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  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

• FIG. 1 shows a diagram of MAD7 comprising one or more nuclear localization signals (NLS).
• Figure 2 shows editing frequency at the DNMT1 locus in and post-transfection cell viability of T-cell leukemic cells following treatment comprising one or more guide nucleic acids complexed with MAD7 comprising one or more NLS.
• Figure 3 shows editing frequency at the DNMT1 locus in T-cell leukemic cells using multiple electroporation programs in combination with the SE electroporation buffer.
• Figure 4 shows editing frequency at the DNMT1 locus in T-cell leukemic cells using multiple electroporation programs in combination with the SF electroporation buffer.
• Figure 5 shows editing frequency at the DNMT1 locus in T-cell leukemic cells using multiple electroporation programs in combination with the SG electroporation buffer.
• Figure 6 shows editing frequency at the DNMT1 locus in T-cell leukemic cells using multiple electroporation programs.
• Figure 7 shows editing frequency by type at eight loci in T-cell leukemic cells using multiple guide nucleic acids complexed with MAD7 comprising one or more NLS.
• Figure 8 shows a comparison of editing efficiency between T-cell leukemic cells treated with MAD7 comprising one or more guide nucleic acids targeting the DNMT 1 locus as compared to a control guide nucleic acid binned by editing frequency.
• Figure 9 shows editing frequency by PAM motif in T-cell leukemic cells using multiple guide nucleic acids complexed with MAD7 comprising one or more NLS.
• Figure 10A shows sequence logo plots for multiple guide nucleic acids binned by editing frequency in T-cell leukemic cells using when complexed with MAD7 comprising one or more NLS.
• Figure 10B shows nucleotide and dinucleotide frequency for multiple guide nucleic acids binned by editing frequency in T-cell leukemic cells using when complexed with MAD7 comprising one or more NLS.
• Figure 11 shows trinucleotide AAA or UUU frequency binned by editing frequency in T-cell leukemic cells following treatment with multiple guide nucleic acids complexed with MAD7 comprising one or more NLS.
• Figure 12 shows editing frequency for both INDELs and frameshift mutations at eight loci in T-cell leukemic cells following treatment with multiple guide nucleic acids complexed with MAD7 comprising one or more NLS.
• Figure 13 shows the correlation between INDEL frequency in the gNA validation experiment versus INDEL formation in the gNA screen experiment.
• Figure 14 shows the proportion of frameshift to INDELs at eight loci in T-cell leukemic cells following treatment with multiple guide nucleic acids complexed with MAD7 comprising one or more NLS.
• Figure 15 shows INDEL frequency for gNAs comprising representative spacer sequences complexed with MAD7 comprising one or more NLS in T-cell leukemic cells at predicted off-target sites.
• Figure 16 shows INDEL frequency for gNAs comprising representative spacer sequences complexed with MAD7 comprising one or more NLS in T-cell leukemic cells at predicted off-target sites.
• Figure 17 shows INDEL frequency at the AAVS1 locus in T-cell leukemic cells following treatment with a gNA:MAD7 complex.
• Figure 18 shows GFP insertion efficiency at the AAVS 1 locus and cell viability following treatment for multiple primer constructs.
• Figure 19 shows GFP insertion efficiency at the AAVS 1 locus with increasing concentrations of donor template (e.g., HDRT) and variable homology arm length.
• donor template e.g., HDRT
• Figure 20 shows CAR insertion efficiency at the AAVS 1 locus and cell viability with increasing concentrations of donor template and variable homology arm length.
• Figure 21 shows CAR insertion efficiency (A) at the AAVS 1 locus and cell viability (B) in primary T-cells.
• CRISPR is an abbreviation of Clustered Regularly Interspaced Short Palindromic Repeats. In a palindromic repeat, the sequence of nucleotides is the same in both directions.
• CRISPR-associated system Small clusters of Cas (CRISPR-associated system) genes are located next to CRISPR sequences.
• the CRISPR/Cas system is a prokaryotic immune system that can confer resistance to foreign genetic elements such as those present within plasmids and phages providing the prokaryote a form of acquired immunity.
• RNA harboring a spacer sequence assists Cas (CRISPR-associated) proteins to recognize and cut exogenous DNA.
• CRISPR sequences are found in approximately 50% of bacterial genomes and nearly 90% of sequenced archaea has selected for efficient and robust metabolic and regulatory networks that prevent unnecessary metabolite biosynthesis and optimally distribute resources to maximize overall cellular fitness.
• CRISPR-Cas driven genome editing and engineering has dramatically impacted biology and biotechnology in general.
