WO2024047587A1 - Compositions cas-phi et méthodes d'utilisation - Google Patents
Compositions cas-phi et méthodes d'utilisation Download PDFInfo
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- WO2024047587A1 WO2024047587A1 PCT/IB2023/058639 IB2023058639W WO2024047587A1 WO 2024047587 A1 WO2024047587 A1 WO 2024047587A1 IB 2023058639 W IB2023058639 W IB 2023058639W WO 2024047587 A1 WO2024047587 A1 WO 2024047587A1
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Classifications
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
Definitions
- the present invention generally relates to systems, methods and compositions used for the control of gene expression involving sequence targeting, that may use vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof.
- CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
- the present invention also generally relates to use of AAV vectors for the delivery of large payloads, such as CRISPR proteins (e.g., Cas-phi), guide RNAs, CRISPR-Cas or CRISPR systems. Additionally, the present invention relates to methods for developing or designing CRISPR-Cas systems-based therapies or therapeutics.
- CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
- Adeno-associated virus (AAV) vectors have been proposed for gene delivery of CRISPR/Cas9 components for in vivo studies and therapeutic applications.
- AAV vectors provide stable gene expression with low risk of mutagenic integration events.
- AAV vectors can be engineered to target tissues of interest in vivo and are currently used in human clinical trials.
- gene delivery of S. pyogenes dCas9 in vivo is challenging because the size of the S. pyogenes dCas9 and an effector domain exceeds the packaging limits of standard AAV vectors. Accordingly, there exists a long felt and unmet medical need for alternative CRISPR systems, compositions and methods for use in in vivo studies and therapeutic applications.
- compositions and methods comprising CRISPR and a catalytically deficient Cas-phi protein, which is smaller in size than a S.
- the present disclosure provides a composition comprising: a) a Cas-phi polypeptide or a polynucleotide sequence encoding the Cas-phi polypeptide, wherein the Cas-phi polypeptide comprises at least one RuvC domain and wherein the at least one RuvC domain is nuclease inactive and the RuvC domain comprises at least one mutation relative to a wildtype RuvC domain; and b) a polynucleotide sequence encoding a guide RNA (gRNA) that can specifically hybridize to a target nucleic sequence and to the Cas phi polypeptide to form a complex.
- gRNA guide RNA
- the composition comprises: a) a Cas-phi polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, or a polynucleotide sequence encoding the Cas-phi polypeptide, wherein the Cas-phi polypeptide comprises at least one RuvC domain and wherein the at least one RuvC domain is nuclease inactive and the RuvC domain comprises at least one mutation relative to a wildtype RuvC domain; and b) a polynucleotide sequence encoding a guide RNA (gRNA) that can specifically hybridize to a target nucleic sequence and to the Cas phi polypeptide to form a complex.
- gRNA guide RNA
- the Cas-phi polypeptide is a Cas-phi-1, a Cas-phi-2 or a Cas- phi-3 polypeptide.
- the at least one mutation is i) D394R, D394A or D394N; ii) E606R, E606A or E606Q; iii) D695R, D695A or D695N; iv) D394A and E606A; v) D394A and D695A; vi) D394A, E606A and D695A; or vii) D394R, E606R and D695R, numbered in accordance to SEQ ID NO: 4.
- the Cas-phi polypeptide is a Cas-phi-2, and wherein the at least one mutation is i) D394A; ii) D394A and E606A; iii) D394A and D695A; or iv) D394A, E606A and D695A, numbered in accordance to SEQ ID NO: 4.
- the at least one mutation is i) D413A or D413N; ii) E618A or E618Q; iii) D708A or D708N; iv) D413A and E618A; v) D413A and D708A; or vi) D413A, E618A and D708A, numbered in accordance to SEQ ID NO: 6.
- the Cas-phi polypeptide is a Cas-phi-3, and wherein the at least one mutation is i) D413A; ii) D413A and E618A; iii) D413A and D708A; or iv) D413A, E618A and D708A, numbered in accordance to SEQ ID NO: 6.
- the Cas-phi polypeptide is a Cas-phi-1, and wherein the at least one mutation is D371A, numbered in accordance to SEQ ID NO: 2.
- the Cas-phi polypeptide further comprises a deletion of a RuvC domain in comparison to a wildtype Cas-phi polypeptide, wherein the deletion of the RuvC domain is a RuvC-II domain deletion and/or a RuvC-III domain deletion.
- the Cas-phi polypeptide further comprises a deletion of a zinc ribbon domain in comparison to a wildtype Cas-phi polypeptide.
- the Cas-phi polypeptide comprises the amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 160, 162, 164, 166, 168, 170, 172, 174, 176, 282, 284, 286, 288 or 290.
- the Cas-phi polypeptide comprises an amino acid sequence at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 160, 162, 164, 166, 168, 170, 172, 174, 176, 282, 284, 286, 288 or 290.
- the Cas-phi polypeptide or the polynucleotide sequence encoding the Cas-phi polypeptide is fused directly or indirectly to at least one effector domain or a polynucleotide sequence encoding the effector domain. In some embodiments, the Cas-phi polypeptide is fused indirectly to the at least one effector domain though a nuclear localization sequence and/or a linker sequence.
- the at least one effector domain is fused to i) the N-terminus of the Cas-phi polypeptide, ii) the C-terminus of the Cas-phi polypeptide, or iii) both the N- terminus and the C-terminus of the Cas-phi polypeptide. In some embodiments, the at least one effector domain fused to the N-terminus of the Cas-phi polypeptide and the C-terminus of the Cas-phi polypeptide are different. In some embodiments, the at least one effector domain fused to the N-terminus of the Cas-phi polypeptide and the C-terminus of the Cas-phi polypeptide are the same.
- the at least one effector domain comprises an effector domain derived from a mini VPR, p65 NF- ⁇ transactivating subunit (p65), VP160, SET7, RTA, histone acetyltransferase p300, VPR, MyoDl, TET1 hydroxylase catalytic domain, LSDI, Cmi, AD2, CR3, GATA4, p53, MEF2C, TAX, PPARy, SET9, KRAB, DNMT3A, DNMT1, KRAB-MeCP2, SIN3A, Mxi1, SID4x or Dnmt3a3L or a combination thereof.
- p65 p65 NF- ⁇ transactivating subunit
- VP160 SET7
- RTA histone acetyltransferase p300
- VPR MyoDl
- TET1 hydroxylase catalytic domain LSDI, Cmi, AD2, CR3, GATA4, p53
- the target nucleic acid sequence of b) is a regulatory region of a gene.
- the regulatory region is a promoter or an enhancer.
- the target nucleic sequence of b) encodes a gene product, and wherein the gene product is A4GALT, AAGAB, ABCD1, ACSL4, ACTC1, ACVRL1, ADNP, AFF2, AHDC1, AKT3, ALX4, ANK2, ANKRD11, ANOS1, AP1S2, APC, AR, ARCN1, ARHGEF9, ARID1A, ARID1B, ARID2, ARSE, ARX, ASH1L, ASXL1, ASXL3, ATP7A, ATP8A2, ATRX, AUTS2, AVPR2, AXIN2, BAG3, BCL11A, BCLAF1, BCOR, BMP4, BMPR1A, BMPR2, BRAF, BRCA1, BRCA2, BRIP1, BRWD3, BTK, CACNA
- the target nucleic sequence of b) encodes a gene product, and wherein the gene product is MT-TL1, KCNQ2, DEAF1, SSBP1, KCNQ1, HNF1B, KAT6B, CDK8, MN1, COL4A1, CDKL5, VAPB, NALCN, TTR, RAC2, GJB2, MYO3A, MEIS2, BRCA2, NARS1, AIRE, GABRG3, RAD51, GATA6, PDX1, ETV6, BCL11B, CHEK2, WARS1, KAT6B, KCNQ1, PRNP, MAT1A, HCN4, DSG2, MAFB, ZSWIM6, WT1, NIPBL, COL9A3, MYH7, SMAD4, IL6ST, CAPN3, KCNK18, DDX3X, SCAMP5, APC, CEBPA, RBM20, PMS2, BEST1, HCN1, PKD1, MSH2, RAD50, EYA1, KCNQ2, PRK
- composition comprising: a) a modified Cas-phi polypeptide comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 2
- This disclosure also provides a vector comprising: the nucleic acid sequence encoding the Cas-phi polypeptide of (a) and the polynucleotide sequence encoding the gRNA of (b).
- the gRNA is operatively linked to a promoter recognized by RNA polymerase III.
- the gRNA is operatively linked to a human U6 promoter.
- the vector is a viral vector.
- the viral vector is an adeno-associated virus (AAV) vector.
- the vector is suitable for delivery via a non-viral delivery system.
- the non-viral delivery system is a lipid nanoparticle or an exosome.
- This disclosure also provides a viral particle comprising the vector of the disclosure.
- the viral particle is a recombinant AAV (rAAV) particle.
- the rAAV particle is an AAV9, AAV-PHP.eB, AAV-DJ, AAV2, MyoAAV, AAV1, AAV5, AAV6 or AAV8.
- This disclosure also provides a population of viral particles comprising a plurality of viral particles of the disclosure.
- This disclosure also provides a pharmaceutical composition comprising any one of the vector, the viral particle or the population of viral particles of the disclosure, and a pharmaceutically acceptable carrier, vehicle or diluent.
- This disclosure also provides a cell comprising any one of the vector or the viral particle of the disclosure. In some embodiments, the cell is a mammalian cell or an insect cell.
- This disclosure also provides a method of modifying the expression of a target gene in a population of cells comprising: contacting a population of cells comprising a target nucleic sequence encoding the target gene with any one of the vector, the viral particle, the population or the pharmaceutical composition of the disclosure, thereby modifying the expression of the target gene.
- the expression of the target gene is increased in the plurality of the modified population of cells in comparison to a population of cells contacted with any one of the vector, the viral particle, the population or the pharmaceutical composition of the disclosure; and a gRNA that does not specifically hybridize to the target nucleic acid sequence, but binds to the Cas-phi polypeptide to form a complex.
- the expression of the target gene is increased by at least about 1.10-fold, at least about 1.15-fold, at least about 1.20-fold, at least about 1.25-fold, at least about 1.30-fold, at least about 1.35- fold, at least about 1.40-fold, at least about 1.45-fold, at least about 1.50-fold, at least about 1.55-fold, at least about 1.60-fold, at least about 1.65-fold, at least about 1.70-fold, at least about 1.75-fold, at least about 1.80-fold, at least about 1.85-fold, at least about 1.90-fold, at least about 1.95-fold or at least about 2.0-fold.
- the target gene is A4GALT, AAGAB, ABCD1, ACSL4, ACTC1, ACVRL1, ADNP, AFF2, AHDC1, AKT3, ALX4, ANK2, ANKRD11, ANOS1, AP1S2, APC, AR, ARCN1, ARHGEF9, ARID1A, ARID1B, ARID2, ARSE, ARX, ASH1L, ASXL1, ASXL3, ATP7A, ATP8A2, ATRX, AUTS2, AVPR2, AXIN2, BAG3, BCL11A, BCLAF1, BCOR, BMP4, BMPR1A, BMPR2, BRAF, BRCA1, BRCA2, BRIP1, BRWD3, BTK, CACNA1A, CACNA1C, CAMK2A, CAMK2B, CAMTA1, CASK, CASZ1, CCNQ, CDC42BPB, CDH1, CDKL5, CDKN1C, CFC1, CHAMP1, CHD2,
- the expression of the target gene is decreased in the plurality of the modified population of cells in comparison to a population of cells contacted with any one of the vector, the viral particle, the population or the pharmaceutical composition, and a gRNA that does not specifically hybridize to the target nucleic acid sequence, but binds to the Cas-phi polypeptide to form a complex.
- the expression of the target gene is decreased by at least about 1.10-fold, at least about 1.15-fold, at least about 1.20-fold, at least about 1.25-fold, at least about 1.30-fold, at least about 1.35-fold, at least about 1.40-fold, at least about 1.45-fold, at least about 1.50-fold, at least about 1.55-fold, at least about 1.60-fold, at least about 1.65-fold, at least about 1.70-fold, at least about 1.75-fold, at least about 1.80-fold, at least about 1.85-fold, at least about 1.90-fold, at least about 1.95-fold or at least about 2.0-fold.
- the target gene is MT-TL1, KCNQ2, DEAF1, SSBP1, KCNQ1, HNF1B, KAT6B, CDK8, MN1, COL4A1, CDKL5, VAPB, NALCN, TTR, RAC2, GJB2, MYO3A, MEIS2, BRCA2, NARS1, AIRE, GABRG3, RAD51, GATA6, PDX1, ETV6, BCL11B, CHEK2, WARS1, KAT6B, KCNQ1, PRNP, MAT1A, HCN4, DSG2, MAFB, ZSWIM6, WT1, NIPBL, COL9A3, MYH7, SMAD4, IL6ST, CAPN3, KCNK18, DDX3X, SCAMP5, APC, CEBPA, RBM20, PMS2, BEST1, HCN1, PKD1, MSH2, RAD50, EYA1, KCNQ2, PRKCE, SYT1, GNAS, GSDME, LMX1B, ME
- the population of cells is a eukaryotic population of cells, a mammalian population of cells, an insect population of cells, a human population of cells or a plant population of cells.
- This disclosure also provides a modified population of cells produced by any one of the methods of the disclosure.
- This disclosure provides a method of reducing or eliminating the expression of a gene product in a subject comprising introducing to a cell of a subject the vector, the viral particle, the population or the pharmaceutical composition of the disclosure.
- This disclosure also provides method for treating or alleviating a symptom of a gene product related disorder in a subject comprising the step of introducing to a cell of the subject the vector, the viral particle, the population or the pharmaceutical composition of the disclosure.
- the subject is a human.
- Any of the above aspects can be combined with any other aspect.
