WO2023044510A2 - Crispr gene editing for diseases associated with a gene mutation or single-nucleotide polymorphism (snp) - Google Patents

Crispr gene editing for diseases associated with a gene mutation or single-nucleotide polymorphism (snp) Download PDF

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WO2023044510A2
WO2023044510A2 PCT/US2022/076743 US2022076743W WO2023044510A2 WO 2023044510 A2 WO2023044510 A2 WO 2023044510A2 US 2022076743 W US2022076743 W US 2022076743W WO 2023044510 A2 WO2023044510 A2 WO 2023044510A2
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syndrome
disease
sequence
snp
target sequence
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WO2023044510A3 (en
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Tara MOORE
Louise J. ROBERTSON
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Avellino Lab Usa, Inc.
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present disclosure relates to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) systems, and methods of use thereof for gene editing or for preventing, ameliorating or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas CRISPR associated protein
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas9 CRISPR associated protein 9
  • This highly specific and efficient RNA- guided DNA endonuclease may be of therapeutic importance in a range of genetic diseases.
  • the CRISPR/Cas9 system relies on a single catalytic protein, Cas9 that is guided to a specific DNA sequence by 2 RNA molecules; the tracrRNA and the crRNA (Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 157: 1262- 1278). Combination of the tracrRNA/crRNA into a single guide RNA molecule (sgRNA) (Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS et al. Genome-scale CRISPR-Cas9 knockout screening in human cells.
  • sgRNA single guide RNA molecule
  • This PAM sequence is an invariant part of the DNA target but not present in the sgRNA, while its absence at the 3' end of the genomic target sequence results in the inability of the Cas9 to cleave the DNA target (Westra ER, Semenova E, Datsenko KA, Jackson RN, Wiedenheft B, Severinov K et al. Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition. PLoS Genet 2013; 9: el003742). This distinction is important as the mutation directly in a PAM-specific approach, or nearby SNPs may be targeted. One SNP allele will represent a PAM site, while the other allele does not. This allows us to discriminate between the two chromosomes.
  • the mutation-independent CRISPR method developed (Christie et al., Mol. Ther, 2020) relies on determining the phase of patient-specific SNPs in relation to the disease-causing mutation (Mutation-Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease. Mol Ther 2020;28(8). Doi: 10.1016/j.ymthe.2020.05.002).
  • the allelespecific SNP/SNPs targeted by the gRNA are on the same allele as the disease-causing mutation to remove the mutant allele and leave the wildtype allele untouched.
  • the treatment can be tailored to the individual, by selecting two validated guide RNAs (gRNAs) from a pool of guides targeting the SNP/s and where necessary, a common intronic SNP.
  • gRNAs validated guide RNAs
  • the 10X Genomics method for this analysis is costly and covers the entire genome rather than just the affected genomic region.
  • bioinformatic analysis to be completed after the sequencing.
  • a more efficient method of identifying the SNPs in cis and selecting sgRNAs is desired.
  • the present disclosure describes the potential of utilizing the PAM-generating mutations in introns of a disease causing gene.
  • the PAM-generating mutations are in adjacent introns of a gene having a disease-causing mutation, and the disease-causing mutation is in exon in between the adjacent introns.
  • Cas nuclease may cleave a gene at two intronic sites, between which an exon containing a disease-causing mutation exists, thereby eliminating the disease-causing exon and knocking out the mutated allele.
  • the CRISPR/Cas system utilizing the PAM-generating mutations or SNPs in introns may be used to treat a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject, for example, including an autosomal dominant disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject.
  • SNP single-nucleotide polymorphism
  • the present disclosure is related to methods of identifying the PAM-generating mutations or SNPs in introns using droplet digital polymerase chain reaction (PCR).
  • PCR droplet digital polymerase chain reaction
  • the present disclosure is related to methods of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject, the method comprising detecting phase of SNPs in cis with the gene mutation or SNP associated with the disease in the subject by droplet digital polymerase chain reaction (PCR), and administering to the subject an engineered CRISPR/Cas system.
  • SNP single-nucleotide polymorphism
  • the detecting comprises preparing at least 10,000 droplets, each comprising a first labeled probe for the gene mutation or SNP and a second labeled probe for a SNP that is in cis with the gene mutation or SNP.
  • the first and second probes are labeled with different fluorescent dyes.
  • the methods further comprise detecting the gene mutation or SNP in the subject prior to detecting phase of SNPs.