• CRISPR-Cas editing systems require a polynucleotide guided nuclease, a guide nucleic acid (gNA) e.g. a guide RNA (gRNA)) that directs the nuclease to cut a specific region of the genome, and, optionally, a donor DNA cassette (also referred to herein as a donor template or editing sequence) that can be used to repair the cut dsDNA and thereby incorporate programmable edits at the site of interest.
• gNA guide nucleic acid
• gRNA guide RNA
• a donor DNA cassette also referred to herein as a donor template or editing sequence
• CRISPR/Cas9 One version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to provide useful tools for editing targeted genomes.
• gRNA synthetic guide RNA
• the cell’s genome can be cut/edited at a predetermined location, allowing existing genes to be removed and/or new ones added.
• modulating and “manipulating” of genome editing can mean an increase, a decrease, upregulation, downregulation, induction, a change in editing activity, a change in binding, a change cleavage or the like, of one or more of targeted genes or gene clusters of certain embodiments disclosed herein.
• primers used herein for preparation per conventional techniques can include sequencing primers and amplification primers.
• plasmids and oligomers used in conventional techniques can include synthesized oligomers and oligomer cassettes.
• nucleic acid-guided nuclease systems and methods of use are provided.
• a nuclease system can include transcripts and other elements involved in the expression of an engineered nuclease disclosed herein, which can include sequences encoding a novel engineered nucleic acid-guided nuclease protein and a guide sequence (gRNA) or a novel gRNA as disclosed herein.
• gRNA guide sequence
• nucleic acid- guided nuclease systems can include at least one CRISPR-associated nucleic acid guided nuclease construct, the disclosure of which are provided herein.
• nucleic acid-guided nuclease systems can include at least one known guide sequence (gRNA) or at least one novel gRNA, such as a single gRNA or a dual gRNA.
• gRNA guide sequence
• an engineered nucleic acid-guided nuclease of the instant invention can be used in systems for editing a gene of interest in humans or other species.
• Bacterial and archaeal targetable nuclease systems have emerged as powerful tools for precision genome editing. However, naturally occurring nucleases have some limitations including expression and delivery challenges due to the nucleic acid sequence and protein size.
• novel engineered nucleic acid-guided nuclease constructs disclosed herein can be created for targeting of a targeted gene and/or increased efficiency and/or accuracy of targeted gene editing in a subject.
• Casl2a is a single RNA- guided CRISPR/Cas endonuclease capable of genome editing having differing features when compared to Cas9.
• a Casl2a-based system allow fast and reliable introduction of donor DNA into a genome.
• Casl2a broadens genome editing.
• CRISPR/Casl2a genome editing has been evaluated in human cells as well as other organisms including plants. Several features of the CRISPR/Cas 12a system are different when compared to CRISPR/Cas9.
• Casl2a nuclease recognizes T-rich protospacer adjacent motif (PAM) sequences (e.g. 5’-TTTN-3’ (AsCasl2a, LbCasl2a) and 5’-TTN-3’ (FnCasl2a); whereas, the comparable sequence for SpCas9 is NGG.
• PAM protospacer adjacent motif
• the PAM sequence of Casl2a is located at the 5’ end of the target DNA sequence, where it is at the 3’ end for Cas9.
• Casl2a is capable of cleaving DNA distal to its PAM around the +18/+23 position of the protospacer. This cleavage creates a staggered DNA overhang (e.g.
• Cas9 cleaves close to its PAM after the 3’ position of the protospacer at both strands and creates blunt ends.
• creating altered recognition of nucleases can provide an improvement over Cas9 or Casl2a to improve accuracy.
• Casl2a is guided by a single crRNA and does not require a tracrRNA, resulting in a shorter gRNA sequence than the sgRNA used by Cas9.
• the modified Casl2a nucleases provided herein can also function with a dual gRNA.
• Casl2a displays additional ribonuclease activity that functions in crRNA processing.
• Casl2a is used as an editing tool for different species (e.g. S. cerevisiae), allowing the use of an alternative PAM sequence compared with the one recognized by CRISPR/Cas9.
• Novel nucleases disclosed herein can further recognize the same or alternative PAM sequences. These novel nucleases can provide an alternative system for multiplex genome editing as compared with known multiplex approaches and can be used as an improved system in mammalian gene editing.
• Cas 12a protein-RNA complexes recognize a T-rich PAM and cleavage leads to a staggered DNA double-stranded break.
• Casl2a-type nuclease interacts with the pseudoknot structure formed by the 5 '-handle of crRNA.
• a guide RNA segment composed of a seed region and the 3' terminus, possesses complementary binding sequences with the target
• Casl2a type nucleases characterized to date have been demonstrated to work with a single gRNA and to process gRNA arrays. While Casl2a-type and Cas9 nuclease systems have proven highly impactful, neither system has been demonstrated to function as predictably as is desired to enable the full range of applications envisioned for gene-editing technologies.