- suitable methods and materials are described below. All documents cited herein, including any cross referenced or related patent or application is hereby incorporated herein by reference in its entirety for all purposes, unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention.
- FIGS.1A-1B shows a schematic representation of the Cas-phi gene expression modulation system (“GEMS”).
- GEMS Cas-phi gene expression modulation system
- FIG.1B shows a schematic representation of a gRNA with a targeting region.
- the inclusion of two SapI restriction enzyme sites flanking the unique targeting region in the gRNA scaffold enables efficient swapping of genome-targeting guides.
- FIG.2A shows a schematic representation of the Cas-phi constructs of the disclosure. Three Cas-phi orthologs (Cas-phi-1, Cas-phi-2 and Cas-phi-3) were each used to create three nuclease deficient Cas-phi polypeptide mutant constructs, respectively.
- Activator domains were fused to the N-terminal and C-terminal region of each mutant.
- Various combinations of RuvC domain point mutations, RuvC-III domain deletions and zinc ribbon domain deletions were designed to total nine constructs, representing the minimal Cas-phi protein required to direct upregulation of gene expression. Aspartic acid to alanine mutations in the first RuvC domain have been shown to eliminate nuclease activity. Additional mutations were included to ensure complete nuclease removal.
- FIG.2B shows a sequence alignment of exemplary Cas-phi-2 polypeptides of the disclosure compared to a wildtype Cas-phi-2 polypeptide sequence. Wildtypd “Cas-phi-2” polypeptide (SEQ ID NO: 4) was aligned with three mutant Cas-phi-2 polypeptides: “GEMS_2.1” (SEQ ID NO: 83); “GEMS_2.2” (SEQ ID NO: 85); and “GEMS_2.3” (SEQ ID NO: 87).
- FIG.2C shows a sequence alignment of exemplary Cas-phi-3 polypeptide of the disclosure compared to a wildtype Cas-phi-3 polypeptide sequence.
- Wildtype “Cas-phi-3” polypeptide (SEQ ID NO: 6) was aligned with three mutant Cas-phi-3 polypeptides: “GEMS_2.1” (SEQ ID NO: 89); “GEMS_2.2” (SEQ ID NO: 91); and “GEMS_2.3” (SEQ ID NO: 93).
- FIGS.3A-3B are a series of graphs showing that GEMS upregulates STXBP1 expression in HEK293T cells. STXBP1 mRNA levels were detected using qPCR.
- FIG.3A shows the results using nuclease deficient Cas-phi-2 polypeptides (2.1, 2.2 and 2.3) each bound with one of two different gRNAs (g4, g5).
- FIG.3B shows the results using nuclease deficient Cas-phi-3 polypeptides (3.1, 3.2 and 3.3) each bound with one of two different gRNAs (g1, g5).
- Expression was normalized to housekeeping gene beta-actin (ACTB) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fold change was determined by comparing to a non-targeting control guide for each respective construct (i.e., GEMS 2.1 uses its own GEMS 2.1 non-targeting control and so forth).
- FIGS.4A-4B are a series of graphs showing that GEMS upregulates SYNGAP1 expression in Kelly neuroblastoma cells. SYNGAP1 mRNA levels detected using qPCR.
- FIG.4A shows the results using nuclease deficient Cas-phi-2 polypeptides (2.1 and 2.2) each bound to one of two different gRNAs (g1, g2).
- FIG.4B shows the results using nuclease deficient Cas-phi-3 polypeptides (3.1 and 3.2) with one gRNAs (g3). Expression was normalized to housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
- GPDH housekeeping gene glyceraldehyde-3-phosphate dehydrogenase
- FIGS.5A-5B are a series of graphs showing that GEMS upregulates ASCL1 in HEK293T cells.
- FIG.5A shows the results using nuclease deficient Cas-phi 3 polypeptide (3.1 and 3.3) with one gRNA (g1). ASCL1 mRNA levels detected using qPCR. Expression was normalized to housekeeping gene beta-actin (ACTB) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fold change was determined by comparing to a non-targeting control guide for each respective construct. Error bars represent standard deviation.
- FIG.5B shows that a nuclease deficient Cas-phi-2 polypeptide (2.3) upregulates ASCL1 in HEK293T cells using two versions of the same gRNA.
- FIGS.6A-6B are a series of graphs showing that GEMS2.1 and GEMS3.1 upregulate luciferase expression via the SYNGAP1 promoter in HEK293T cells. Relative luminescence was measured using a plate reader. Luminescence signal from luciferase was normalized to an internal control (Renilla). Fold change was determined by comparing luminance from experimental conditions to the luminance of a non-targeting control guide for each respective construct. The gRNA comprised the mature scaffold (Table 6.2) in all samples. Each GEMS variant comprised the NLS linker 1 (SEQ ID NO: 297).
- FIG.7 shows a schematic representation of the Cas-phi constructs of the disclosure.
- Cas-phi orthologs (Cas-phi-2 and Cas-phi-3) were each used to create five nuclease deficient Cas-phi polypeptide mutant constructs, respectively.
- Cas-phi-3 - GEMS 3.4A, GEMS 3.4N, GEMS 3.5, GEMS 3.6A, GEMS3.6N Activator domains
- FIG.8 is a graph showing GEMS 2.0 variant with only a C-terminal VP64 upregulates SYNGAP1 expression using SYNGAPg7 gRNA in Kelly neuroblastoma cells. SYNGAP1 mRNA levels detected using qPCR. Expression was normalized to housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fold change was determined by comparing to a non-targeting control guide for each respective construct. Error bars represent standard deviation. The gRNA comprised the mature scaffold (Table 6.2) in all samples.
- FIG.9 is a graph showing GEMS 3.1 and 3.4N variants with only a C-terminal VP64 upregulate SYNGAP1 expression using SYNGAPg7 gRNA in HEK293T cells. SYNGAP1 mRNA levels detected using qPCR. Expression was normalized to housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fold change was determined by comparing to a non-targeting control guide for each respective construct. Error bars represent standard deviation. The gRNA comprised the mature scaffold (Table 6.2) in all samples.
- FIG.10 is a graph showing GEMS 3.1 variant with miniVPR upregulates SYNGAP1 expression using SYNGAPg7 gRNA in HEK293T cells. SYNGAP1 mRNA levels detected using qPCR. Expression was normalized to housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fold change was determined by comparing to a non-targeting control guide for each respective construct. Error bars represent standard deviation. The gRNA comprised the mature scaffold (Table 6.2) in all samples. Each GEMS variant comprised the NLS linker 3 (SEQ ID NO: 299).
- FIGS.11A-11B are a series of graphs showing that GEMS 2.0 and 3.0 variants with miniVPR upregulate IL1RN in HeLa cells.
- the gRNA comprised the mature scaffold (Table 6.2) in all samples.
- Each GEMS variant comprised the NLS linker 3 (SEQ ID NO: 299).
- This disclosure provides engineered CRISPR-Cas effector proteins (e.g., Cas-phi) that comprise at least one modification compared to an unmodified CRISPR-Cas effector protein, which preserves binding of the of the CRISPR complex to the target binding site and reduces or eliminates the nuclease activity of the CRISPR-Cas effector protein relative to a wildtype.
- the engineered CRISPR-Cas effector protein is a nuclease deficient Cas-phi polypeptide.
- This disclosure also provides nuclease deficient Cas-phi polypeptides that are fused with an effector domain (e.g., an activator or a repressor protein) at the N-terminus or the C- terminus, or both the N-terminus and the C-terminus to create a gene expression modulation system (“GEMS”).
- GEMS gene expression modulation system
- This disclosure also provides viral vectors (e.g., AAV vectors) for delivery of CRISPR-Cas effector proteins, including nuclease deficient Cas-phi polypeptides.
- AAV vectors e.g., AAV vectors
- the clinical standard for many CRISPR-based therapeutics is Cas9 from S. aureus.
- the size of the smallest Cas9 is 3.1kb, which represents two-thirds of the total 4.7kb packaging capacity for AAV vectors. Therefore, a substantial benefit is to be gained by reducing the size of the DNA targeting module (DTM) used for therapeutic purposes.
- DTM DNA targeting module
- Shorter DTMs allow the opportunity to include additional effector domains (e.g., activator or repressor proteins) that can be packaged within the total 4.7kb packaging capacity for AAV vectors.
- DTMs i.e., nuclease deficient Cas-phi polypeptide
- This disclosure provides DTMs (i.e., nuclease deficient Cas-phi polypeptide) that is about 2.2kb to about 2.7kb in size, which is smaller than a wildtype Cas-phi polypeptide and also smaller than the smallest wildtype Cas9.
- This is advantageous for AAV packaging and the ability to include additional effector domains, which is beneficial for downstream therapeutic purposes.
- the present invention in particular relates to methods for improving CRISPR-Cas systems, such as CRISPR-Cas system based therapy or therapeutics for modulating gene expression.
- CRISPR-Cas system based therapy or therapeutics involve altered gene expression (increased or decreased relative to wildtype), high specificity, high efficacy, and high safety.
- CRISPR-Cas system based therapy or therapeutics involve altered gene expression (increased or decreased relative to wildtype), high specificity, high efficacy, and high safety.
- CRISPR-Cas system based therapy or therapeutics involve altered gene expression (increased or decreased relative to wildtype), high specificity, high efficacy, and high safety.
- compositions herein comprise or the methods herein comprise delivering one or more components of a nuclei acid-targeting system.
- nucleic acid-targeting system refers collectively to transcripts and other elements involved in the expression of or directing the activity of nucleic acid-targeting CRISPR-associated (“Cas”) genes (also referred to herein as an effector protein), including sequences encoding a nucleic acid-targeting Cas (effector) protein and a guide RNA, or other sequences and transcripts from a nucleic acid-targeting CRISPR locus.
- Cas nucleic acid-targeting CRISPR-associated
- one or more elements of a nucleic acid-targeting system is derived from a particular organism comprising an endogenous nucleic acid-targeting CRISPR system.
- a nucleic acid-targeting system is characterized by elements that promote the formation of a nucleic acid-targeting complex at the site of a target sequence.
- 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 RNA promotes the formation of a DNA or RNA-targeting complex.
- a target sequence may comprise 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 “target nucleic acid sequence” or “target DNA” or “target sequence”.
- nucleic acid-targeting complex comprising a guide RNA hybridized to a target sequence and complexed with one or more nucleic acid-targeting effector proteins
- cleavage of one or both RNA strands in or near e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from
- one or more vectors driving expression of one or more elements of a nucleic acid-targeting system are introduced into a host cell such that expression of the elements of the nucleic acid-targeting system direct formation of a nucleic acid-targeting complex at one or more target sites.
- nucleic acid-targeting effector protein and a guide RNA could each be operably linked to separate regulatory elements on separate vectors.
- two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the nucleic acid- targeting system not included in the first vector.
- Nucleic acid-targeting system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5′ with respect to (“upstream” of) or 3′ with respect to (“downstream” of) a second element.
- the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction.
- a single promoter drives expression of a transcript encoding a nucleic acid-targeting effector protein and a guide RNA embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).
- the nucleic acid-targeting effector protein and guide RNA are operably linked to and expressed from the same promoter.
- 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 target, e.g., have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
- the section of the guide sequence through which complementarity to the target sequence is important for cleavage activity is referred to herein as the seed sequence.
- a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides and is comprised within a target locus of interest. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.
- the nucleic acid-targeting effector protein is part of a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the nucleic acid-targeting effector protein).
- the CRISPR effector protein is part of a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme).
- fuse refers to the covalent linkage between two polypeptides in a fusion protein.
- the polypeptides may be fused via a peptide bond, either directly to each other or via a linker.
- fusion protein refers to a protein having at least two polypeptides covalently linked, either directly or via a linker (e.g., an amino acid linker).
- the polypeptides forming a fusion protein may be linked C-terminus to N-terminus, C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus.
- the polypeptides of the fusion protein may be in any order and may include more than one of either or both of the constituent polypeptides.
- fusion protein encompass conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, interspecies homologs, and immunogenic fragments of the antigens that make up the fusion protein.
- a fusion protein may be a protein developed from a fusion gene that is created through a joining of two or more genes originally coding for separate proteins. Translation of this fusion gene may result in a single or multiple polypeptides with functional properties derived from each of the original proteins. Fusion proteins of the disclosure may also comprise additional copies of a component antigen or immunogenic fragment thereof.
- a CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains.
- protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
- epitope tags include histidine (His) tags, V5 tags, FLAG tags (e.g., DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 124), influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
- reporter genes include, but are not limited to, glutathione-S- transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).
- a CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference.
- a tagged CRISPR enzyme is used to identify the location of a target sequence.
- a CRISPR enzyme may form a component of an inducible system.
- the inducible nature of the system would allow for spatiotemporal control of gene editing or gene expression using a form of energy.
- the form of energy may include but is not limited to electromagnetic radiation, sound energy, chemical energy and thermal energy.
- Examples of inducible system include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc), or light inducible systems (Phytochrome, LOV domains, or cryptochrome).
- the CRISPR enzyme may be a part of a Light Inducible Transcriptional Effector (LITE) to direct changes in transcriptional activity in a sequence-specific manner.
- LITE Light Inducible Transcriptional Effector
- the components of a light may include a CRISPR enzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsis thaliana), and a transcriptional activation/repression domain.
- CRISPR enzyme e.g. from Arabidopsis thaliana
- a transcriptional activation/repression domain e.g. from Arabidopsis thaliana
- inducible DNA binding proteins and methods for their use are provided in U.S. 61/736,465 and U.S.61/721,283 and WO 2014/018423 and U.S. Pat. Nos.8,889,418, 8,895,308, US20140186919, US20140242700, US20140273234, US20140335620, WO2014093635, which is hereby incorporated by reference in its entirety.