  • the methods further comprise diagnosing the disease in the subject prior to detecting phase of SNPs.
  • the detecting phase of SNPs excludes sequencing a full genome in a sample from the subject.
  • the methods further comprise obtaining a sample form the subject, and the detecting phase of SNPs from the sample.
  • the administering comprises administering to the subject an engineered CRISPR/Cas system comprising at least one vector comprising at least two different CRISPR targeting RNA (crRNA) sequences or single guide RNA (sgRNA) sequences.
  • crRNA CRISPR targeting RNA
  • sgRNA single guide RNA
  • the present disclosure is related to methods of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject, comprising administering to the subject an engineered CRISPR/Cas system comprising at least one vector comprising (i) a nucleotide molecule encoding Cas nuclease; (ii) a first sgRNA comprising a first crRNA sequence that hybridizes to a nucleotide sequence complementary to a first target sequence, the first target sequence being adjacent to (e.g., the 5 ’-end of) a first protospacer adjacent motif (PAM) at the 3 ’-end side of a disease-causing mutation or SNP in cis, wherein the first target sequence or the first PAM comprises a first ancestral variation or SNP site; and (iii) a second sgRNA comprising a second crRNA sequence that hybridizes to a nucleotide sequence complementary to
  • the second target sequence or the second PAM comprises a second ancestral variation or SNP site.
  • the at least one vector does not have a nucleotide molecule encoding Cas nuclease and a sgRNA sequence that naturally occur together.
  • the disease is an autosomal dominant disease.
  • the disease is selected from the group consisting of Acropectoral syndrome, Acute intermittent porphyria, Adermatoglyphia, Albright's hereditary osteodystrophy, Arakawa's syndrome II, Aromatase excess syndrome, Autosomal dominant cerebellar ataxia, Axenfeld syndrome, Benign hereditary chorea, Bethlem myopathy, Birt-Hogg-Dube syndrome, Boomerang dysplasia, Branchio- oto-renal syndrome, Buschke-Ollendorff syndrome, Camurati-Engelmann disease, Central core disease, Collagen disease, Collagenopathy, types II and XI, Congenital distal spinal muscular atrophy, Congenital stromal corneal dystrophy, Costello syndrome, Currarino syndrome, Darier's disease, Glutl deficiency, Dentatorubral-pallidoluysian atrophy, Dermatopathia pigmentosa reticularis, Dysfibrinogenemia,
  • the disease is an autosomal dominant disease of an eye.
  • the disease may include or excludes corneal dystrophy.
  • the corneal dystrophy is associated with R124H granular corneal dystrophy type 2 mutation.
  • the disease-causing mutation or SNP is in an exon of a gene causing the disease.
  • the first and second PAMs are in different introns surrounding one or more exons containing the disease-causing mutation or SNP.
  • the first PAM comprises the first ancestral variation or SNP site and/or the second PAM comprises the second ancestral variation or SNP site.
  • the first crRNA sequence comprises the first target sequence, and the second crRNA sequence comprises the second target sequence.
  • the first crRNA sequence is from 17 to 24 nucleotide long; and/or the second crRNA sequence is from 17 to 24 nucleotide long.
  • the first and/or second PAMs and the Cas nuclease are from Streptococcus or Staphylococcus. In additional embodiments, the first and second PAMs are both from Streptococcus or Staphylococcus.
  • the Cas nuclease is Cas9 nuclease.
  • each of the first and second PAMs independently consists of NGG or NNGRRT, wherein N is any of A, T, G, and C, and R is A or G.
  • the Cas nuclease is Cpfl nuclease.
  • the Cas nuclease is selected from the group consisting of: Cas9 nuclease, Cpfl nuclease (also known as Cas 12a nuclease), C2cl nuclease (also known as Cas 12b nuclease), C2c2 nuclease (also known as Casl3al nuclease), C2c3 nuclease (also known as Casl2c nuclease), and Cmsl nuclease. In some embodiments, any other Cas nuclease may be used.
  • the administration comprises injecting the engineered CRISPR/Cas system into the subject.
  • the administering comprises introducing the engineered CRISPR/Cas system into a cell containing and expressing a DNA molecule having the target sequence.
  • the disease is associated with the SNP; the first target sequence or the first PAM comprises the first ancestral SNP site; and/or the second target sequence or the second PAM comprises the second ancestral SNP site.
