extracting RNA from the selected DNA source and then converting the RNA into DNA; wherein extracting comprises normalizing the DNA, wherein normalizing comprises balancing quantities of various DNA components in the mixture to ensure more equal representation in an output library; simultaneously digesting the extracted DNA by targeting a restriction enzyme to sites containing PAM sequences to produce DNA fragment
  • the step of ligating may include introducing an engineered polynucleotide sequence that can associate with a CAS9 molecule (scaffold) such that the engineered polynucleotide sequence:
  • the engineered polynucleotide does not contain a 5’ phosphate on the ligated end; wherein the ligation product contains at least one nick; wherein the engineered polynucleotide sequence does not maintain the G- U hydrogen bond of the G at position 1 of the scaffold sequence, wherein the G may be replaced by a different base; wherein the engineered polynucleotide sequence comprises a single gRNA scaffold or crRNA sequence or an adapter sequence for cloning into a vector containing either a single gRNA scaffold or crRNA sequence simultaneously digesting and
  • polynucleotide is made double stranded by the polymerase thereby preventing rehybridization to the polynucleotide to the beads.
  • a method for generating DNA templates for production of a CRISPR/Cas9 guide RNA library may include digesting a polynucleotide sample into digested products in the presence of a ligase and an adaptor, wherein the digested products are chemically prevented from reversing the digestion and are ligated to the adaptor in a single reaction vessel.
  • the digesting and ligating include digesting the polynucleotide sample at protospacer adjacent motifs (PAM) to form fragments with a first restriction enzyme (RE) in the presence of a ligase and first adapter, wherein the first adapter sequence comprises a constant region containing a sequence that a CRISPR enzyme can bind to and/or cloning site sequence, and lacks sufficient sequence similarity to the first RE recognition sequence such that ligation of the adapter to one or both ends of the fragments produces an intermediate product which lacks binding sites recognized by the first RE.
  • PAM protospacer adjacent motifs
  • RE restriction enzyme
  • the digesting and ligating may include digesting the second polynucleotide with the type II restriction enzyme to form a third polynucleotide encoding a CRISPR sgRNA or CRISPR crRNA, wherein the type II restriction enzyme cuts the DNA at a site that is 17 to 27 base pairs from the end of the first polynucleotide.
  • the digesting and ligating may include performing the digestion and ligation steps in a single reaction vessel. In one example, the steps of digesting and ligating may include unphosphorylated adapters resulting in nicked products.
  • the polynucleotide sample may include normalized cDNA. The method described above may further including bead purification.
  • the strand displacing polymerase may be used to elute the library from a solid support and simultaneously repair any nicks in the library.
  • a G-U wobble base pair in the guide RNA stem loop may be replaced by G-V.
  • the method may further include digesting and ligating that includes the enzymatic addition of a methyl moiety.
  • a first polynucleotide may include a sequence encoding for a constant region of one of a CRISPR single guide RNA (sgRNA) and CRISPR targeting RNA (crRNA).
  • the sequence may include a non-palindromic recognition site recognized by a type II restriction enzyme, wherein the non-palindromic recognition site may be configured such that a second polynucleotide operably linked to the first polynucleotide may be cleaved 17 to 27 base pairs from the recognition site by the type II restriction enzyme, the type II restriction enzyme configured to add a methyl moiety to the recognition site such that methylation of the recognition site prevents subsequent cleavage of the second polynucleotide.
  • a first polynucleotide may include a sequence encoding for a constant region of one of a CRISPR single guide RNA (sgRNA) and CRISPR targeting RNA (crRNA).
  • sgRNA CRISPR single guide RNA
  • crRNA CRISPR targeting RNA
  • the sequence may include a non-palindromic recognition site configured to be recognized by a type II restriction enzyme, wherein the non-palindromic recognition site is oriented such that a second polynucleotide operably linked an end adjacent to the non-palindromic recognition site, the first polynucleotide configured to be cleaved 17 to 27 base pairs from the recognition site by the type II restriction enzyme, and the type II restriction enzyme may be configured to add a methyl moiety to the recognition site such that methylation of the recognition site prevents subsequent cleavage of the second polynucleotide.
  • a first polynucleotide may include a sequence encoding for a constant region of one of a CRISPR single guide RNA (sgRNA) and CRISPR targeting RNA (crRNA), and an end configured to operably link a second polynucleotide, the sequence may include: a non- palindromic recognition site recognized by a type II restriction enzyme, wherein the non- palindromic recognition site is configured to position and orient the type II restriction enzyme to cleave the second polynucleotide 17 to 27 base pairs from the recognition site; wherein the type II restriction enzyme is configured to add a methyl moiety to the recognition site such that the methyl moiety prevents subsequent cleavage of the second polynucleotide.
  • sgRNA CRISPR single guide RNA
  • crRNA CRISPR targeting RNA
  • a polynucleotide may include a sequence encoding for at least part of a protein binding segment of an RNA component of a CRISPR complex.
