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  • A01K2267/0306—Animal model for genetic diseases
• • A—HUMAN NECESSITIES
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
• • C—CHEMISTRY; METALLURGY
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the sgRNA of item 30 further comprising one or more linker segments located between the first hairpin forming segment and additional hairpin-forming segments, and/or between the additional hairpin-forming segments.
• sgRNA of any one of items 1 to 31 , the nucleic acid of item 32, the vector of any one of items 33 to 49, the composition of item 51 or 52 and/or the system of any one of items 53 to 56 for preventing or treating a condition associated with a target polynucleotide in a subject.
• sgRNA of any one of items 1 to 31 , the nucleic acid of item 32, the vector of any one of items 33 to 49, the composition of item 51 or 52 and/or the system of any one of items 53 to 56 for the preparation of a medicament for preventing or treating a condition associated with a target polynucleotide in a subject.
• the isolated polypeptide of item 81 comprising a first NLS N-terminal to the first domain and a second NLS C-terminal to the second domain.
• a system comprising the vector of item 105 and a further vector comprising a nucleotide sequence encoding an sgRNA. 120.
• a method of modifying a target polynucleotide in a cell comprising contacting the cell with the polypeptide according to any one of items 74 to 103, the nucleic acid of item 104, the vector of any one of items 105 to 1 12, the host cell of item 1 15, and/or the composition of item 1 16 or 1 17, and/or the system of item 1 18 or 1 19.
• polypeptide according to any one of items 74 to 103, the nucleic acid of item 104, the vector of any one of items 105 to 1 12, the host cell of item 115, and/or the composition of item 116 or 117, and/or the system of item 1 18 or 1 19, for the preparation of a medicament for modifying a target polynucleotide in a cell.
• FIG. 2 Screening for active sgRNAs targeting genes affecting liver function in mouse cells
• (a) Surveyor assays to determine St1 Cas9 activity programmed with various sgRNAs targeting Pck1.
• Neuro-2a cells were transiently transfected with a single vector (0.5pg) driving the expression of St1 Cas9 and its sgRNA.
• Surveyor assays were performed 3 days later to determine the frequency of indels, as indicated at the base of each lane.
• An expression vector encoding EGFP (-) was used as a negative control
• (b) Same as in (a) but targeting Pcsk9.
• (c) Same as in (a) but targeting Hpd.
• mice per group (n) and rAAV doses (vg) is indicated (f) Same as in (e) but body weight was measured daily. Solid lines designate the mean and error bars are represented by shaded areas and denote s.e.m. (g) Same as in (f) but glycemia was monitored in non-fasted mice (h) Same as in (e) but succinylacetone levels in urine were determined 15 days following NTBC removal. Samples were collected from the indicated treatment groups over a 24 hours period using metabolic cages.
• FIG. 4 Alternative rAAV-St1 Cas9 vector architectures can further improve potency
• v2 Schematic representations of the second-generation rAAV-St1Cas9 (v2) vector of similar size to the parent AAV genome ( ⁇ 4.7kb). Annotated is the human thyroxine binding globulin (TBG) promoter, synthetic polyadenylation sequence (SpA) and hU6 promoter. Arrows indicate the direction of transcriptional unit.
• Neonatal (2 days old) Fab-'- mice were injected with 2E1 1 vg rAAV8-St1 Cas9 v2 targeting Hpd exon 13 (G5) or saline into the retro-orbital sinus and killed 13 days post injection.
• FIG. 5 Engineered CRISPR1 -StCas9 system drives robust gene editing in human cells
• FIG. 10 Amino acid sequence alignment of St1 Cas9 from different strains (SEQ ID NOs: 16-19 corresponding to LMD_9, LMGJ 831 1 , CNRZ_1066, and TH1477, respectively). Identical residues are highlighted in black. The position of the WED and PAM-interacting domain (PI) are indicated by arrows. This region of the protein has diverged the most as compared to the N-terminal segment. In SaCas9, the PAM duplex is sandwiched between the WED and PI domains5. Alignment was performed with Clustal Omega 6 and ESPript 7 .
• FIG. 21 Converting St1 Cas9 LMD-9 to a cytosine base editor (CBE).
• St1 BE4max programmed with sgRNAs targeting NNAGAA and NNGGAA PAMs in human cells.
• K562 cells were transiently transfected with a single vector (1 pg) driving the expression of St1 BE4max LMD-9 and its sgRNA.
• Quantification of base editing from sanger sequencing reads was performed 3 days later using EditRTM software. Numbers in each box indicate the % of C to T conversions.
• FIGs. 25a-25b Nucleotide sequence of NLS-St1Cas9 LMD-9/CNRZ1066 Hybrid-NLS (SEQ ID NO: 37).