  • the target sequence or the PAM comprises a plurality of mutation or SNP sites.
  • the subject is human.
  • the methods described herein further comprises, prior to administering to the subject the engineered CRISPR/Cas system, obtaining genomic or sequence information of the subject; and selecting the first crRNA sequence and/or the second crRNA sequence based on the genomic or sequence information of the subject.
  • the genomic or sequence information of the subject includes whole or partial genome sequence information of the subject.
  • the first crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves at a first cleaving site that is adjacent to the first ancestral variation or SNP site; and/or the second crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves at a second cleaving site that is adjacent to the second ancestral variation or SNP site.
  • the first crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves only at the first cleaving site that is adjacent to the first ancestral variation or SNP site; and/or the second crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves only at the second cleaving site that is adjacent to the second ancestral variation or SNP site.
  • the first crRNA sequence is configured to reduce cleaving of the genome of the subject at a site other than a first cleaving site compared to other crRNA sequences hybridizing to the nucleotide sequence complementary to the first target sequences; and/or the second crRNA sequence is configured to reduce cleaving of the genome of the subject at a site other than a second cleaving site compared to other crRNA sequences hybridizing to the nucleotide sequence complementary to the second target sequences.
  • the first crRNA sequence is configured to reduce cleaving of a gene, in trans, that corresponds to a gene causing the disease in cis compared to other crRNA sequences hybridizing to the nucleotide sequence complementary to the first target sequences; and/or the second crRNA sequence is configured to reduce cleaving of a gene, in trans, that corresponds to the gene causing the disease in cis compared to other crRNA sequences hybridizing to the nucleotide sequence complementary to the second target sequences.
  • the selected first crRNA sequence is configured to cause cleaving at a first cleaving site, within genome of the subject, that is adjacent to the first ancestral variation or SNP site; and/or the selected second crRNA sequence is configured to cause cleaving at a second cleaving site, within the genome of the subject, that is adjacent to the second ancestral variation or SNP site.
  • the selected first crRNA sequence is configured to cause cleaving only at the first cleaving site; and/or the selected second crRNA sequence is configured to cause cleaving only at the second cleaving site.
  • the first crRNA sequence hybridizes to the nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence in trans not being adjacent to the 5 ’-end of a PAM; and/or the second crRNA sequence hybridizes to the nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence not being adjacent to the 5 ’-end of a PAM.
  • a method of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject includes administering to the subject an engineered CRISPR/Cas system comprising: (i) a Cas nuclease; (ii) a first sgRNA comprising a first CRISPR targeting RNA (crRNA) sequence that hybridizes to a nucleotide sequence complementary to a first target sequence, the first target sequence being adjacent to a first protospacer adjacent motif (PAM) at 3 ’-end side of a disease-causing mutation or SNP in cis, wherein the first target sequence or the first PAM comprises a first ancestral variation or SNP site; and (iii) a second sgRNA comprising a second crRNA sequence that hybridizes to a nucleotide sequence complementary to a second target sequence, the second target sequence being adjacent to a second PAM at 5
  • crRNA CRISPR targeting
  • an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) system includes (i) a Cas nuclease; (ii) a first sgRNA comprising a first CRISPR targeting RNA (crRNA) sequence that hybridizes to a nucleotide sequence complementary to a first target sequence, the first target sequence being adjacent to a first protospacer adjacent motif (PAM) at 3 ’-end side of a disease-causing mutation or SNP in cis, wherein the first target sequence or the first PAM comprises a first ancestral variation or SNP site; and (iii) a second sgRNA comprising a second crRNA sequence that hybridizes to a nucleotide sequence complementary to a second target sequence, the second target sequence being adjacent to a second PAM at 5 ’-end side of the disease-causing mutation or SNP in cis.
  • Figure 1 illustrates an example of a sgRNA sequence, nucleotide and amino acid sequences of Cas9 nuclease from Streptococcus pyogenes (Spy) and Staphylococcus aureus (Sau).
  • Figure 2 illustrates an example of a dual-cut approach using intronic PAM sites.
  • Two separate guides are introduced, and Cas9 generates a double stranded break (DSB) at two sites. Repair of this doubly cut region will result in an excision of the region between the two breaks.
  • the deletion encompasses the exonic coding region of the gene shown by the yellow boxes in this figure.