  • the sequence may include a non-palindromic recognition sequence for a type II restriction enzyme configured to cleave at least 17 nucleotides outside of the non-palindromic recognition sequence, the non-palindromic recognition sequence being oriented such that the type II restriction enzyme may be configured such it cannot cleave within the sequence encoding for at least part of a protein binding segment of an RNA component of a CRISPR complex.
  • a polynucleotide encoding at least part of a protein binding segment of an RNA component of a CRISPR complex may include a non-palindromic recognition sequence for a type II restriction enzyme having methyltransferase activity and configured to cleave at least 17 nucleotides outside of the non-palindromic recognition sequence.
  • the recognition sequence may include a nucleotide capable of being methylated by the type II restriction enzyme, wherein methylation of the nucleotide prevents cleavage of DNA by the restriction enzyme.
  • the recognition sequence may be oriented such that, in the presence of the restriction enzyme, the DNA cleavage domain of the restriction enzyme may be positioned outside of the sequence encoding for at least part of the protein binding segment of the RNA component of the CRISPR complex.
  • An example of the invention may include a polynucleotide comprising a sequence encoding for a constant region.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the constant region comprising a protein binding segment of an RNA component of a CRISPR complex.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the sequence comprising a non-palindromic recognition sequence for a first restriction enzyme having methyltransferase activity and configured to cleave at least 17 nucleotides outside of the non-palindromic recognition sequence.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with at least one base of the non-palindromic recognition sequence being configured to be methylated by the first restriction enzyme, thereby blocking endonuclease activity of the first restriction enzyme.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the RNA component of the CRISPR complex being configured to form a secondary structure comprising the non-palindromic recognition sequence.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with at least a portion of the polynucleotide being double stranded.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with at least one base of the non-palindromic recognition sequence having an attached methyl group.
  • the example of the invention may also include one or more steps, functions, or structures set forth above further comprising a sequence such that ligation of the polynucleotide to a second polynucleotide cleaved by a second restriction enzyme does not restore a recognition sequence of the second restriction enzyme.
  • Another example of the invention may include a method for generating a library of polynucleotides.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with providing a first adapter.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first adapter comprising a sequence encoding for a protein binding segment of an RNA component of a CRISPR complex.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first adapter further comprising a second recognition sequence for a second restriction enzyme.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with combining a polynucleotide sample with a first restriction enzyme, the first adapter, and a first ligase.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the polynucleotide sample comprising a first recognition sequence for the first restriction enzyme.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first recognition sequence comprising a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first restriction enzyme being configured to cleave the first recognition sequence to produce a cleaved fragment.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first ligase being configured to operably linking the first adapter to the cleaved fragment to generate an intermediate polynucleotide.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the intermediate polynucleotide not containing the first recognition sequence.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with combining the intermediate polynucleotide with the second restriction enzyme, a second adapter, and a second ligase.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the second restriction enzyme having methyltransferase activity.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the second restriction enzyme being configured to cleave the
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the methyl group inhibiting activity of the second restriction enzyme.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the second ligase being configured to operably linking the methylated polynucleotide to the second adapter to generate a final polynucleotide.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first adapter comprising a plurality of first adapters.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with at least a portion of the plurality of first adapters being operably linked to a plurality of cleaved fragments to form a plurality of intermediate polynucleotides.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the plurality of intermediate polynucleotides being digested with the second restriction enzyme to form a plurality of methylated polynucleotides encoding a plurality of RNA components of CRISPR complexes.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with at least one of the plurality of methylated polynucleotides encoding a variable region different from the other methylated polynucleotides.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with cleavage and operably linking in step (b) being carried out in two separate steps.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with cleavage and operably linking in step (c) being carried out in two separate steps.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with step (b) and step (c) being combined to create a single step.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with step (b) and step (c) being performed in a single reaction vessel.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first restriction enzyme comprising at least one of Hpall, Mspl,
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first ligase and the second ligase comprising the same ligase.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the second adapter comprising at least a portion of a promoter sequence.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the methylated polynucleotide further comprising at least one sequence configured for molecular cloning.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with at least one of the adapters lacking a 5’ phosphate.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the methylated polynucleotide comprising at least one nick.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first adapter being purified using a solid support operably linked to a capture polynucleotide at a 3’ end.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first adapter further comprising a 5’ overhang configured to hybridize to the capture polynucleotide.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the second recognition sequence comprising a non-palindromic recognition sequence.
  • Another example of the invention may include a kit for generating a library of
  • polynucleotides encoding for RNA components of CRISPR complexes are polynucleotides encoding for RNA components of CRISPR complexes.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with a polynucleotide comprising a sequence encoding for a constant region.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the sequence comprising a protein binding segment of an RNA component of a CRISPR complex.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the sequence further comprising a non-palindromic recognition sequence for a restriction enzyme having methyltransferase activity.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the restriction enzyme being configured to cleave at least 17 nucleotides outside of the non-palindromic recognition sequence.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with at least one base of the non-palindromic recognition sequence being configured to be methylated by the restriction enzyme, thereby blocking the endonuclease activity of the restriction enzyme.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the kit further comprising at least one ligase.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the kit further comprising the restriction enzyme.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the kit further comprising a restriction enzyme configured to cleave a recognition sequence comprising a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the kit further comprising a second polynucleotide configured to be ligated.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the kit further comprising at least one buffer to provide operable conditions for both the restriction enzyme and at least one ligase.