- a recombination template is also provided.
- a recombination template may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide.
- a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a nucleic acid-targeting effector protein as a part of a nucleic acid-targeting complex.
- the CRISPR system comprises (i) a CRISPR protein or a polynucleotide encoding a CRISPR effector protein and (ii) one or more polynucleotides engineered to: complex with the CRISPR protein to form a CRISPR complex; and to complex with the target sequence.
- the therapeutic is for delivery (or application or administration) to a eukaryotic cell, either in vivo or ex vivo.
- the CRISPR protein is a Cas-phi from Biggiephage.
- the CRISPR protein further comprises one or more nuclear localization sequences (NLSs) capable of driving the accumulation of the CRISPR protein to a detectible amount in the nucleus of the cell of the organism.
- NLSs nuclear localization sequences
- the CRISPR protein comprises one or more mutations.
- the CRISPR protein has one or more mutations in a catalytic domain, and wherein the protein further comprises a functional domain.
- the CRISPR system is comprised within a delivery system, optionally: a vector system comprising one or more vectors, optionally wherein the vectors comprise one or more viral vectors, optionally wherein the one or more viral vectors comprise one or more lentiviral, adenoviral or adeno-associated viral (AAV) vectors; or a particle or lipid particle, optionally wherein the CRISPR protein is complexed with the polynucleotides to form the CRISPR complex.
- the system, complex or protein is for use in a method of modifying an organism or a non-human organism by manipulation of the expression of a gene encoded by a target sequence in a genomic locus of interest.
- the polynucleotides encoding the sequence encoding or providing the CRISPR system are delivered via liposomes, particles, cell penetrating peptides, exosomes, microvesicles, or a gene-gun. In some embodiments, a delivery system is included.
- the delivery system comprises: a vector system comprising one or more vectors comprising the engineered polynucleotides and polynucleotide encoding the CRISPR protein, optionally wherein the vectors comprise one or more viral vectors, optionally wherein the one or more viral vectors comprise one or more lentiviral, adenoviral or adeno-associated viral (AAV) vectors; or a particle or lipid particle, containing the CRISPR system or the CRISPR complex.
- the CRISPR protein has one or more mutations in a catalytic domain, and wherein the enzyme further comprises a functional domain.
- a recombination/repair template is provided.
- gRNA molecule refers to a guide RNA that is capable of targeting a CRISPR nuclease or a nuclease-deficient CRISPR-associated protein to a target nucleic acid.
- gRNA molecule refers to a guide ribonucleic acid or to a nucleic acid encoding a gRNA.
- sequence identity refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of residues, e.g. nucleotides or amino acids.
- an “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical residues which are shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, i.e. the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100. Percentage identity can be calculated using the alignment program Clustal Omega, available at ebi.ac.uk/Tools/msa/clustalo using default parameters. See, Sievers et al., “Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega” (2011 October 11) Molecular Systems Biology 7:539.
- a regulatory sequence e.g., a promoter
- a regulatory sequence is considered to be “operatively linked” when it is in a functional location and orientation in relation to a nucleic acid sequence it regulates to control transcriptional initiation and/or expression of that sequence.
- self-complementary when referring to an AAV vector refers to an AAV vector comprising a nucleic acid (i.e., a DNA) that forms a dimeric inverted repeat molecule that spontaneously anneals, resulting in earlier and more robust transgene expression compared with conventional single-strand (ss) AAV genomes.
- scAAV self- complementary AAV
- scAAV self- complementary AAV
- the terms “treat,” “treating” or “treatment of” mean that the severity of the subject's condition is reduced, at least partially improved or stabilized and/or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
- the terms “prevent,” “preventing” and “prevention” refer to prevention and/or delay of the onset of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the compositions and/or methods described herein.
- the prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s).
- a nucleic acid sequence provided herein is a nucleic acid sense strand (e.g., 5' to 3' strand), or in the context of a viral sequences a plus (+) strand.
- a nucleic acid sequence is a nucleic acid antisense strand (e.g., 3' to 5' strand), or in the context of viral sequences a minus (-) strand.
- a “therapeutically effective amount” is the amount of a vector, viral particles, a population of viral particles or a pharmaceutical composition provided herein that is effective to treat or prevent a disease or disorder in a subject or to ameliorate a sign or symptom thereof.
- the “therapeutically effective amount” may vary depending, for example, on the disease and/or symptoms of the disease, severity of the disease and/or symptoms of the disease or disorder, the age, weight, and/or health of the patient to be treated, and the judgment of the prescribing physician.
- wildtype is a term of the art understood by skilled persons and means the typical form of an organism, strain, protein, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
- the methods may comprise delivering one or more components of a CRISPR-Cas system to a target locus.
- a nucleic acid-targeting system may comprise one or more components of a CRISPR-Cas system.
- the present invention relates to methods for increasing the expression of a gene product encoded by a target sequence using a CRISPR-Cas system, such as CRISPR-Cas system based therapy or therapeutics.
- the present invention relates to methods for decreasing the expression of a gene product encoded by a target sequence using a CRISPR-Cas system, such as CRISPR-Cas system based therapy or therapeutics.
- a CRISPR-Cas system such as CRISPR-Cas system based therapy or therapeutics.
- the CRISPR-Cas system comprises a CRISPR effector as defined herein elsewhere.
- the methods of the present invention in particular involve optimization of selected parameters or variables associated with the CRISPR-Cas system and/or its functionality, as described herein further elsewhere.
- optimization of the CRISPR-Cas system in the methods as described herein may depend on the target(s), such as the therapeutic target or therapeutic targets, the mode or type of CRISPR-Cas system modulation, such as CRISPR-Cas system based therapeutic target(s) modulation, modification, or manipulation, as well as the delivery of the CRISPR-Cas system components.
- One or more targets may be selected, depending on the genotypic and/or phenotypic outcome.
- one or more therapeutic targets may be selected, depending on (genetic) disease etiology or the desired therapeutic outcome.
- the (therapeutic) target(s) may be a single gene, locus, or other genomic site, or may be multiple genes, loci or other genomic sites.
- CRISPR-Cas system activity such as CRISPR-Cas system design may involve target disruption, such as target mutation, such as leading to gene knockout.
- CRISPR-Cas system activity such as CRISPR-Cas system design may involve replacement of particular target sites, such as leading to target correction.
- CISPR-Cas system design may involve removal of particular target sites, such as leading to target deletion.
- CRISPR-Cas system activity may involve modulation of target site functionality, such as target site activity or accessibility, leading for instance to (transcriptional and/or epigenetic) gene or genomic region activation or gene or genomic region silencing.
- target site functionality such as target site activity or accessibility
- modulation of target site functionality may involve CRISPR effector mutation (such as for instance generation of a catalytically inactive CRISPR effector) and/or functionalization (such as for instance fusion of the CRISPR effector with a heterologous functional domain, such as a transcriptional activator or repressor), as described herein elsewhere.
- Example functional domains suitable for use in the embodiments disclosed herein are discussed in further detail below.
- CRISPR-Cas protein and corresponding guide molecule.
- said CRISPR-Cas protein Cas-phi.
- the CRISPR-Cas system does not require the generation of customized proteins to target specific sequences but rather a single Cas protein can be programmed by guide molecule to recognize a specific nucleic acid target, in other words the Cas enzyme protein can be recruited to a specific nucleic acid target locus of interest using said guide molecule.
- CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
- SPIDRs Sacer Interspersed Direct Repeats
- the CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556 [1989]), and associated genes.
- SSRs interspersed short sequence repeats
- the CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]).
- SRSRs short regularly spaced repeats
- the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al., [2000], supra).
- the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J.
- CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575 [2002]; and Mojica et al., [2005]) including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomon
- CRISPR-Cas Enzyme [095] The terms “CRISPR-Cas protein”, “CRISPR protein”, “Cas protein”, “Cas effector protein”, “CRISPR enzyme”, and “Cas enzyme” may be used interchangeably herein.
- a CRISPR-Cas protein In its unmodified form, a CRISPR-Cas protein is a catalytically active protein. This implies that upon formation of a nucleic acid-targeting complex (comprising a guide RNA hybridized to a target sequence one or both DNA strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence is modified (e.g. cleaved).
- sequence(s) associated with a target locus of interest refers to sequences near the vicinity of the target sequence (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence, wherein the target sequence is comprised within a target locus of interest).
- the unmodified catalytically active Cas-phi protein targets and cleaves foreign genomes in bacterial cells. Unlike other compact Cas proteins that preferentially cut single-strantded DNA, Cas-phi is able to cut double-stranded DNA.
- Cas ⁇ or “Cas12j”
- the present invention encompasses the use of a Cas-phi polypeptide.
- Cas-phi polypeptides are also referred to as “Cas ⁇ ” or “Cas12j” polypeptide.
- Cas-phi is a family of Cas proteins encoded in the Biggiephage clade.
- Cas-phi has three RuvC domains (RuvC-I, RuvC-II and RuvC-III) and a zinc ribbon domain.
- Cas-phi contains a C-terminal RuvC domain with remote homology to that of the TnpB nuclease superfamily from which type V CRISPR-Cas proteins are thought to have evolved (Pausch et. Al., CRISPR-Cas ⁇ from huge phages is a hypercompact genome editor, 2020). Cas-phi has a small size of ⁇ 70– 80 kDa, about half the size of the Cas9 and Cas12a. [099] Orthologs of Cas-phi [0100] The terms “orthologue” (also referred to as “ortholog” herein) and “homologue” (also referred to as “homolog” herein) are well known in the art.
- a “homologue” of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homologue of. Homologous proteins may but need not be structurally related, or are only partially structurally related.
- An “orthologue” of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of.
- Orthologous proteins may but need not be structurally related, or are only partially structurally related. Homologs and orthologs may be identified by homology modelling (see, e.g., Greer, Science vol.228 (1985) 1055, and Blundell et al.
- the Cas-phi polypeptide is derived from an organism from Biggiephage clade.
- the amino acid sequence of the Cas-phi polypeptide corresponds to Cas-phi-1 (SEQ ID NO: 2), Cas-phi-2 (SEQ ID NO: 4; PDB: 7LYT, 7M5O, 7LYS) or Cas-phi-3 (SEQ ID NO: 6; PDB: 7ODF).
- Amino acid sequences of exemplary Cas- phi polypeptides of the disclosure and polynucleotide sequences encoding the Cas-phi polypeptides are shown in Table 1.
- the Cas-phi polypeptide has a sequence homology or sequence identity of at least 60%, more particularly at least 70%, such as at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95%, with Cas- phi-1 polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
- the Cas-phi polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 2.
- the Cas-phi polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 2.
- the Cas-phi polypeptide is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 1.
- the Cas-phi polypeptide is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 1.
- the Cas-phi polypeptide has a sequence homology or sequence identity of at least 60%, more particularly at least 70%, such as at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95%, with Cas- phi-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 4.
- the Cas-phi polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 4.
- the Cas-phi polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 4.
- the Cas-phi polypeptide is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 3.
- the Cas-phi polypeptide is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 3.
- the Cas-phi polypeptide has a sequence homology or sequence identity of at least 60%, more particularly at least 70%, such as at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95%, with Cas- phi-3 polypeptide comprising the amino acid sequence of SEQ ID NO: 6.
- the Cas-phi polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 6.
- the Cas-phi polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 6.
- the Cas-phi polypeptide is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 5.
- the Cas-phi polypeptide is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 5.
- any one of the Cas-phi polypeptides includes truncated forms of the Cas-phi polypeptide, whereby the sequence identity is determined over the length of the truncated form.
- RuvC-1 domain is shown in bold font
- RuvC-II domain is shown in underline, italicized font
- RuvC-III domain is shown in underline font
- zinc ribbon domain is shown in bold, italicized font
- point mutations are shown in bold, underline font.
- Exemplary Wildtype Cas-phi polypeptide sequences [0117] Modified Cas-phi polypeptides [0118]
- the disclosure provides a Cas-phi polypeptide that is mutated with respect to a corresponding wild-type enzyme, such that the mutated CRISPR-Cas protein lacks the ability to cleave one or both DNA strands of a target locus containing a target sequence.
- one or more nuclease domains of the Cas-phi polypeptide are mutated to produce a mutated Cas-phi polypeptide that is nuclease deficient.
- one or more RuvC domains of the Cas-phi polypeptide are mutated to produce a mutated Cas-phi polypeptide that is nuclease deficient.
- one or more RuvC domains of the Cas-phi polypeptide are deleted to produce a truncated Cas-phi polypeptide that is nuclease deficient.
- one or more zinc ribbon domains are deleted to produce a mutated Cas-phi polypeptide that is nuclease deficient.
- the Cas-phi polypeptide may be mutated with respect to a corresponding wild-type enzyme such that the mutated Cas-phi polypeptide lacks substantially all DNA cleavage activity.
- the Cas-phi polypeptide is engineered and comprises at least one mutation that reduces or eliminates nuclease activity.
- a Cas-phi polypeptide may be considered to substantially lack all DNA and/or RNA cleavage activity when the cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the nucleic acid cleavage activity of the non-mutated form of the enzyme; an example can be when the nucleic acid cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form.
- the Cas-phi polypeptide may be modified to have diminished nuclease activity e.g., nuclease inactivation of at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% as compared with the wild type enzyme; or to put in another way, a Cas-phi enzyme having advantageously about 0% of the nuclease activity of the non-mutated or wild type Cas-phi enzyme or CRISPR enzyme, or no more than about 3% or about 5% or about 10% of the nuclease activity of the non-mutated or wild type Cas-phi enzyme.
- the Cas-phi polypeptide is used as a generic DNA binding protein (or “DNA binding module” or “DTM”).
- the mutations may be artificially introduced mutations or gain- or loss-of-function mutations.