  • Figure 3 illustrates an embodiment in which a sgRNA utilizing a flanking SNP within the PAM site is designed in the first intron. Additionally, a sgRNA common to both the wild-type and mutant allele is designed in the second intron. In the wild-type allele the single sgRNA causes NHEJ in the second intron, which may have no functional effect. However, in the mutant allele, the sgRNA utilizing the flanking SNP derived PAM and the common sgRNA result in a large deletion that results in a knockout of the mutant allele.
  • FIG. 4 illustrates all SNPs in TGFBI with a MAF of >10% that generate a novel PAM.
  • the numbered boxes indicate the exons within TGFBI.
  • the hotspots in TGFBI, where multiple diseasecausing mutations are found, are shown by the red boxes.
  • the blue arrows indicate the position of a SNP that generates a novel PAM.
  • the novel PAM is shown for each arrow, with the required variant highlighted in red.
  • Figure 5 depicts experimental results from using an exemplary lymphocyte cell line derived from a patient with a R124H granular corneal dystrophy type 2 mutation that was nucleofected with CRISPR/Cas9 and sgRNA.
  • the guide utilized the novel PAM that is generated by the rs3805700 SNP. This PAM is present on the same chromosome as the patients R124H mutation but does not exist on the wild-type chromosome.
  • single clones were isolated to determine whether indels had occurred. Six of the single clones had the unedited wild-type chromosome, indicating stringent allele-specificity of this guide.
  • Figure 6 shows the results from a dual-guide approach. Two CRISPR plasmids were transfected into the LCLs, one tagged with mCherry the other tagged with GFP. Positive cells were sorted for both mCherry and GFP, collecting 2.6% of the total population. The cells were then allowed to repair and expand, and the genomic DNA was isolated.
  • Figure 7 on the right, illustrates that using the original clonal isolation of single alleles, a 565bp deletion encompassing both PAM sites was confirmed. The deletion is shown in red with the PAM sites highlighted in blue. On the left, Figure 7 also illustrates the two guides cutting at their target sites, the region between these cuts being excised upon repair, and the genomic region after repair.
  • Figures 8-23 illustrate exemplary common guides in intronic regions of TGFBI gene.
  • Figure 24 illustrates a flowchart for exemplary personalized gene editing
  • the present disclosure is related to methods of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject, comprising detecting phase of SNPs in cis with the gene mutation or SNP associated with the disease in the subject by droplet digital polymerase chain reaction (ddPCR), and administering to the subject an engineered CRISPR/Cas system.
  • SNP single-nucleotide polymorphism
  • ddPCR refers to droplet digital polymerase chain reaction.
  • ddPCR one or more PCR amplifications are performed, wherein each reaction is separated into a plurality of water-oil emulsion droplets, so that PCR amplification of the target sequence may occur in each individual droplet.
  • the ddPCR may measure absolute quantities by counting nucleic acid molecules encapsulated in discrete, vohimetrically defined, water-in-oil droplet partitions that support PCR amplification (Hinson et al., 2011, Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem.
  • a single ddPCR reaction may be comprised of at least 20,000 partitioned droplets per well.
  • a “droplet” or “water-in-oil droplet” refers to an individual partition of the droplet digital PCR assay.
  • a droplet supports PCR amplification of template molecule(s) using homogenous assay chemistries and workflows similar to those widely used for real-time PCR applications (Hinson et al., 2011, Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84:1003-1011).
  • Droplets may be read as either positive or negative for specific fluorescent signals and the fraction of positive droplets (and calculations using Poisson statistics), allowing quantification of the target in the sample.
  • the digital droplet system may be useful for determining phase of SNPs.
  • a SNP-specific assay may be run in the same ddPCR reaction as the disease-causing mutation specific assay, each with different fluorescent signals. If the SNP lies in cis with the disease-causing mutation, the droplet will be positive for both assays in a significantly higher portion than if they lie on different alleles (A Rapid Molecular Approach for Chromosomal Phasing. PLoS One 2015;10:e0118270. Doi: 10.1371/joumal.pone.0H8270). This may also be checked via restriction digest between the two sites, which will destroy co-partioning.
  • Allele-specific probes may also be designed, for example, as shown in Mutation- Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease. Mol Ther 2020;28(8). Doi: 10.1016/j.ymthe.2020.05.002 and adapted to the ddPCR platform.