• Casl2a-like nucleases and engineered gRNAs disclosed herein are contemplated for use in bacteria, and other prokaryotes.
• engineered designer nucleases are contemplated for use in eukaryotes such as yeast, mammals, e.g., human as well as of use in birds and fish, or cells derived from same.
• off-targeting rates for nuclease constructs disclosed herein can be reduced compared to a control, e.g., a native sequence, for improved editing. Off-targeting rates can be readily tested.
• nuclease constructs disclosed herein can share conserved encoded motifs of known nucleases. In other embodiments, nuclease constructs disclosed herein do not share conserved encoded peptide motifs with known nucleases.
• the CRISPR nuclease comprises a Type V nuclease. In certain embodiments, provided herein are compositions, methods, and/or kits wherein the CRISPR nuclease comprises a Type V-A, V-B, V-C, V-D, or V-E CRISPR nuclease.
• compositions, methods, and/or kits wherein the CRISPR nuclease comprises a Type V-A nuclease.
• Naturally occurring type V-A CRISPR nucleases comprise a RuvC-like nuclease domain but lack an HNH endonuclease domain, and recognize a 5 ’ T -rich PAM located immediately upstream from the target nucleotide sequence, the orientation determined using the non-target strand (i.e., the strand not hybridized with the spacer sequence) as the coordinate.
• These CRISPR nucleases cleave a double-stranded DNA to generate a staggered double-stranded break rather than a blunt end.
• the cleavage site is distant from the PAM site (e.g., separated by at least 10, 11, 12, 13, 14, or 15 nucleotides downstream from the PAM on the non-target strand and/or separated by at least 15, 16, 17, 18, or 19 nucleotides upstream from the sequence complementary to PAM on the target strand).
• a type V-A CRISPR nuclease comprises Cpfl.
• Cpfl proteins are known in the art and are described, e.g., in U.S. Patent Nos. 9,790,490 and 10,113,179.
• Cpfl orthologs can be found in various bacterial and archaeal genomes.
• the Cpfl protein is derived from Francisella novicida U112 (Fn), Acidaminococcus sp.
• BV3L6 (As), Lachnospiraceae bacterium ND2006 (Lb), Lachnospiraceae bacterium MA2020 (Lb2), Candidatus Methanoplasma termitum (CMt), Moraxella bovoculi 237 (Mb), Porphyromonas crevioricanis (Pc), Prevotella disiens (Pd), Francisella tularensis 1 , Francisella tularensis subsp.
• CMt Candidatus Methanoplasma termitum
• Moraxella bovoculi 237 Mb
• Porphyromonas crevioricanis Pc
• Pd Prevotella disiens
• Francisella tularensis 1 Francisella tularensis subsp.
• a type V-A CRISPR nuclease comprises AsCpfl or a variant thereof.
• a type V-A CRISPR nucleases comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A CRISPR nucleases comprises the amino acid sequence set forth in SEQ ID NO: 3 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A CRISPR nuclease comprises LbCpfl or a variant thereof.
• a type V-A CRISPR nucleases comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4 of International (PCT) Application Publication No. WO 2021158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 4 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A CRISPR nuclease comprises FnCpfl or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 5 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A CRISPR nuclease comprises Prevotella hryantii Cpfl (PbCpfl) or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 6 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 6 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A CRISPR nuclease comprises Proteocatella sphenisci Cpfl (PsCpfl) or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 of International (PCT) Application Publication No. WO 2021158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 7 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A CRISPR nuclease comprises Anaerovibrio sp. RM50 Cpfl (As2Cpfl) or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8 of International (PCT) Application Publication No. WO 2021158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 8 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A CRISPR nuclease comprises Moraxella caprae Cpfl (McCpfl) or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 9 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A CRISPR nuclease comprises Lachnospiraceae bacterium COE1 Cpfl (Lb3Cpfl) or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10 of International (PCT) Application Publication No. WO 2021158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 10 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A CRISPR nuclease comprises Eubacterium coprostanoli genes Cpfl (EcCpfl) or a variant thereof.
• a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11 of International (PCT) Application Publication No. WO 2021158918.
• a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 11 of International (PCT) Application Publication No. WO 2021/158918.
• a type V-A CRISPR nuclease is not Cpfl. In certain embodiments, a type V-A CRISPR nuclease is not AsCpfl.
• a type V-A CRISPR nuclease comprises a Type V-A nuclease described in U.S. Patent No. 9,982,279.