- the CRISPR-Cas protein may be additionally modified.
- the term “modified” with regard to a CRISPR-Cas protein generally refers to a CRISPR-Cas protein having one or more modifications or mutations (including point mutations, truncations, insertions, deletions, chimeras, fusion proteins, etc.) compared to the wild type Cas protein from which it is derived.
- derived is meant that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as known in the art or as described herein.
- the additional modifications and/or truncations of the CRISPR-Cas protein may or may not cause an altered functionality.
- modifications which do not result in an altered functionality include for instance codon optimization for expression into a particular host, or providing the nuclease with a particular marker (e.g. for visualization).
- the Cas-phi polypeptide comprises a mutation of at least one residue, including but not limited to positions E107, Q127, D134, D141, K145, N195, K265, K278, D329, N333, D337, T340, D342, T357, K367, K371, K373, Q397, E446, N497, K522, K527, K528, E569, Q368, K642, I665, E674, A682, N693, N702, T709, E717 or E718 numbered according to Cas-phi-2 polypeptide (SEQ ID NO: 4) or any corresponding ortholog.
- the Cas-phi polypeptide comprises a mutation of at least one residue, including but not limited to E107R, Q127R, D134R, D141R, K145R, N195R, K265R, K278R, D329R, N333R, D337R, T340R, D342R, T357R, K367R, K371R, K373R, Q397R, E446R, N497R, K522R, K527R, K528R, E569R, Q368R, K642R, I665R, E674R, A682R, N693R, N702R, T709R, E717R or E718R, numbered according to Cas-phi-2 polypeptide (SEQ ID NO: 4) or any corresponding ortholog.
- SEQ ID NO: 4 Cas-phi-2 polypeptide
- the Cas- phi polypeptide comprises a mutation of E107R/E606Q, E107R/E606Q, Q127R/E606Q, Q127R/E606Q, D134R/E606Q, D134R/E606Q, D141R/E606Q, D141R/E606Q, K145R/E606Q, K145R/E606Q, N195R/E606Q, N195R/E606Q, K265R/E606Q, K265R/E606Q, K278R/E606Q, K278R/E606Q, D329R/E606Q, D329R/E606Q, N333R/E606Q, N333R/E606Q, D337R/E606Q, D337R/E606Q, T340R/E606Q, T340R/E606Q, D342R/E606Q, D342R/E
- the Cas-phi polypeptide has reduced or no catalytic and/or nuclease activity.
- Cas-phi polypeptide mutations may include but are not limited to one or more mutations in the catalytic RuvC domain (e.g. RuvC-I, RuvC-II or RuvC-III).
- the Cas-phi polypeptide comprises at least one mutation in the RuvC-I domain, the RuvC-II domain, the RuvC-III domain or a combination thereof.
- the Cas-phi polypeptide comprises at least one mutation in the RuvC-I domain.
- the Cas-phi polypeptide comprises at least one mutation in the RuvC-I domain and the RuvC-II domain. In some embodiments, the Cas-phi polypeptide comprises at least one mutation in the RuvC-I domain, the RuvC-II domain and the RuvC-III domain. [0127] Exemplary RuvC domains are shown in Table 2. [0128] Table 2. Cas-phi polypeptide RuvC and Zinc Ribbon Domains [0129] In some embodiments, the Cas-phi polypeptide comprises a mutation of at least one residue in at least one Ruv-C domain, including but not limited to positions D371, numbered according to Cas-phi-1 polypeptide (SEQ ID NO: 2) or any corresponding ortholog.
- the Cas-phi polypeptide comprises a mutation of at least one residue in at least one Ruv-C domain, including but not limited to D371A numbered according to Cas-phi-1 polypeptide (SEQ ID NO: 2) or any corresponding ortholog. [0130] In some embodiments, the Cas-phi polypeptide comprises a mutation of at least one residue in at least one Ruv-C domain, including but not limited to positions D394, E606 and D695 numbered according to Cas-phi-2 polypeptide (SEQ ID NO: 4) or any corresponding ortholog.
- the Cas-phi polypeptide comprises a mutation of at least one residue in at least one Ruv-C domain, including but not limited to D394A, D394N, D394R, E606A, E606Q, E606R, D695N, D695R and D695A numbered according to Cas-phi-2 polypeptide (SEQ ID NO: 4) or any corresponding ortholog.
- the Cas- phi polypeptide comprises a mutation of D394A; D394A and E606A; D394A and D695A; D394A, E606A and D695A; or D394R, E606R and D695R, numbered according to Cas-phi- 2 polypeptide (SEQ ID NO: 4) or any corresponding ortholog.
- the Cas-phi polypeptide comprises a mutation of at least one residue in at least one Ruv-C domain, including but not limited to positions D413, E618 and D708 numbered according to Cas-phi-3 polypeptide (SEQ ID NO: 6) or any corresponding ortholog.
- the Cas-phi polypeptide comprises a mutation of at least one residue in at least one Ruv-C domain, including but not limited to D413A, D413N, E618A, E618Q, D708N and D708A numbered according to Cas-phi-3 polypeptide (SEQ ID NO: 6) or any corresponding ortholog.
- the Cas-phi polypeptide comprises a mutation of D413A; D413A and E618A; D413A and D708A; or D413A, E618A and D708A, numbered according to Cas-phi-3 polypeptide (SEQ ID NO: 6) or any corresponding ortholog.
- the Cas-phi polypeptide comprises a deletion of at least one RuvC domain according to a Cas-phi-1 polypeptide, a Cas-phi-2 polypeptide, a Cas-phi-3 polypeptide or any corresponding ortholog.
- Cas-phi polypeptide deletions may include but are not limited to one or more deletions in the catalytic RuvC domain (e.g. RuvC-I, RuvC-II or RuvC-III).
- the Cas-phi polypeptide comprises at least one deletion in the RuvC-I domain, the RuvC-II domain, the RuvC-III domain or a combination thereof.
- the Cas-phi polypeptide comprises a deletion of the RuvC-III domain. In some embodiments, the Cas-phi polypeptide comprises a deletion of the RuvC-II and RuvC-III domain. [0133] In some embodiments, the Cas-phi polypeptide comprises a deletion of at least one zinc ribbon domain according to a Cas-phi-1 polypeptide, a Cas-phi-2 polypeptide, a Cas-phi- 3 polypeptide or any corresponding ortholog. [0134] Amino acid sequences of exemplary mutant Cas-phi polypeptides and polynucleotide sequences encoding the same are shown in Table 3.1 and 3.2.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.1” or “1.1”) comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 77.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.1” or “1.1”) comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 77.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.1” or “1.1”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 76.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.1” or “1.1”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 76.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.2” or “1.2”) comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 79.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.2” or “1.2”) comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 79.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.2” or “1.2”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 78.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.2” or “1.2”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 78.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.3” or “1.3”) comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 81.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.3” or “1.3”) comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 81.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.3” or “1.3”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 80.
- the Cas-phi-1 mutant polypeptide (“CAS-PHI 1.3” or “1.3”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 80.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.1” or “2.1”) comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 83.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.1” or “2.1”) comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 83.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.1” or “2.1”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 82.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.1” or “2.1”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 82.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.2” or “2.2”) comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 85.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.2” or “2.2”) comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 85.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.2” or “2.2”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 84.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.2” or “2.2”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 84.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.3” or “2.3”) comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 87.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.3” or “2.3”) comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 87.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.3” or “2.3”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 86.
- the Cas-phi-2 mutant polypeptide (“CAS-PHI 2.3” or “2.3”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 86.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 160.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 160.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.4A” or “2.4A”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 159.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.4A” or “2.4A”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 159.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 162.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 162.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.4N” or “2.4N”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 161.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.4N” or “2.4N”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 161.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 164.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 164.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.5” or “2.5”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 163.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.5” or “2.5”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 163.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 166.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 166.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.6A” or “2.6A”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 165.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.6A” or “2.6A”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 165.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 168.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 168.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.6N” or “2.6N”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 167.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.6N” or “2.6N”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 167.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 170.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 170.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.7” or “2.7”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 169.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.7” or “2.7”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 169.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 172.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 172.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.8” or “2.8”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 171.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.8” or “2.8”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 171.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 174.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 174.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.9” or “2.9”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 173.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.9” or “2.9”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 173.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 176.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 176.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.11” or “2.11”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 175.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.11” or “2.11”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 175.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 178.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 178.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.12” or “2.12”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 177.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.12” or “2.12”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 177.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 180.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 180.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.13” or “2.13”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 179.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.13” or “2.13”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 179.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 182.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 182.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.14” or “2.14”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 181.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.14” or “2.14”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 181.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 184.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 184.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.15” or “2.15”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 183.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.15” or “2.15”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 183.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 186.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 186.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.16” or “2.16”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 185.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.16” or “2.16”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 185.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 188.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 188.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.17” or “2.17”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 187.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.17” or “2.17”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 187.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 190.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 190.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.18” or “2.18”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 189.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.18” or “2.18”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 189.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 192.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 192.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.19” or “2.19”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 191.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.19” or “2.19”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 191.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 194.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 194.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.21” or “2.21”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 193.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.21” or “2.21”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 193.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 196.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 196.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.22” or “2.22”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 195.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.22” or “2.22”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 195.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 198.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 198.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.23” or “2.23”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 197.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.23” or “2.23”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 197.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 200.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 200.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.24” or “2.24”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 199.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.24” or “2.24”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 199.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 202.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 202.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.25” or “2.25”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 201.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.25” or “2.25”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 201.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 204.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 204.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.26” or “2.26”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 203.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.26” or “2.26”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 203.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 206.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 206.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.27” or “2.27”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 205.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.27” or “2.27”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 205.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 208.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 208.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.28” or “2.28”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 207.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.28” or “2.28”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 207.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 210.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 210.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.29” or “2.29”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 209.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.29” or “2.29”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 209.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 212.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 212.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.31” or “2.31”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 211.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.31” or “2.31”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 211.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 214.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 214.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.32” or “2.32”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 213.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.32” or “2.32”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 213.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 216.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 216.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.33” or “2.33”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 215.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.33” or “2.33”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 215.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 218.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 218.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.34” or “2.34”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 217.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.34” or “2.34”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 217.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 220.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 220.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.35” or “2.35”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 219.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.35” or “2.35”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 219.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 222.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 222.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.36” or “2.36”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 221.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.36” or “2.36”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 221.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 224.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 224.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.37” or “2.37”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 223.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.37” or “2.37”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 223.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 226.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 226.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.38” or “2.38”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 225.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.38” or “2.38”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 225.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 228.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 228.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.39” or “2.39”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 227.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.39” or “2.39”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 227.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 230.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 230.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.41” or “2.41”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 229.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.41” or “2.41”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 229.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 232.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 232.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.42” or “2.42”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 231.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.42” or “2.42”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 231.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 234.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 234.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.43” or “2.43”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 233.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.43” or “2.43”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 233.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 236.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 236.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.44” or “2.44”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 235.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.44” or “2.44”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 235.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 238.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 238.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.45” or “2.45”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 237.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.45” or “2.45”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 237.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 240.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 240.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.46” or “2.46”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 239.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.46” or “2.46”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 239.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 242.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 242.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.47” or “2.47”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 241.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.47” or “2.47”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 241.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 244.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 244.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.48” or “2.48”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 243.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.48” or “2.48”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 243.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 246.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 246.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.49” or “2.49”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 245.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.49” or “2.49”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 245.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 248.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 248.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.51” or “2.51”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 247.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.51” or “2.51”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 247.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 250.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 250.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.52” or “2.52”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 249.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.52” or “2.52”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 249.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 252.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 252.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.53” or “2.53”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 251.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.53” or “2.53”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 251.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 254.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 254.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.54” or “2.54”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 253.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.54” or “2.54”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 253.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 256.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 256.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.55” or “2.55”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 255.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.55” or “2.55”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 255.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 258.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 258.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.56” or “2.56”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 257.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.56” or “2.56”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 257.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 260.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 260.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.57” or “2.57”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 259.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.57” or “2.57”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 259.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 262.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 262.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.58” or “2.58”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 261.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.58” or “2.58”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 261.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 264.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 264.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.59” or “2.59”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 263.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.59” or “2.59”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 263.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 266.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 266.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.61” or “2.61”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 265.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.61” or “2.61”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 265.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 268.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 268.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.62” or “2.62”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 267.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.62” or “2.62”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 267.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 270.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 270.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.63” or “2.63”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 269.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.63” or “2.63”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 269.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 272.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 272.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.64” or “2.64”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 271.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.64” or “2.64”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 271.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 274.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 274.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.65” or “2.65”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 273.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.65” or “2.65”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 273.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 276.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 276.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.66” or “2.66”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 275.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.66” or “2.66”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 275.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 278.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 278.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.67” or “2.67”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 277.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.67” or “2.67”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 277.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 280.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 280.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.68” or “2.68”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 279.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “2.68” or “2.68”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 279.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.1” or “3.1”) comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 89.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.1” or “3.1”) comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 89.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.1” or “3.1”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 88.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.1” or “3.1”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 88.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.2” or “3.2”) comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 91.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.2” or “3.2”) comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 91.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.2” or “3.2”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 90.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.2” or “3.2”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 90.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.3” or “3.3”) comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 93.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.3” or “3.3”) comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 93.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.3” or “3.3”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 92.
- the Cas-phi-3 mutant polypeptide (“CAS-PHI 3.3” or “3.3”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 92.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 282.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 282.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “3.4A” or “3.4A”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 281.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “3.4A” or “3.4A”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 281.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 284.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 284.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “3.4N” or “3.4N”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 283.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “3.4N” or “3.4N”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 283.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 286.
- the Cas-phi- 2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 286.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “3.5” or “3.5”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 285.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “3.5” or “3.5”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 285.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 288.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 288.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “3.6A” or “3.6A”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 287.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “3.6A” or “3.6A”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 287.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 290.