  • detecting phase of SNPs in cis comprises preparing at least 10,000, 15,000, 20,000, or 25,000 droplets, each comprising a first labeled probe for the gene mutation or SNP and a second labeled probe for a SNP that is in cis with the gene mutation or SNP.
  • the first and second probes are labeled with different fluorescent dyes.
  • suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes are described in the 1996 Molecular Probes Handbook by Richard P. Haugland.
  • Suitable fluorescent labels also include, but are not limited to, green fluorescent protein (GFP; Chalfie, et al., Science 263(5148): 802-805, 1994); and EGFP; Clontech — Genbank Accession Number U55762), blue fluorescent protein (BFP; Quantum Biotechnologies, Inc.; Stauber, R. H. Biotechniques 24(3):462-471 (1998); Heim, R. and Tsien, R. Y. Curr. Biol. 6: 178-182 (1996)), enhanced yellow fluorescent protein (EYFP; Clontech Laboratories, Inc.), luciferase (Ichiki, et al., J. Immunol.
  • GFP green fluorescent protein
  • BFP blue fluorescent protein
  • EYFP enhanced yellow fluorescent protein
  • EYFP Clontech Laboratories, Inc.
  • luciferase Ichiki, et al., J. Immunol.
  • the labels descried herein include: Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.), Sulfo-Cyanine 3, Sulfo-Cyanine 5, Sulfo-Cyanine 5.5, Sulfo-Cyanine 7, Sulfo- Cyanine 7.5 (Lumi)
  • Tandem conjugate protocols for Cy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, Cy7APC are known. Additional labels are available from commercial sources such as BD Biosciences, Beckman Coulter, AnaSpec, Invitrogen, Cell Signaling Technology, Millipore, eBioscience, Santa Cruz Biotech, Abeam, LiCor, and Sigma-Aldrich.
  • the methods described herein comprises administering to the subject an engineered CRISPR/Cas system comprising at least one vector comprising (i) a nucleotide molecule encoding Cas nuclease; (ii) a first sgRNA comprising a first CRISPR targeting RNA (crRNA) sequence that hybridizes to a nucleotide sequence complementary to a first target sequence, the first target sequence being adjacent to the 5 ’-end of a first protospacer adjacent motif (PAM) at 3 ’-end side of a disease-causing mutation or SNP in cis, wherein the first target sequence or the first PAM comprises a first ancestral variation or SNP site; and (iii) a second sgRNA comprising a second crRNA sequence that hybridizes to a nucleotide sequence complementary to a second target sequence, the second target sequence being adjacent to the 5 ’-end of a second PAM at 5 ’-end side of the disease-causing mutation or
  • crRNA C
  • the present disclosure is related to methods of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject comprising altering expression of the gene product of the subject by the methods described above, wherein the gene comprises a mutant or SNP mutant sequence.
  • the disease is associated with the SNP; the first target sequence or the first PAM comprises the first ancestral SNP site; and/or the second target sequence or the second PAM comprises the second ancestral SNP site.
  • the target sequence comprises a plurality of mutation or SNP sites.
  • the subject is human.
  • the first crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves at a first cleaving site that is adjacent to the first ancestral variation or SNP site; and/or the second crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves at a second cleaving site that is adjacent to the second ancestral variation or SNP site.
  • the first crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves only at the first cleaving site; and/or the second crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves only at the second cleaving site.
  • being “in cis'” with the disease-causing mutation or SNP refers to being on the same molecule of DNA or chromosome as the disease-causing mutation
  • being “in trans” with the disease-causing mutation or SNP refers to being on a different molecule of DNA or chromosome as the disease-causing mutation or SNP.
  • the first crRNA sequence hybridizes to the nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence not being adjacent to the 5 ’-end of a PAM; and/or the second crRNA sequence hybridizes to the nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence not being adjacent to the 5 ’-end of a PAM.
  • the first and/or the second target sequences in trans with the diseasecausing mutation or SNP may remain intact without any cleavage (e.g., the Cas nuclease does not cleave the first and/or the second target sequences in trans with the disease-causing mutation or SNP).
  • This approach may permit expression of a gene that is in trans with the disease-causing mutation or SNP and does not include a disease-causing mutation or SNP.
  • This approach may also reduce or eliminate any adverse impacts associated with knocking out both the gene that includes the diseasecausing mutation or SNP and the gene that does not include the disease-causing mutation or SNP in a subject.