• a Type VA CRISPR nuclease polypeptide used in compositions and methods herein can be represented by a polypeptide that includes a sequence that has at least 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98% sequence identity with SEQ ID NO: 1 SEQ ID NO: 1 wherein the Type VA CRISPR nuclease polypeptide further comprises at least one, two, three, four, five or six nuclear localization sequences (NLS), each of which can be at or near the amino end or carboxy end of the CRISPR nuclease polypeptide; and/or one or more purification tags; in addition, a cleavage sequence can be provided to remove portions of a protopeptide.
• NLS nuclear localization sequences
• the term “at or near” an N-terminus or a C-terminus includes where the nearest amino acid of the NLS to the N- or C-terminus is within 300 amino acids, in some cases within 200 amino acids, from the N- or C-terminus of the polypeptide (e.g., a core polypeptide such as one of the CRISPR nucleases described herein, to which the NLS or NLSs is attached).
• the polypeptide e.g., a core polypeptide such as one of the CRISPR nucleases described herein, to which the NLS or NLSs is attached.
• a Type V CRISPR nuclease polypeptide e.g., Type Va CRISPR polypeptide
• a CRISPR nuclease polypeptide comprising one or more NLSs and, in some cases, a purification tag and/or a cleavage site, comprises a sequence at least 60, 70, 80, 85, 90, 95, 98, 99%, or 100%, identical, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98% identical to any one of SEQ ID NOs: 109-112.
• a Type V, e.g., VA CRISPR nuclease polypeptide comprises at least 1-30, 1-20, 1- 15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 2-30, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 3-30, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, or 3-5, preferably 1-10, more preferably 2-10, even more preferably 3-10 NLSs, each of which is at or near the N-terminus or the C-terminus of the polypeptide, in preferred embodiments at or near the N-terminus.
• At least two, or at least three, of the NLSs have different mechanisms, that is, different mechanisms by which they localize an attached polypeptide to a nucleus.
• Such mechanisms are well-known in the art; see, e.g., Lu et al. Cell Commun Signal (2021) 19:60 hftpsV/doLorg/IO.1186/s 12964 -021 -0074 ! -y.
• Suitable NLS, purification tag, and cleavage site sequences can be as described elsewhere herein, e.g., in sections labled Nuclear Localization Signals, Purification Tags, and Cleavage Sites.
• Nucleotide sequences coding for SEQ ID NO: 1 can include sequences with less than
• a nucleotide sequence coding for SEQ ID NO: 1 can also include nucleic acid sequences coding for one or more NLS at the N-terminus and/or C-terminus, as described herein, and/or a tag such as a purification tag at the N-terminus, as described herein.
• compositions comprising a first polynucleotide coding for a polypeptide comprising a nucleic acid-guided nuclease comprising a CRISPR Type V nuclease polypeptide, wherein the polynucleotide has less than 75% sequence identity to SEQ ID NO: 22, such as wherein the nuclease polypeptide comprises at least 1, 2, 3, 4, or 5 NLSs, wherein each of the NLSs is at or near the N-terminus or the C-terminus of the nuclease polypeptide.
• NLSs can be any of those described herein.
• the first polynucleotide can comprise a sequence coding for a purification tag, such as a purification tag described herein, and/or cleavage site, such as a cleavage site described herein.
• the first polynucleotide codes for a polypeptide comprising a sequence at least 60, 70, 80, 85, 90, 95, 98, 99%, or 100%, identical, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98% identical to any one of SEQ ID NOs: 109-112, such as SEQ ID NO: 109, or SEQ ID NO:
• the first polynucleotide comprises a sequence at least 50, 60, 70, 80, 90, 95, 97, or 99% identical, or 100% identical , preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98% identical to SEQ ID NO: 113.
• the composition further comprises a second polynucleotide coding for a gNA or portion thereof, wherein the gNA, e.g., gRNA, comprises a spacer sequence that targets a target nucleotide sequence within a polynucleotide, or a polynuclotide coding for the gNA, e.g., gRNA, wherein the gNA, e.g., gRNA is compatible with the Type V CRISPR nuclease.
• the first and second polynucleotides are the same.
• the composition can further comprise a third polynucleotide comprising a donor template.
• a vector comprising one of the polynucleotide compositions of this paragraph.
• a cell comprising one of the polynucleotide compositions of this paragraph, e.g., a human cell, such as an immune cell, for example a T cell, or a stem cell, such as an iPSC.
• a method comprising inserting any one of the polynucleotide compositions of this paragraph into a cell. In certain embodiments inserting the composition comprises electroporation.
• Exemplary nucleotide sequences coding for SEQ ID NO: 1 can include, e.g., SEQ ID NOs: 23-42:

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