- the Cas-phi-2 mutant polypeptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 290.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “3.6N” or “3.6N”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence of SEQ ID NO: 289.
- the Cas-phi-2 mutant polypeptide (CAS-PHI “3.6N” or “3.6N”) is encoded by a polynucleotide sequence comprising, consisting essentially of or consisting of a nucleic acid sequence of SEQ ID NO: 289.
- Fusion proteins may without limitation include for instance fusions with heterologous domains or functional domains (e.g., localization signals, catalytic domains, etc.).
- various different modifications may be combined (e.g., a mutated nuclease which is catalytically inactive and which further is fused to a functional domain, such as for instance to induce DNA methylation or another nucleic acid modification, such as including without limitation a break (e.g. by a different nuclease (domain)), a mutation, a deletion, an insertion, a replacement, a ligation, a digestion, a break or a recombination).
- altered functionality includes without limitation an altered specificity (e.g., altered target recognition, increased (e.g., “enhanced” Cas proteins) or decreased specificity, or altered PAM recognition), altered activity (e.g., increased or decreased catalytic activity, including catalytically inactive nucleases or nickases), and/or altered stability (e.g., fusions with destabilization domains).
- altered specificity e.g., altered target recognition, increased (e.g., “enhanced” Cas proteins) or decreased specificity, or altered PAM recognition
- altered activity e.g., increased or decreased catalytic activity, including catalytically inactive nucleases or nickases
- stability e.g., fusions with destabilization domains.
- Suitable heterologous domains include without limitation a nuclease, a ligase, a repair protein, a methyltransferase, (viral) integrase, a recombinase, a transposase, an argonaute, a cytidine deaminase, a retron, a group II intron, a phosphatase, a phosphorylase, a sulpfurylase, a kinase, a polymerase, an exonuclease, etc. Examples of all these modifications are known in the art.
- a “modified” nuclease as referred to herein, and in particular a “modified” Cas or “modified” CRISPR-Cas system or complex preferably still has the capacity to interact with or bind to the polynucleic acid (e.g., in complex with the guide molecule).
- the Cas-phi polypeptide may have associated (e.g., via fusion protein or suitable linkers) one or more functional domains, including for example, one or more domains from the group comprising, consisting essentially of, or consisting of deaminase activity, methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g., light inducible).
- one or more functional domains including for example, one or more domains from the group comprising, consisting essentially of, or consisting of deaminase activity, methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g., light inducible).
- Suitable linkers for use in the methods of the present invention are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
- the linker is used to separate the Cas-phi polypeptide and the effector domain by a distance sufficient to ensure that each protein retains its required functional property.
- Preferred peptide linker sequences adopt a flexible extended conformation and do not exhibit a propensity for developing an ordered secondary structure.
- the linker can be a chemical moiety which can be monomeric, dimeric, multimeric or polymeric.
- the linker comprises amino acids.
- Typical amino acids in flexible linkers include Gly, Asn and Ser. Accordingly, in particular embodiments, the linker comprises a combination of one or more of Gly, Asn and Ser amino acids. Other near neutral amino acids, such as Thr and Ala, also may be used in the linker sequence.
- Exemplary linkers are disclosed in Maratea et al. (1985), Gene 40: 39-46; Murphy et al. (1986) Proc. Nat'l. Acad. Sci. USA 83: 8258-62; U.S. Pat. Nos.4,935,233; and 4,751,180.
- GlySer linkers GS, GGS, GGGS or GSG can be used.
- GGS, GSG, GGGS or GGGGS linkers can be used in repeats of 3 (such as (GGS)3 (SEQ ID NO: 146), (GGGGS)3 (SEQ ID NO: 94)) or 5 (SEQ ID NO: 95), 6 (SEQ ID NO: 96), 7 (SEQ ID NO: 97), 9 (SEQ ID NO: 98) or even 12 (SEQ ID NO: 99) or more, to provide suitable lengths.
- 3 such as (GGS)3 (SEQ ID NO: 146), (GGGGS)3 (SEQ ID NO: 94)
- 5 SEQ ID NO: 95
- 6 SEQ ID NO: 96
- 7 SEQ ID NO: 97
- 9 SEQ ID NO: 98
- 12 SEQ ID NO: 99
- the linker comprises (GGGGS)3 (SEQ ID NO: 94) (GGGGS)6 (SEQ ID NO: 96), (GGGGS)9 (SEQ ID NO: 98), (GGGGS)12 (SEQ ID NO: 99), (GGGGS)1 (SEQ ID NO: 147), (GGGGS)2 (SEQ ID NO: 100), (GGGGS)4 (SEQ ID NO: 101), (GGGGS)5 (SEQ ID NO: 102), (GGGGS)7 (SEQ ID NO: 103), (GGGGS)8 (SEQ ID NO: 104), (GGGGS)10 (SEQ ID NO: 105), or (GGGGS)11 (SEQ ID NO: 106).
- the linker comprises LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 107). In some embodiments, the linker comprises GSEASGSGRA (SEQ ID NO: 290) In some embodiments, the linker comprises an XTEN linker. [0292] NLSs [0293] In some embodiments, the Cas-phi polypeptide further comprises one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, vector comprises one or more NLSs not naturally present in the Cpf1 effector protein.
- NLSs nuclear localization sequences
- the NLS is present in the vector 5′ and/or 3′ of the Cpf1 effector protein sequence
- the RNA-targeting effector protein comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g., zero or at least one or more NLS at the amino-terminus and zero or at one or more NLS at the carboxy terminus).
- an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C- terminus.
- Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 108); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 109)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 110) or RQRRNELKRSP (SEQ ID NO: 111); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 112); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 113) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 114) and
- the one or more NLSs are of sufficient strength to drive accumulation of the DNA/RNA-targeting Cas protein in a detectable amount in the nucleus of a eukaryotic cell.
- strength of nuclear localization activity may derive from the number of NLSs in the nucleic acid- targeting effector protein, the particular NLS(s) used, or a combination of these factors.
- the Cas-phi polypeptide further comprises one or more nuclear localization sequences fused with a linker (“NLS linker”).
- an “NLS linker 1” comprises the amino acid sequence of KRPAATKKAGQAKKKKGS (SEQ ID NO: 297).
- an “NLS linker 2” comprises the amino acid sequence of KRPAATKKAGQAKKKKGSEASGSGRA (SEQ ID NO: 298).
- an “NLS linker 1” comprises the amino acid sequence of KRPAATKKAGQAKKKKGGGGSGGGGSGGGGS (SEQ ID NO: 299).
- NLS nuclear localization signal sequence
- NLS nuclear localization signal sequence
- an N- and C-terminal nuclear localization signal sequence (NLSs) can also function as linker.
- Detection of accumulation in the nucleus may be performed by any suitable technique.
- a detectable marker may be fused to the nucleic acid-targeting protein, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g., a stain specific for the nucleus such as DAPI).
- Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay.
- Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of nucleic acid-targeting complex formation (e.g., assay for DNA or RNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by DNA or RNA-targeting complex formation and/or DNA or RNA-targeting Cas protein activity), as compared to a control not exposed to the nucleic acid-targeting Cas protein or nucleic acid-targeting complex, or exposed to a nucleic acid- targeting Cas protein lacking the one or more NLSs.
- an assay for the effect of nucleic acid-targeting complex formation e.g., assay for DNA or RNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by DNA or RNA-targeting complex formation and/or DNA or RNA-targeting Cas protein activity
- a control not exposed to the nucleic acid-targeting Cas protein or nucleic acid-targeting complex or exposed to a nu
- the positioning of the one or more effector domain on the inactivated Cas-phi polypeptide is one which allows for correct spatial orientation for the effector domain to affect the target with the attributed functional effect.
- the effector domain is a transcription activator (e.g., VP64 or p65)
- the transcription activator is placed in a spatial orientation which allows it to affect the transcription of the target.
- a transcription repressor will be advantageously positioned to affect the transcription of the target
- a nuclease e.g., Fok1
- This may include positions other than the N-/C-terminus of the CRISPR enzyme.
- the effector domain is a transcription activator. In some embodiments, the effector domain is a transcription repressor.
- Exemplary transcription activators domains and repressor domains for use in the methods of the present invention are well known to those of skill in the art and include, but are not limited to those listed in Table 4. [0301] Table 4. Exemplary activator and repressor domains [0302] Exemplary fusion Cas-phi polypeptides include but are not limited to those listed in Table 5.1.
- the CRISPR-Cas system may comprise one or more guide molecules.
- the guide molecule or guide RNA of a Class 2 type V CRISPR-Cas protein comprises a tracr-mate sequence (encompassing a “direct repeat” in the context of an endogenous CRISPR system) and a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system).
- the guide molecule may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence.
- a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
- target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target RNA sequence and a guide sequence promotes the formation of a CRISPR complex.
- guide molecule guide RNA
- sgRNA sgRNA
- gRNA gRNA
- guide molecule or guide RNA specifically encompasses RNA-based molecules having one or more chemically modifications (e.g., by chemical linking two ribonucleotides or by replacement of one or more ribonucleotides with one or more deoxyribonucleotides), as described herein.
- Guide Sequence As used herein, the term “guide sequence” or “targeting sequence” in the context of a CRISPR-Cas system, comprises any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence.
- 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., the Burrows Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
- 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., the Burrows Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina,
- a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.
- the ability of a guide sequence (within a nucleic acid-targeting guide RNA) to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence may be assessed by any suitable assay (as described in EP3009511 or US2016208243).
- the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid-targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within or in the vicinity of the target nucleic acid sequence, such as by Surveyor assay as described herein.
- preferential targeting e.g., cleavage
- cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at or in the vicinity of the target sequence between the test and control guide sequence reactions.
- Other assays are possible, and will occur to those skilled in the art.
- a guide sequence, and hence a nucleic acid-targeting guide RNA may be selected to target any target nucleic acid sequence.
- the guide sequence or spacer length of the guide molecules is from 15 to 50 nt.
- the spacer length of the guide RNA is at least 15 nucleotides. In certain embodiments, the spacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.
- the spacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19, or 20 nt, from 20
- the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 3940, 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, or 100 nt.
- a guide sequence may be selected to target any target sequence.
- the target sequence is a sequence within a gene transcript or mRNA.
- the target sequence is a sequence within a genome of a cell.
- the guide sequence is an RNA sequence of between 10 to 50 nt in length, but more particularly of about 20-30 nt advantageously about 20 nt, 23 nt, 24 nt or 25 nt in length. In some embodiments, the guide sequence is about 20 nt in length.
- Exemplary guide RNA spacer sequences include but are not limited to those shown in Table 6.1.
- Exemplary Cas-phi sgRNA scaffold sequences are shown in Table 6.2.
- the guide molecule comprises non-naturally occurring nucleic acids and/or non-naturally occurring nucleotides and/or nucleotide analogs, and/or chemically modifications. Preferably, these non-naturally occurring nucleic acids and non- naturally occurring nucleotides are located outside the guide sequence.
- Non-naturally occurring nucleic acids can include, for example, mixtures of naturally and non-naturally occurring nucleotides. Non-naturally occurring nucleotides and/or nucleotide analogs may be modified at the ribose, phosphate, and/or base moiety.
- a guide nucleic acid comprises ribonucleotides and non-ribonucleotides. In one such embodiment, a guide comprises one or more ribonucleotides and one or more deoxyribonucleotides.
- the guide comprises one or more non-naturally occurring nucleotide or nucleotide analog such as a nucleotide with phosphorothioate linkage, a locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2′ and 4′ carbons of the ribose ring, or bridged nucleic acids (BNA).
- LNA locked nucleic acid
- BNA bridged nucleic acids
- modified nucleotides include 2′-O-methyl analogs, 2′-deoxy analogs, or 2′-fluoro analogs.
- modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine.
- Examples of guide RNA chemical modifications include, without limitation, incorporation of 2′-O-methyl (M), 2′-O- methyl 3′phosphorothioate (MS), S-constrained ethyl(cEt), or 2′-O-methyl 3′thioPACE (MSP) at one or more terminal nucleotides.
- M 2′-O-methyl
- MS 2′-O-methyl 3′phosphorothioate
- cEt S-constrained ethyl
- MSP 2′-O-methyl 3′thioPACE
- a guide RNA comprises ribonucleotides in a region that binds to a target RNA and one or more deoxyribonucletides and/or nucleotide analogs in a region that binds to Cas-phi.
- the guide comprises a modified Cpf1 crRNA, having a 5′-handle and a guide segment further comprising a seed region and a 3′-terminus.
- the modified guide can be used with a Cpf1 of any one of Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1); Francisella tularensis subsp. Novicida U112 Cpf1 (FnCpf1); L.
- bacterium MA2020 Cpf1 Lb2Cpf1; Porphyromonas crevioricanis Cpf1 (PcCpf1); Porphyromonas macacae Cpf1 (PmCpf1); Candidatus Methanoplasma termitum Cpf1 (CMtCpf1); Eubacterium eligens Cpf1 (EeCpf1); Moraxella bovoculi 237 Cpf1 (MbCpf1); Prevotella disiens Cpf1 (PdCpf1); or L. bacterium ND2006 Cpf1 (LbCpf1).
- the target sequence may be associated with a PAM (protospacer adjacent motif) or PFS (protospacer flanking sequence or site); that is, a short sequence recognized by the CRISPR complex.
- PAM protospacer adjacent motif
- PFS protospacer flanking sequence or site
- the target sequence should be selected such that its complementary sequence in the RNA duplex (also referred to herein as the non-target sequence) is upstream or downstream of the PAM.
- engineering of the PAM Interacting (PI) domain may allow programing of PAM specificity, improve target site recognition fidelity, and increase the versatility of the CRISPR-Cas protein, for example as described for Cas9 in Kleinstiver et al. Nature.2015 Jul.