  • the example of the invention may also include one or more steps, functions, or structures set forth above further comprising a solid support configured to secure polynucleotides.
  • the example of the invention may also include one or more steps, functions, or structures set forth above further comprising a strand displacing polymerase.
  • Another example of the invention may include a method for removing a polynucleotide immobilized on a solid support.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with providing a first polynucleotide attached to a solid support at the 3’ end.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with providing a second polynucleotide hybridized to least a portion of the first polynucleotide.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the second polynucleotide extending past the 5’ end of the first polynucleotide.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with providing a third polynucleotide hybridized to least a portion of the second polynucleotide.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with all three polynucleotides being immobilized on the solid support.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with contacting the hybridized second and third polynucleotides with a polymerase having strand displacement activity.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the second polynucleotide serving as a template for extension by the polymerase.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the 3’ end of the third nucleotide being extended and the first polynucleotide being displaced by the polymerase.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with detaching the second and third polynucleotides from the solid support.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with inhibiting rehybridization of the first and second polynucleotides.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with providing the polymerase having 5’ to 3’ exonuclease activity.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with displacing and degrading hybridized polynucleotides during strand extension.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the second polynucleotide and third polynucleotide comprising the same polynucleotide.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with providing the 3’ end of the second polynucleotide being configured to connect to the 5’ end of the third polynucleotide by a phosphodiester bond.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with providing the first, second, and third polynucleotides each comprise a plurality of polynucleotides.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the support being a bead.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with providing the support being a magnetic bead.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the polynucleotides being separated by at least one of: magnetic separation, precipitation, hybridization, antibody isolation, and co-precipitation.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the plurality of first polynucleotides comprising a polynucleotide that includes a sequence different from the other first polynucleotides.
  • Another example of the invention may include a system to remove polynucleotides immobilized on a solid supports.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with a solid support.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with a first polynucleotide configured to attach to a solid support at the 3’ end.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with a second polynucleotide configured to hybridize to least a portion of the first polynucleotide.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with a second polynucleotide configured to extend past the 5’ end of the first polynucleotide.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with a strand displacing polymerase.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first polynucleotide being covalently attached to the solid support.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the first polynucleotide being attached to the solid support by affinity binding.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the solid support being a bead.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with the solid support being a magnetic bead.
  • the example of the invention may also include one or more steps, functions, or structures set forth above combined with buffers that provide conditions for both hybridization and strand displacement to occur.

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Abstract

L'invention concerne un procédé amélioré pour la production rapide et efficace de banques de brins guides. L'invention concerne également des kits comprenant des réactifs appropriés pour la mise en œuvre du procédé. Des modes de réalisation peuvent comprendre un polynucléotide comprenant une séquence codant pour une région constante comprenant un segment de liaison de protéine d'un composant ARN d'un complexe CRISPR. La séquence peut comprendre une séquence de reconnaissance non palindromique pour une première enzyme de restriction ayant une activité méthyltransférase et conçue pour cliver au moins 17 nucléotides à l'extérieur de la séquence de reconnaissance non palindromique, au moins une base de la séquence de reconnaissance non palindromique étant conçue pour être méthylée par la première enzyme de restriction, ce qui permet de bloquer l'activité endonucléase de la première enzyme de restriction.
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US6197557B1 (en) * 1997-03-05 2001-03-06 The Regents Of The University Of Michigan Compositions and methods for analysis of nucleic acids
EP2250283A2 (fr) * 2008-02-12 2010-11-17 Nugen Technologies, Inc. Procédés et composition pour amplification isotherme d'acide nucléique
US20110269194A1 (en) * 2010-04-20 2011-11-03 Swift Biosciences, Inc. Materials and methods for nucleic acid fractionation by solid phase entrapment and enzyme-mediated detachment
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US8980553B2 (en) * 2011-04-02 2015-03-17 New England Biolabs, Inc. Methods and compositions for enriching either target polynucleotides or non-target polynucleotides from a mixture of target and non-target polynucleotides
US10253321B2 (en) * 2013-05-01 2019-04-09 Dna2.0, Inc. Methods, compositions and kits for a one-step DNA cloning system
US11279926B2 (en) * 2015-06-05 2022-03-22 The Regents Of The University Of California Methods and compositions for generating CRISPR/Cas guide RNAs
US10669539B2 (en) * 2016-10-06 2020-06-02 Pioneer Biolabs, Llc Methods and compositions for generating CRISPR guide RNA libraries
US20210308171A1 (en) * 2018-08-07 2021-10-07 The Broad Institute, Inc. Methods for combinatorial screening and use of therapeutic targets thereof

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