• SV40 NLS is uppercase and underlined SEQ ID NOs: 23-24);
• St1Cas9 hybrid sequence is in uppercase italic (SEQ ID NO: 38);
• linker regions are in lowercase (linkers flanking St1Cas9 hybrid, SEQ ID NOs: 26-27).
• FIGs. 27a-27b Nucleotide sequence of NLS-St1 Cas9 LMD-9/TH 1477 Hybrid-NLS (SEQ ID NO: 41).
• SV40 NLS is uppercase and underlined SEQ ID NOs: 23-24);
• St1 Cas9 hybrid sequence is in uppercase italic (SEQ ID NO: 42);
• linker regions are in lowercase (linkers flanking St1Cas9 hybrid, SEQ ID NOs: 26-27).
• (a) and (b) denote the two strands of the stem portion, created when the single strand folds back onto itself to create a two-strand hybrid or duplex structure.
• the (a) and (b) portions are at least partially complementary to each other to enable formation of the stem portion.
• the sgRNA further comprises one or more additional hairpin-forming segments located 3' to the first hairpin-forming segment.
• the sgRNA further comprises one or more linker segments located between the first hairpin-forming segment and additional hairpin-forming segments, and/or between the additional hairpin-forming segments.
• one or more linker regions may be used to connect any of the domains described herein.
• the domain comprising the PAM-interacting domain is derived from LMD-9 (e.g., SEQ ID NO: 260, or a functional fragment of any thereof, or a functional variant of any thereof) and is specific for NNAGAA and NNGGAA PAMs.
• the domain comprising the PAM-interacting domain is derived from CNRZ1066 (e.g., SEQ ID NO: 262, or a functional fragment of any thereof, or a functional variant of any thereof) and is specific for NNACAA PAMs.
• MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001 ; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METFIODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CFIROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, "Chromatin" (P. M. Wassarman and A. P.
• a non-conservative substitution includes one that changes an amino acid of one group with another amino acid of another group (e.g., an aliphatic amino acid for a basic, a cyclic, an aromatic or a polar amino acid; a basic amino acid for an acidic amino acid, a negatively charged amino acid (aspartic acid or glutamic acid) for a positively charged amino acid (lysine, arginine or histidine) etc.
• an amino acid of one group e.g., an aliphatic amino acid for a basic, a cyclic, an aromatic or a polar amino acid; a basic amino acid for an acidic amino acid, a negatively charged amino acid (aspartic acid or glutamic acid) for a positively charged amino acid (lysine, arginine or histidine) etc.
• “Complement” or “complementary” as used herein refers to Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. "Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.
• one or more targeted (site-specific) nucleases create a double-stranded break in the target sequence (e.g., cellular chromatin) at a predetermined site.
• a "donor" polynucleotide having homology to the nucleotide sequence in the region of the break, may be introduced into the cell if desired. The presence of the double-stranded break has been shown to facilitate integration of the donor sequence.
• Promoter means a synthetic or naturally-derived nucleic acid molecule which is capable of conferring, modulating or controlling (e.g., activating, enhancing and/or repressing) expression of a nucleic acid in a cell.
• a promoter may comprise one or more specific transcriptional regulatory sequences to further enhance or repress expression and/or to alter the spatial expression and/or temporal expression of same.
• a promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription.
• a promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
• Adeno-associated virus or “AAV” as used interchangeably herein refers to a small virus belonging to the genus Dependovirus of the Parvoviridae family that infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response.
• Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population).
• the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .
• Pharmaceutical compositions, which exhibit large therapeutic indices, are preferred.
• the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
• the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
• the sgRNAs and the nuclease are then used together to introduce the desired modification(s) (i.e., gene-editing events) by NHEJ or HDR within the genome of one or more target cells.
• the desired modification(s) include specific point mutation(s) or insertions/deletion(s)
• one or more donor or patch nucleic acids comprising the desired modification(s) are provided to introduce the modification(s) by HDR.
• CRISPR nucleases require the presence of a sgRNA and a protospacer adjacent motif (PAM) on the targeted gene.
• PAM protospacer adjacent motif
• the PAM immediately follows (i.e., is adjacent to) the sgRNA target sequence in the targeted polynucleotide gene sequence.
• the PAM is located at the 3’ end or 5’ end of the sgRNA target sequence (depending on the CRISPR nuclease used) but is not included in the sgRNA guide sequence.
• the PAM for a Cas9 protein used in accordance with the present disclosure is a NGG trinucleotide-sequence (Cas9).
• the PAM for a Cpf1 CRISPR nuclease used in accordance with the present disclosure is a TTTN nucleotide sequence.