- Vectors Provided herein are vectors for delivery of the engineered CRISPR-Cas effector proteins disclosed herein (e.g., nuclease deficient Cas-phi polypeptides) to host cells.
- a vector comprising: a nucleic acid sequence encoding a Cas-phi polypeptide disclosed herein and a polynucleotide sequence encoding a gRNA.
- the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. It is a replicon, such as a plasmid, viral expression cassette, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements.
- Vectors include, but are not limited to, nucleic acid molecules that are single- stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, or no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
- plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
- viral vector Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)).
- viruses e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)
- Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
- Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
- vectors e.g., non-episomal mammalian vectors
- Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
- certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
- Vectors that are used for expression in a eukaryotic cell can be referred to herein as “eukaryotic expression vectors.”
- Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
- the vectors provided herein are plasmids or viral expression cassettes that comprise additional nucleic acid sequences.
- the vectors provided herein may be used to generate recombinant virus particles to serve as viral vectors for gene delivery.
- the vectors provided herein are formulated for use with via non-viral delivery systems.
- plasmids comprising any of the vector nucleic acid sequences disclosed herein.
- Viral and non-viral based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids sequences encoding engineered CRISPR-Cas effector proteins disclosed herein and/or gRNAs to cells in culture, or in a host organism.
- Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
- Non-viral vector delivery systems include DNA plasmids, RNA, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
- Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, exosomes, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
- Lipofection is described in e.g., US 5,049,386, US 4,946,787; and US 4,897,355.
- Some lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
- Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those described in, for example, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
- a vector provided herein is non-integrating. In some embodiments, a vector provided herein is non-replicating.
- Recombinant expression vectors can comprise a nucleic acid sequence encoding a Cas-phi polypeptide disclosed herein (and a polynucleotide sequence encoding a gRNA) in a form suitable for expression of the nucleic acid sequence in a host cell, which means that the recombinant expression vectors include one or more regulatory elements. Such regulatory elements may be selected on the basis of the host cells to be used for expression and operatively linked to the nucleic acid sequence to be expressed.
- operatively linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
- Advantageous vectors include lentiviruses and adeno-associated viruses, and specific types of such vectors can also be selected for targeting particular types of cells.
- regulatory element includes promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
- IRES internal ribosomal entry sites
- regulatory elements e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences.
- Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
- a tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporal- dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.
- a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.
- pol III promoters include, but are not limited to, U6 and H1 promoters.
- pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFa promoter.
- RSV Rous sarcoma virus
- CMV cytomegalovirus
- PGK phosphoglycerol kinase
- enhancer elements such as Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE); CMV enhancers; the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol.8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit ⁇ -globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc.
- WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
- CMV enhancers the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol.8(1), p.466-472, 1988)
- SV40 enhancer the intron sequence between exon
- a vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., CRISPR transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, gRNAs, etc.). Exemplary promoters are disclosed in WO 2011/028929. [0331] In some embodiments, bicistronic vectors are used for expression of a gRNA and a Cas-phi polypeptide disclosed herein. In some embodiments, a vector comprises a CBh promoter. In some embodiments, a vector comprises an RNA polymerase III promoter, such as a U6 promoter.
- a vector comprises a nucleic acid sequence encoding a Cas-phi polypeptide disclosed herein and a polynucleotide sequence encoding a gRNA, wherein the gRNA is operatively linked to a promoter recognized by RNA polymerase III. In some embodiments, the gRNA is operatively linked to a human U6 promoter.
- a vector provided herein further comprises an artificial intron. In some embodiments, a vector provided herein further comprises a chimeric intron.
- a vector provided herein further comprises or encodes a woodchuck hepatitis virus post-transcriptional element (WPRE).
- WPRE woodchuck hepatitis virus post-transcriptional element
- a vector comprises or encodes a hepatitis B virus posttranscriptional regulatory element (HBVPRE) and/or a RNA transport element (RTE).
- HBVPRE hepatitis B virus posttranscriptional regulatory element
- RTE RNA transport element
- the WPRE or HBVPRE sequence is any of the WPRE or HBVPRE sequences disclosed in US 6,136,597 or US 6,287,814.
- a vector provided herein further comprises or encodes a WPRE3 or a wsl3 regulatory element.
- a WPRE3 comprises or consists of GATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGC TCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTA TGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATC GCCGCCTGCCTTGCCCGCTGCTGGACAGGGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGT GTT (SEQ ID NO: 144).
- a vector provided herein further comprises or encodes a polyadenylation (polyA) signal sequence.
- a “polyadenylation signal sequence” refers to a DNA sequence that when transcribed regulates the addition of a polyA tail to the mRNA transcript.
- a polyA signal sequence is a SV40, human, bovine or rabbit polyA signal sequence.
- a polyA signal sequence is a SV40 polyA signal sequence.
- a polyA signal sequence is a ⁇ -globin polyA signal sequence.
- a polyA signal sequence is a human growth hormone polyA signal sequence or a bovine growth hormone polyA signal sequence.
- a SV40 polyA signal sequence comprises or consists of AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAA TAAAGCATTTTTTTCACTGC (SEQ ID NO: 145).
- a vector provided herein further comprises or encodes a Kozak sequence (for example, a DNA sequence transcribed to an RNA Kozak sequence).
- a vector comprises a Kozak sequence upstream of the transgene.
- the Kozak sequence is encoded by GCCACC.
- the Kozak sequence (e.g., RNA Kozak sequence) comprises or consists of ACCAUGG, GCCGCCACCAUGG (SEQ ID NO: 141), CCACCAUG or CCACCAUGG.
- a vector provided herein further comprises a TATA transcriptional regulatory activation site (see, e.g., Francois et al., (2005) J. Virol. 79(17):11082–11094).
- Vectors can be designed for expression of nucleic acid sequences disclosed herein in prokaryotic or eukaryotic cells.
- nucleic acid sequences can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
- a recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
- Vectors may be introduced and propagated in a prokaryote or prokaryotic cell.
- a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g., amplifying a plasmid as part of a viral vector packaging system).
- a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism.
- Fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus of the recombinant protein.
- Such fusion vectors may serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
- a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
- Such enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
- Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988.
- a vector is a yeast expression vector.
- a vector drives protein expression in insect cells using baculovirus expression vectors.
- Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al., 1983. Mol. Cell. Biol.3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
- a vector is capable of driving expression of one or more nucleic acid sequences in mammalian cells using a mammalian expression vector.
- mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J.6: 187-195).
- the expression vector's control functions are typically provided by one or more regulatory elements.
- promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art.
- suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
- the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
- tissue-specific regulatory elements are known in the art.
- suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev.1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol.43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989.
- a host cell is transiently or non-transiently transfected with one or more vectors comprising one or more nucleic acid sequences disclosed herein.
- a cell is transfected as it naturally occurs in a subject.
- a cell that is transfected is taken from a subject.
- the cell is derived from cells taken from a subject, such as a cell line.
- a cell line A wide variety of cell lines for tissue culture are known in the art and exemplified herein elsewhere. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, VA)).
- ATCC American Type Culture Collection
- VA Manassas, VA
- cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc, PC-3, TF1, CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS- M6A, BS-C-1 monkey kidney epithelial, BALB
- a cell transfected with one or more vectors comprising one or more nucleic acid sequences disclosed herein is used to establish a new cell line comprising one or more vector-derived sequences.
- a vector provided herein is a viral vector.
- a viral vector is a retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV) or herpes simplex virus (HSV) vector.
- Retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol.176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol.65:2220-2224 (1991)).
- MiLV murine leukemia virus
- GaLV gibbon ape leukemia virus
- SIV Simian immunodeficiency virus
- HAV human immunodeficiency virus
- a viral vector is an adeno-associated virus (AAV) vector.
- AAV vector comprises a first AAV inverted terminal repeat (ITR) located upstream of the promoter polynucleotide sequence and a second AAV ITR located downstream of the transgene polynucleotide sequence. ITRs are sequences that mediate AAV proviral integration and packaging of AAV DNA into virions.
- an AAV vector comprises a first AAV ITR and a second AAV ITR flanking the polynucleotide sequences to be packaged into a recombinant AAV (rAAV) particle.
- the first AAV ITR is an AAV2 ITR and the second AAV ITR is an AAV2 ITR.
- AAV expression cassettes and related plasmids provided herein can be used in production of rAAV particles.
- an AAV vector provided herein is self-complementary.
- an AAV vector provided herein is single-stranded.
- a viral particle also referred to as a virion
- a viral particle is a retrovirus, lentivirus, adenovirus, AAV or HSV particle.
- the viral particle is a recombinant AAV (rAAV) particle.
- the rAAV particle is an AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh.74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, TM-AAV6, AAV-PHP.A, AAV- PHP.B, AAV-PHP.S, AAV-PHPeB, AAV-DJ, AAV-CAP.B10, AAV
- the rAAV particle comprises an AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh.74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, TM-AAV6, AAV-PHP.A, AAV- PHP.B, AAV-PHP.S, AAV-PHPeB, AAV-DJ, AAV-CAP.B10, AAV2-r3.45, AAV2-LSS, AAV2PFG, AAV2-PPS, AAV
- the rAAV particle is an AAV capsid with tissue tropism such as muscle specific, cardiac cells specific or skeletal muscle specific or CNS tissue or cell type specific serotype or AAV capsid particle.
- tissue tropism such as muscle specific, cardiac cells specific or skeletal muscle specific or CNS tissue or cell type specific serotype or AAV capsid particle.
- Provided herein is a population of viral particles comprising a plurality of viral particles disclosed herein.
- a population of rAAV particles comprising a plurality of rAAV particles disclosed herein.
- a cell comprising any of the vectors or viral particles disclosed herein. In some embodiments, the cell is a mammalian cell.
- a mammalian cell is a HEK293 cell.
- the cell is an insect cell.
- the insect cell is a Spodoptera frugiperda cell (for example, the Sf9 or ExpiSf9TM cell lines).
- the Sf9 insect cell line (Thermo Fisher Scientific, Waltham, MA) is a clonal isolate derived from the parental S. frugiperda cell line IPLB-Sf-21-AE.
- ExpiSf9TM cells are a non-engineered derivative of Sf9 insect cells that have been adapted for high-density suspension growth.
- the cell is a C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc, PC-3, TF1, CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T
- compositions comprising any of the vectors, viral particles, nucleic acid molecules, populations of viral particles disclosed herein, and a pharmaceutically acceptable carrier, vehicle or diluent.
- “Pharmaceutically acceptable” refers to a material that is not toxic or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects.
- a pharmaceutically acceptable material has one or more benefits that outweigh any undesirable biological effect that the material may have. Undesirable biological effects may include, for example, excessive toxicity, irritation, allergic response, and other problems and complications.
- the carrier will typically be a liquid.
- the carrier may be either solid or liquid.
- a pharmaceutical composition may comprise other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, adjuvants and/or diluents.
- a pharmaceutical composition comprises at least one pharmaceutically acceptable carrier, excipient, and/or vehicle, for example, solvents, buffers, solutions, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, and absorption delaying agents.
- the pharmaceutically acceptable carrier, excipient, and/or vehicle comprises saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, or a combination thereof.
- the pharmaceutically acceptable carrier, excipient, and/or vehicle comprises phosphate buffered saline, sterile saline, lactose, sucrose, calcium phosphate, dextran, agar, pectin, peanut oil, sesame oil, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), or a suitable mixture thereof.
- the compositions disclosed herein further comprise emulsifying or wetting agents, or pH buffering agents.
- a pharmaceutical composition further comprises one or more other pharmaceutical ingredients, such as one or more preservatives or chemical stabilizers.
- preservatives and chemical stabilizers include, but are not limited to, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, and albumin.
- compositions disclosed herein further comprise antibacterial agents and/or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal; isotonic agents, such as sugars and sodium chloride; and/or agents delaying absorption, such as aluminum monostearate and gelatin.
- antibacterial agents and/or antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal
- isotonic agents such as sugars and sodium chloride
- agents delaying absorption such as aluminum monostearate and gelatin.
- a pharmaceutical composition is in a form of an injectable solution or dispersion, such as an aqueous solution or dispersion.
- a pharmaceutical composition is a sterile powder for the extemporaneous preparation of sterile injectable solutions or dispersions.
- Dispersions may be prepared in water, glycerol, liquid polyethylene glycols, oils, or any combination thereof. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the pharmaceutical compositions provided herein.
- a pharmaceutical composition comprises or consists of a sterile saline (e.g., pharmaceutical grade saline) solution and a vector.
- a pharmaceutical composition comprises or consists of a vector and sterile water (e.g., pharmaceutical grade water).
- a pharmaceutical composition comprises or consists of a vector and phosphate-buffered saline (PBS) (e.g., pharmaceutical grade PBS).
- PBS phosphate-buffered saline
- a pharmaceutical composition provided herein comprises a vector and one or more excipients.
- an excipient is water, a salt solution, an alcohol, a polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose or polyvinylpyrrolidone.
- pharmaceutical compositions provided herein comprise a lipid moiety.
- a vector may be introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
- vector complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
- a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.
- a lipid moiety is selected to increase distribution of a pharmaceutical agent to neurons.
- pharmaceutical compositions comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Some delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds.
- compositions comprise a nanoparticle-based delivery system.
- PLGA polylactic-co-glycolic acid
- PBAE poly ( ⁇ -amino esters)
- PEI polyethylenimine
- pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver a vector to specific tissues or cell types.
- pharmaceutical compositions include liposomes coated with a tissue-specific or a cell-specific antibody.
- pharmaceutical compositions comprise a cosolvent system.
- cosolvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
- cosolvent systems are used for hydrophobic compounds.
- a non-limiting example of such a cosolvent system is the VPD cosolvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80 and 65% w/v polyethylene glycol 300.
- the proportions of such cosolvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics.
- a nanoparticle comprising a vector disclosed herein.
- a nanoparticle is a lipid nanoparticle.
- a nanoparticle is a solid lipid nanoparticle (SLN).
- a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
- aqueous solution such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
- other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives).
- injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like.
- pharmaceutical compositions for injection are prepared in unit dosage form, e.g., in ampoules or in multi- dose containers.
- compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- exemplary solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
- vectors may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations.
- compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
- a pharmaceutical composition is suitable or formulated for systemic administration.
- a pharmaceutical composition is suitable or formulated for intravenous administration.
- a pharmaceutical composition is suitable or formulated for intracerebroventricular injection, intrathecal injection, intracarotid artery injection, or intraparenchymal injection.
- a method of producing a rAAV particle comprising: (i) culturing a cell comprising a vector or an AAV expression cassette disclosed herein under conditions allowing for packaging the rAAV particle; and (ii) harvesting the cultured host cell or culture medium for collection of the rAAV particle.
- a method of producing a rAAV particle comprises providing to a cell: (a) a vector (i.e., a nucleic acid template comprising an AAV expression cassette) comprising two AAV ITRs located 5' and 3' to the polynucleotide sequences desired to be packaged into the rAAV particle, and (b) AAV sequences sufficient for replication of the nucleic acid template and encapsidation into AAV protein capsids (e.g., AAV rep sequences and AAV cap sequences encoding the AAV capsid subunits, also referred to as “helper functions”).
- helper functions e.g., helper functions
- the AAV rep and cap sequences will not be flanked by AAV ITRs, to prevent rescue and/or packaging of these sequences.
- the vector (nucleic acid template), rep sequences, cap sequences, and any other helper functions required for producing the rAAV particles disclosed herein may be delivered to the packaging host cell using any appropriate genetic element. Further details on methods of preparing rAAV particles are provided in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY; Fisher et al, J. Virol., 70:520-532 (1993) and US 5,478,745.
- the nucleic acid template and AAV rep and cap sequences are provided under conditions such that virus vector comprising the nucleic acid template packaged within the AAV protein capsid is produced in the cell.
- the method can further comprise the step of collecting the virus vector from the cell.
- the virus vector can be collected from the medium and/or by lysing the cells.
- the cell can be a cell that is permissive for AAV viral replication. Any suitable cell known in the art may be employed.
- the cell is a mammalian cell (e.g., a HEK293 cell).
- the cell can be a trans-complementing packaging cell line that provides functions deleted from a replication-defective helper virus, e.g., HEK293 cells or other E1a trans-complementing cells.
- the helper sequences may be embedded in a chromosome or maintained as a stable extrachromosomal element.
- rAAV particles are produced using the triple transfection method, as described in US 6,001,650.
- the rAAVs are produced by transfecting a host cell with an AAV vector (i.e., AAV expression cassette) to be packaged into rAAV particles, an AAV helper function vector, and an accessory function vector.
- An AAV helper function vector encodes the “AAV helper function” sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation.
- AAV helper function vectors include pHLP19 and pRep6cap6 vector, described in US 6,001,650 and US 6,156,303, respectively.
- the accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (i.e., “accessory functions”).
- the accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly.
- Viral- based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (e.g., other than herpes simplex virus type-1) and vaccinia virus.
- helper viruses such as adenovirus, herpesvirus (e.g., other than herpes simplex virus type-1) and vaccinia virus.
- rAAVs are produced using recombinant baculovirus vectors. Production of rAAVs using baculovirus vectors is described in, for example, Urabe et al.
- a baculovirus vector genome is derived from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), Bombyx mori nuclear polyhedrosis virus (BmNPV), Helicoverpa armigera (HearNPV) or Spodoptera exigua MNPV.
- AcMNPV Autographa californica multicapsid nucleopolyhedrovirus
- BmNPV Bombyx mori nuclear polyhedrosis virus
- HearNPV Helicoverpa armigera
- Spodoptera exigua MNPV a baculovirus vector genome is derived from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), Bombyx mori nuclear polyhedrosis virus (BmNPV), Helicoverpa armigera (HearNPV) or Spodoptera exigua MNPV.
- Baculovirus vectors are used to produce
- the Sf9 or ExpiSf9TM Spodoptera frugiperda cell lines are used to produce rAAVs.
- methods of the disclosure comprise co-infecting insect cells with populations of recombinant baculoviruses (rBVs) to produce rAAV disclosed herein. At least two populations of rBVs may be used in the methods of the disclosure. Methods for generating recombinant baculovirus are known in the art (see, e.g., the Bac-to-Bac® Baculovirus Expression System (Thermo Fisher Scientific, Waltham, MA)).
- a rAAV particle produced by the methods provided herein comprises an AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh.74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV- LK03, AAV7m8, AAV Anc80, TM-AAV6, AAV-PHP.A, AAV-PHP.B, AAV-PHP.S, AAV- PHPeB, AAV-DJ, AAV-CAP.B10, AAV2-r3.45, AAV2-LSS, A
- Methods of Using Vectors comprising: contacting a population of cells comprising a target nucleic sequence encoding the target gene with a vector, a viral particle, a population of viral particles, or a pharmaceutical composition disclosed herein, thereby modifying the expression of the target gene.
- the expression of the target gene is increased in the plurality of the modified population of cells in comparison to a population of cells contacted with a vector, a viral particle, a population of viral particles, or a pharmaceutical composition disclosed herein; and a gRNA that does not specifically hybridize to the target nucleic acid sequence, but binds to the Cas-phi polypeptide to form a complex.
- the expression of the target gene is increased by at least about 1.10-fold, at least about 1.15-fold, at least about 1.20-fold, at least about 1.25-fold, at least about 1.30-fold, at least about 1.35- fold, at least about 1.40-fold, at least about 1.45-fold, at least about 1.50-fold, at least about 1.55-fold, at least about 1.60-fold, at least about 1.65-fold, at least about 1.70-fold, at least about 1.75-fold, at least about 1.80-fold, at least about 1.85-fold, at least about 1.90-fold, at least about 1.95-fold or at least about 2.0-fold.
- Exemplary target genes with increased expression in the plurality of modified the modified population of cells include, but are not limited to those shown in Table 7.
- Table 7 Target genes for upregulation.
- the expression of the target gene is decreased in the plurality of the modified population of cells in comparison to a population of cells contacted with a vector, a viral particle, a population of viral particles, or a pharmaceutical composition disclosed herein; and a gRNA that does not specifically hybridize to the target nucleic acid sequence, but binds to the Cas-phi polypeptide to form a complex.
- the expression of the target gene is decreased by at least about 1.10-fold, at least about 1.15-fold, at least about 1.20-fold, at least about 1.25-fold, at least about 1.30-fold, at least about 1.35- fold, at least about 1.40-fold, at least about 1.45-fold, at least about 1.50-fold, at least about 1.55-fold, at least about 1.60-fold, at least about 1.65-fold, at least about 1.70-fold, at least about 1.75-fold, at least about 1.80-fold, at least about 1.85-fold, at least about 1.90-fold, at least about 1.95-fold or at least about 2.0-fold.
- Exemplary target genes with decreased expression in the plurality of modified the modified population of cells include, but are not limited to those shown in Table 8.
- Table 8 Target genes for repression.
- Provided herein is a method of reducing or eliminating the expression of a gene product in a subject comprising introducing to a cell of a subject a therapeutically effective amount of a vector, a viral particle, a population of viral particles, or a pharmaceutical composition disclosed herein.
- a method for treating or alleviating a symptom of a gene product related disorder in a subject comprising the step of introducing to a cell of the subject a therapeutically effective amount of a vector, a viral particle, a population of viral particles, or a pharmaceutical composition disclosed herein.
- a vector provided herein increases or decreases mRNA expression. Levels of mRNA expression may be measured by a Northern blot, a nuclease protection assay (NPA), in situ hybridization or reverse transcription-polymerase chain reaction (RT-PCR).
- NPA nuclease protection assay
- RT-PCR reverse transcription-polymerase chain reaction
- a vector provided herein increases or decreases protein expression.
- the subject is human. In some embodiments, the subject is less than 2 years old. In some embodiments, the subject is between about 2 years old and about 18 years old. In some embodiments, the subject is older than 18 years.
- the vector, viral particle, population of viral particles or pharmaceutical composition is administered to the subject via intracerebroventricular injection, intrathecal injection, intracarotid artery injection, or intraparenchymal injection. [0388] In some embodiments, the vector, viral particle, population of viral particles or pharmaceutical composition is administered to the subject in a single dose.
- EXAMPLE 1 Construction of Nuclease Deficient Cas-Phi Polypeptides [0391] Cas-phi targets genomic loci by recognizing a 5’ proto-spacer adjacent motif (PAM) (FIG.1A) sequence and has been shown to be effective at DNA cleaving in human cell lines (Pausch et al., Science 369, 333–337 (2020)).
- PAM proto-spacer adjacent motif
- Nuclease deficient Cas-phi (dCas-phi) polypeptides were constructed for the use in the novel gene expression modulation system (GEMS), which was designed to shuttle transcriptional activators or repressors to specific genomic loci and modulate gene expression (FIG.1A). Aspartic acid to alanine mutations of the first RuvC domain (RuvC-I) have been shown to decrease nuclease activity. Additional Cas-phi point mutations and RuvC domain deletions and truncations were added to further eliminate nuclease activity.
- GEMS novel gene expression modulation system
- FIG.2A shows a schematic diagram of the nuclease deficient Cas-phi polypeptide mutants that were constructed.
- FIGS. 2B-2C show amino acid sequence alignments of the nuclease deficient Cas-phi polypeptide mutants with a wildtype Cas-phi-2 polypeptide and wildtype Cas-phi-3 polypeptide, respectively.
- GEMS 1.1 mutant (2520 bp in length) comprises a D371A mutation in the RuvC-I domain in comparison to a wildtype Cas-Phi-1 polypeptide.
- GEMS 1.2 mutant (2394 bp in length) comprises a D371A mutation in the RuvC-I domain and a RuvC-III domain deletion in comparison to a wildtype Cas-Phi-1 polypeptide.
- GEMS 1.3 (2316 bp in length) comprises a D371A mutation in the RuvC-I domain, a RuvC-III domain deletion and a zinc ribbon deletion in comparison to a wildtype Cas-Phi-1 polypeptide.
- Cas-phi-2 mutations [0397] “GEMS 2.1” mutant (2670 bp in length) comprises a D394A in the RuvC-I domain, a E606A mutation in the RuvC-II domain, and a D695A mutation in the RuvC-III domain in comparison to a wildtype Cas-Phi-2 polypeptide.
- GEMS 2.2” mutant (2478 bp in length) comprises a D394A in the RuvC-I domain, a E606A mutation in the RuvC-II domain, and a RuvC-III domain deletion in comparison to a wildtype Cas-Phi-2 polypeptide.
- GEMS 2.3” mutant (2400 bp in length) comprises a D394A in the RuvC-I domain, a E606A mutation in the RuvC-II domain, a RuvC-III domain deletion and a zinc ribbon deletion in comparison to a wildtype Cas-Phi-2 polypeptide.
- Cas-phi-3 mutations [0401] “GEMS 3.1” mutant (2697 bp in length) comprises a D413A in the RuvC-I domain, a E618A mutation in the RuvC-II domain, and a D708A mutation in the RuvC-III domain in comparison to a wildtype Cas-Phi-3 polypeptide.
- GEMS 3.2 (2582 bp in length) comprises a D413A in the RuvC-I domain, a E618A mutation in the RuvC-II domain, and a D708A mutation in the RuvC-III domain and a RuvC-III domain deletion in comparison to a wildtype Cas-Phi-3 polypeptide.
- GEMS 3.3 (2225 bp in length) comprises a D413A in the RuvC-I domain, a RuvC-II domain deletion and a RuvC-III domain deletion in comparison to a wildtype Cas- Phi-3 polypeptide.
- the AAV vector was digested with XbaI and AfeI to clone in the Cas-phi gRNA scaffold downstream of a U6 promoter using NEBuilder HiFi DNA Assembly (New England Biolabs). Constructs were further digested with EcoRI and NheI to clone in the GEMS sequences (dead-Cas-phi-fusion versions) downstream of a CMV promoter using the In-Fusion HD Cloning kit (Takara). The unique targeting site of 18-23 bases in the sgRNA scaffold was flanked by SapI enzyme site enabling easy swapping of guide RNAs to target unique promoter sequences (FIG.1B).
- EXAMPLE 2 Use of Nuclease Deficient Cas-Phi Polypeptides for Targeted Upregulation of Gene Expression
- Each of the “GEMS” constructs were tested in AAVpro HEK293T cells, Kelly neuroblastoma cells or HeLa cells by targeting various genes.
- Two separate gRNAs each, for Cas-phi-2 and Cas-phi-3 using their respective PAM sequences (Cas-phi-2 PAM: TBN, where ‘B’ is T, C or G and ‘N’ is A, T, C, or G;
- Cas-phi-3 PAM VTTY, where ‘V’ is A, G, or C, and ‘Y’ is C or T) (Table 6.1).
- gRNAs for all three constructs of Cas-phi-2 mutants (GEMS 2.1, GEMS 2.2, GEMS 2.3) and all three constructs of Cas-phi-3 mutants (GEMS 3.1, GEMS 3.2, GEMS 3.3) were constructed.
- gRNAs for all additional “GEMS” constructs were constructed (Table 6.1).
- the sgRNA scaffold was engineered to remove or include additional bases to improve targeting (Table 6.2)
- Transfection [0412] Human embryonic kidney (HEK) 293T cells (AAVpro HEK293T) or Kelly neuroblastoma cells were seeded at a density of 200k/cells per well in a 12-well plate in 1 ml of DMEM media.