• the St1 Cas9 may be used, which corresponds to the PAM sequences NNAGAA and NNGGAA.
• Table 1 Non-exhaustive list of CRISPR-nuclease systems from different species (see. Mohanraju, P. et al complicat PMID 27493190; Shmakov, S et al complicat PMID: 26593719; and Zetsche, B. et al complicat PMID: 26422227). Also included are engineered variants recognizing alternative PAM sequences (see Kleinstiver, BP. et al., (Nature biotech 2015) PMID: 26524662 and Kleinstiver, BP. et al., (Nature 2015)).
• the “CRISPR nuclease recognition sequence” may thus include a crRNA sequence only (e.g., for AsCpfl activity, such as the CRISPR nuclease recognition sequence UAAUUUCUAC UCUUGUAGAU (SEQ ID NO: 38)) or may include additional sequences (e.g., tracrRNA sequence necessary for Cas9 activity).
• RNA motifs necessary for CRISPR nuclease binding and activity may be provided separately (e.g., (i) RNA guide sequence-crRNA CRISPR recognition sequence” (also known as crRNA) in one RNA molecule and (ii) a tracrRNA CRISPR recognition sequence on another, separate RNA molecule.
• target region in the context of sgRNAs and CRISPR system of the present disclosure are used herein interchangeably and refers to the region of the target gene, which is targeted by the CRISPR/nuclease-based system, without the PAM. It refers to the sequence corresponding to the nucleotides that precede the PAM (i.e., in 5’ or 3’ of the PAM, depending of the CRISPR nuclease) in the genomic DNA. It is the sequence that is included into a sgRNA expression construct (e.g., vector/plasmid/AAV).
• a sgRNA expression construct e.g., vector/plasmid/AAV
• the sgRNA of the present disclosure comprises a“sgRNA guide sequence” or has a “sgRNA target sequence” which corresponds to the target sequence on the gene of interest or target polynucleotide sequence that is followed or preceded by a PAM sequence (is adjacent to a PAM).
• the sgRNA may comprise a "G” at the 5' end of its polynucleotide sequence. The presence of a“G” in 5’ is preferred when the sgRNA is expressed under the control of the U6 promoter (Taeyoung KooJungjoon Lee and Jin-Soo Kim Mol Cells. 2015 Jun 30; 38(6): 475-481).
• the sgRNA of the present disclosure comprises 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, more preferably 2 mismatches, or less, and even more preferably no mismatch, with the corresponding sgRNA target gene sequence (less the PAM).
• the sgRNA nucleic acid sequence is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identical to the sgRNA target polynucleotide sequence in the gene of interest.
• the smaller the number of nucleotides in the sgRNA guide sequence the smaller the number of mismatches tolerated.
• the binding affinity is thought to depend on the sum of matching sgRNA-DNA combinations.
• the recombinant CRISPR nuclease that may be used in accordance with the present disclosure is i) derived from a naturally occurring Cas; and ii) has a nuclease (or nickase) activity to introduce a DSB (or two SSBs in the case of a nickase) in cellular DNA when in the presence of appropriate sgRNA(s).
• CRISPR nuclease proteins are (or are derived from) proteins normally expressed in bacteria, it may be advantageous to modify their nucleic acid sequences for optimal expression in eukaryotic cells (e.g., mammalian cells) when designing and preparing CRISPR nuclease recombinant proteins.
• donor or patch nucleic acids of the present disclosure used to introduce specific modifications in the target polynucleotide may use codon degeneracy (e.g., to introduce new restriction sites for enabling easier detection of the targeted modification).
• codon chart (Table 2) may be used, in a site-directed mutagenic scheme, to produce nucleic acids encoding the same or slightly different amino acid sequences of a given nucleic acid:
• K562 were obtained from the ATCC (CCL-243) and maintained at 37 °C under 5% CO2 in RPMI medium supplemented with 10% FBS, penicillin-streptomycin and GlutaMAXTM.
• Neuro-2a were obtained from the ATCC and maintained at 37 °C under 5% C02 in DMEM medium supplemented with 10% FBS, penicillin-streptomycin and GlutaMAXTM. All cell lines are tested for absence of mycoplasma contamination. Cells (2x10 5 per transfection) were transfected using the Amaxa 4D-Nucleofector (Lonza) per manufacturer's recommendations.
• St1 Cas9 strain variants that display unique PAM preferences are also functional as CBEs.
• LMD-9/LMG18311 hybrid- and LMD-9/CNRZ1066 hybrid-based St1 BE4max are potent base editors at NNGCAA and NNACAA PAMs, respectively (Fig. 22).
• Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759-771 (2015).

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