- Cells were transfected with GEMS constructs with either a gene-specific guide or a non-targeting control 24 hours after seeding. Transfection was achieved with 1 or 2 ⁇ g of plasmid using X-tremeGENE HP DNA transfection reagent (Roche; for HEK cells) or Lipofectamine LTX (Thermo; for Kelly cells) at a 3:1 ratio (3 ⁇ l reagent for every 1 ⁇ g plasmid). Plasmids were thoroughly mixed with 100 ⁇ l OptiMEM and the transfection reagent and allowed to sit at RT for 20 minutes before adding 100 ⁇ l of the mixture dropwise to each well. Cells were harvested 48 hours after transfection.
- HeLa cells were seeded at a density of 160k/cells per well in a 12-well plate in 1 ml of EMEM media. Cells were transfected with GEMS constructs with either a gene-specific guide or a non-targeting control 24 hours after seeding. Transfection was achieved with 1ug of plasmid using X-tremeGENE HP DNA transfection reagent at a 2:1 ratio (2 ⁇ l reagent for every 1 ⁇ g plasmid). Plasmids were thoroughly mixed with 100 ⁇ l OptiMEM and the transfection reagent and allowed to sit at RT for 20 minutes before adding 100 ⁇ l of the mixture dropwise to each well. RNA was extracted 48 hours after transfection for qPCR analysis.
- RNA isolation and quantitative reverse-transcription PCR [0414] RNA was isolated using the Quick-RNA miniprep kit (Zymo) following the manufacturer’s protocol and quantified using Qubit RNA BR reagents (Thermo Fisher). Following RNA isolation, cDNA was prepared using qScript cDNA Synthesis kit (Quantabio) using the manufacturer’s protocol. qPCR was performed with PerfeCTa SYBR Green SuperMix Reagent (Quantabio) and gene-specific primers. Fold change in mRNA expression of the gene of interest was normalized to ACTB or GAPDH expression using the ⁇ CT method.
- Luciferase reporter assay [0417] Human embryonic kidney (HEK) 293T cells (AAVpro HEK293T) were seeded at a density of 20k/cells per well in a 96-well plate in 75 ⁇ l of DMEM media.
- Luciferase expression was measured 48-hours post transfection by following the manufacturer’s protocol for Promega’s Dual-Glo Luciferase Assay System (E2940). Luminance was read by a plate reader (Agilent Cytation 5) and fold change was calculated by dividing the average signal from the targeting guide by the signal from a non-targeting control. [0418] Each of the fusion Cas-phi “GEMS” constructs were tested in AAVpro HEK293T cells by targeting STXBP1 (NCBI Gene id: 6812) gene. Fold-change of target gene expression over non-targeting guides was measured (FIGS.3A-3B).
- GEMS 2.3 and GEMS 3.3 constructs consistently resulted in upregulation of STXBP1 over non targeting guide controls (Table 9).
- Table 9 Fold change per STXBP1-targeting replicate
- the fusion Cas-phi GEMS variants can upregulate SYNGAP1 (Gene id: 8831) in a neuroblastoma cell line (FIGS.4A-4B; Table 10), demonstrating the ability of the variants to upregulate gene expression across different cell types.
- a subset of fusion Cas-phi GEMS variants from Table 5.2 were constructed and tested using a luciferase reporter assay.
- the promoter sequence of human SYNGAP1 (Gene id: 8831) was cloned upstream of the luciferase coding sequence in a reporter vector and was co-transfected with a GEMS mutants expression vector in HEK293T cells. Binding of GEMS mutants at the promoter results in an increase in luciferase expression, which is measured as a relative increase in luminescence compared to a non-targeting control.
- a composition comprising: a) a Cas-phi polypeptide or a polynucleotide sequence encoding the Cas-phi polypeptide, wherein the Cas-phi polypeptide comprises at least one RuvC domain and wherein the at least one RuvC domain is nuclease inactive and the RuvC domain comprises at least one mutation relative to a wildtype RuvC domain; and b) a polynucleotide sequence encoding a guide RNA (gRNA) that can specifically hybridize to a target nucleic sequence and to the Cas phi polypeptide to form a complex.
- gRNA guide RNA
- composition of embodiment 1, wherein the Cas-phi polypeptide is a Cas-phi-1, a Cas-phi-2 or a Cas-phi-3 polypeptide.
- Embodiment 3 The composition of embodiment 2, wherein the Cas-phi polypeptide is a Cas-phi-2, and wherein the at least one mutation is i) D394A; ii) D394A and E606A; iii) D394A and D695A; or iv) D394A, E606A and D695A, numbered in accordance to SEQ ID NO: 4.
- Embodiment 5 The composition of embodiment 2, wherein the Cas-phi polypeptide is a Cas-phi-1, and wherein the at least one mutation is D371A, numbered in accordance to SEQ ID NO: 2. [0437] Embodiment 6.
- Embodiment 7 The composition of any one to embodiments 1-2, wherein the Cas-phi polypeptide further comprises a deletion of a zinc ribbon domain in comparison to a wildtype Cas-phi polypeptide.
- composition of any one of embodiments 1-7, wherein the Cas-phi polypeptide comprises the amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42 or 44.
- Embodiment 9 The composition of any one of embodiments 1-7, wherein the Cas-phi polypeptide comprises an amino acid sequence at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42 or 44.
- Embodiment 11 The composition of embodiment 10, wherein the at least one effector domain is fused to i) the N-terminus of the Cas-phi polypeptide, ii) the C-terminus of the Cas-phi polypeptide, or iii) both the N-terminus and the C-terminus of the Cas-phi polypeptide.
- composition of embodiment 11, wherein the at least one effector domain fused to the N-terminus of the Cas-phi polypeptide and the C-terminus of the Cas-phi polypeptide are different.
- Embodiment 13 The composition of embodiment 11, wherein the at least one effector domain fused to the N-terminus of the Cas-phi polypeptide and the C-terminus of the Cas-phi polypeptide are the same.
- Embodiment 14 Embodiment 14.
- composition of any one of embodiments 10-13, wherein the at least one effector domain comprises an effector domain derived from a p65 NF- ⁇ transactivating subunit (p65), VP160, SET7, RTA, histone acetyltransferase p300, VPR, MyoDl, TET1 hydroxylase catalytic domain, LSDI, Cmi, AD2, CR3, GATA4, p53, MEF2C, TAX, PPARy, SET9, KRAB, DNMT3A, DNMT1, KRAB-MeCP2, SIN3A, Mxi1, SID4x or Dnmt3a3L or a combination thereof.
- p65 p65 NF- ⁇ transactivating subunit
- VP160 VP160
- SET7 histone acetyltransferase p300
- VPR MyoDl
- TET1 hydroxylase catalytic domain LSDI, Cmi, AD2,
- composition of any one of embodiments 1-14, wherein the target nucleic acid sequence of b) is a regulatory region of a gene.
- the target nucleic acid sequence of b is a regulatory region of a gene.
- Embodiment 16 The composition of embodiment 15, wherein the regulatory region is a promoter or an enhancer.
- Embodiment 18 The composition of any one of embodiments 1-16, wherein the target nucleic sequence of b) encodes a gene product, and wherein the gene product is MT- TL1, KCNQ2, DEAF1, SSBP1, KCNQ1, HNF1B, KAT6B, CDK8, MN1, COL4A1, CDKL5, VAPB, NALCN, TTR, RAC2, GJB2, MYO3A, MEIS2, BRCA2, NARS1, AIRE, GABRG3, RAD51, GATA6, PDX1, ETV6, BCL11B, CHEK2, WARS1, KAT6B, KCNQ1, PRNP, MAT1A, HCN4, DSG2, MAFB, ZSWIM6, WT1, NIPBL, COL9A3, MYH7, SMAD4, IL6ST, CAPN3, KCNK18, DDX3X, SCAMP5, APC, CEBPA, RBM20, PMS2, BEST1, HCN1, PKD1, MSH
- Embodiment 19 A vector comprising: the nucleic acid sequence encoding the Cas-phi polypeptide of (a) and the polynucleotide sequence encoding the gRNA of (b) of any one of embodiments 1-18.
- Embodiment 20 The vector of embodiment 19, wherein the gRNA is operatively linked to a promoter recognized by RNA polymerase III.
- Embodiment 21 The vector of embodiment 20, wherein the gRNA is operatively linked to a human U6 promoter.
- Embodiment 22 The vector of any one of embodiments 19-21, wherein the vector is a viral vector.
- Embodiment 23 Embodiment 23.
- Embodiment 24 The vector of any one of embodiments 1-21, wherein the vector is suitable for delivery via a non-viral delivery system.
- Embodiment 25 The vector of embodiment 24, wherein the non-viral delivery system is a lipid nanoparticle or an exosome.
- Embodiment 26 A viral particle comprising the vector of any one of embodiments 19-23.
- Embodiment 27 The viral particle of embodiment 26, wherein the viral particle is a recombinant AAV (rAAV) particle.
- Embodiment 28 Embodiment 28.
- Embodiment 29 A population of viral particles comprising a plurality of viral particles of any one of embodiments 26-28.
- Embodiment 30 A pharmaceutical composition comprising the vector of any one of embodiments 19-25, the viral particle of any one of embodiments 26-28 or the population of embodiment 29, and a pharmaceutically acceptable carrier, vehicle or diluent.
- Embodiment 31 A cell comprising the vector of any one of embodiments 19-25 or the viral particle of any one of embodiments 26-28.
- Embodiment 32 The cell of embodiment 31, wherein the cell is a mammalian cell or an insect cell.
- Embodiment 33 A method of modifying the expression of a target gene in a population of cells comprising: contacting a population of cells comprising a target nucleic sequence encoding the target gene with the vector of any one of embodiments 19-25, the viral particle of any one of embodiments 26-28, the population of embodiment 29 or the pharmaceutical composition of embodiment 30, thereby modifying the expression of the target gene.
- Embodiment 34 Embodiment 34.
- the target gene is A4GALT, AAGAB, ABCD1, ACSL4, ACTC1, ACVRL1, ADNP, AFF2, AHDC1, AKT3, ALX4, ANK2, ANKRD11, ANOS1, AP1S2, APC, AR, ARCN1, ARHGEF9, ARID1A, ARID1B, ARID2, ARSE, ARX, ASH1L, ASXL1, ASXL3, ATP7A, ATP8A2, ATRX, AUTS2, AVPR2, AXIN2, BAG3, BCL11A, BCLAF1, BCOR, BMP4, BMPR1A, BMPR2, BRAF, BRCA1, BRCA2, BRIP1, BRWD3, BTK, CACNA1A, CACNA1C, CAMK2A, CAMK2B, CAMTA1, CASK, CASZ1, CCNQ, CDC42BPB, CDH1, CDKL5, CDKN1C, CFC1, CH
- Embodiment 37 The method of embodiment 33, wherein the expression of the target gene is decreased in the plurality of the modified population of cells in comparison to a population of cells contacted with the vector of any one of embodiments 19-25, the viral particle of any one of embodiments 26-28, the population of embodiment 29 or the pharmaceutical composition of embodiment 30; and a gRNA that does not specifically hybridize to the target nucleic acid sequence, but binds to the Cas-phi polypeptide to form a complex.
- Embodiment 38 Embodiment 38.
- the target gene is MT-TL1, KCNQ2, DEAF1, SSBP1, KCNQ1, HNF1B, KAT6B, CDK8, MN1, COL4A1, CDKL5, VAPB, NALCN, TTR, RAC2, GJB2, MYO3A, MEIS2, BRCA2, NARS1, AIRE, GABRG3, RAD51, GATA6, PDX1, ETV6, BCL11B, CHEK2, WARS1, KAT6B, KCNQ1, PRNP, MAT1A, HCN4, DSG2, MAFB, ZSWIM6, WT1, NIPBL, COL9A3, MYH7, SMAD4, IL6ST, CAPN3, KCNK18, DDX3X, SCAMP5, APC, CEBPA, RBM20, PMS2, BEST1, HCN1, PKD1, MSH2, RAD50, EYA1, KCNQ2, PRKCE, SYT1, GNAS, GSDME,
- Embodiment 40 The method of any one of embodiments 33-39, wherein the population of cells is a eukaryotic population of cells, a mammalian population of cells, an insect population of cells, a human population of cells or a plant population of cells.
- Embodiment 41 A modified population of cells produced by any one of the methods of embodiments 33-40.
- Embodiment 42 A method of reducing or eliminating the expression of a gene product in a subject comprising introducing to a cell of a subject the vector of any one of embodiments 19-25, the viral particle of any one of embodiments 26-28, the population of embodiment 29 or the pharmaceutical composition of embodiment 30.
- Embodiment 43 Embodiment 43.
- a method for treating or alleviating a symptom of a gene product related disorder in a subject comprising the step of introducing to a cell of the subject the vector of any one of embodiments 19-25, the viral particle of any one of embodiments 26- 28, the population of embodiment 29 or the pharmaceutical composition of embodiment 30.
- Embodiment 44 The method of embodiment 42 or 43, wherein the subject is a human.
Abstract
Sont divulgués dans la présente invention des systèmes, des méthode et des compositions utilisés pour la régulation de l'expression génique impliquant un ciblage de séquence, qui peuvent utiliser des systèmes vectoriels associés à de courtes répétitions palindromiques groupées et régulièrement espacées (CRISPR) ainsi que des composants de celles-ci. La présente invention concerne également de manière générale l'utilisation de vecteurs AAV pour l'administration de grandes charges utiles, telles que des protéines CRISPR (p. ex., Cas-phi), des ARN guides, des CRISPR-Cas ou des systèmes CRISPR. En outre, la présente invention concerne des méthodes de développement ou de conception de thérapies ou de thérapies basées sur des systèmes CRISPR-Cas.
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