  • the first crRNA sequence hybridizes to the nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence not being adjacent to the 5 ’-end of a PAM; and the second crRNA sequence hybridizes to the nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence being adjacent to the 5 ’-end of a PAM.
  • the first target sequence in trans with the disease-causing mutation or SNP may remain intact without any cleavage while the second target sequence in trans with the disease-causing mutation or SNP may be cleaved (e.g., the Cas nuclease cleaves the first target sequence in trans with the disease-causing mutation or SNP but does not cleave the second target sequence in trans with the disease-causing mutation or SNP).
  • the first crRNA sequence hybridizes to the nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence being adjacent to the 5 ’-end of a PAM; and the second crRNA sequence hybridizes to the nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence not being adjacent to the 5 ’-end of a PAM.
  • the second target sequence in trans with the diseasecausing mutation or SNP may remain intact without any cleavage while the first target sequence in trans with the disease-causing mutation or SNP is cleaved (e.g., the Cas nuclease cleaves the second target sequence in trans with the disease -causing mutation or SNP but does not cleave the first target sequence in trans with the disease-causing mutation or SNP).
  • Said “nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP” herein has the identical nucleotide sequence as the nucleotide sequence complementary to the first target sequence in cis with the disease-causing mutation or SNP.
  • nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP and said “the first target sequence in trans with the disease -causing mutation or SNP,” however, may be located on a different molecule of DNA or chromosome where the same disease-causing mutation or SNP is absent (thus are in trans with the disease-causing mutation or SNP).
  • said “nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP” herein has the identical nucleotide sequence as the nucleotide sequence complementary to the second target sequence in cis with the disease-causing mutation or SNP.
  • the engineered CRISPR/Cas system described herein may comprise at least one vector comprising (i) a nucleotide molecule encoding Cas nuclease described herein, and (ii) a plurality of sgRNA targeting intronic sites surrounding one or more exons containing a disease- associate mutation or SNP of interest as described herein.
  • the sgRNA may comprise a target sequence adjacent to the 5 ’-end of a protospacer adjacent motif (PAM), and/or hybridize to a first target sequence complementary to a second target sequence adjacent to the 5’ end of the PAM.
  • the target sequence or the PAM may comprise the ancestral variation or SNP in an intronic site.
  • the ancestral variation or SNP in the intronic site does not cause a disease.
  • sgRNA may comprise a target sequence adjacent to a PAM site located in the flanking intron that is common to both wild-type and mutant alleles in tandem with a sgRNA adjacent to a PAM site that is specific to the mutant allele.
  • the Cas nuclease and the sgRNA do not naturally occur together. The sequence of this PAM site is specific to the Cas nuclease being used.
  • the PAM comprises the mutation or SNP site.
  • the PAM consists of a PAM selected from the group consisting of NGG and NNGRRT, wherein N is any of A, T, G, and C, and R is A or G.
  • the disease-causing mutation or SNP is in an exon of a gene associated with the disease, and the first and second PAMs are in different introns surrounding one or more exons containing the disease-causing mutation or SNP.
  • first and second CRISPR targeting RNA (crRNA) sequences hybridize to nucleotide sequences complementary to first and second target sequences, the first target sequence being adjacent to the 5 ’-end of a first protospacer adjacent motif (PAM) at 3 ’-end side of a disease-causing mutation or SNP in cis, and the second target sequence being adjacent to the 5 ’-end of a first protospacer adjacent motif (PAM) at 5’- end side of a disease-causing mutation or SNP in cis.
  • PAM protospacer adjacent motif
  • the first and second PAMs are located on opposite sides of one or more exons containing the disease-causing mutation or SNP.
  • an “intron” means a section of DNA occurring between two adjacent exons within a gene which is removed during pre-mRNA splicing and does not code for any amino acids constituting the gene product.
  • An “intronic site” is a site within an intron.
  • An “exon” means a section of DNA occurring in a gene which codes for one or more amino acids in the gene product.
  • the constitutively spliced exon known so far has 6 nucleotides or more, and the alternatively spliced exon has 3 nucleotides or more, which is equivalent to 1 or 2 amino acids or more depending on the frame that the mRNA is read in.
  • An “exonic site” is a site within an exon.
  • the first PAM comprises the first mutation or SNP site and/or the second PAM comprises the second mutation or SNP site.
  • the first crRNA sequence comprises the first target sequence
  • the second crRNA sequence comprises the second target sequence.
  • each of the first crRNA sequence and the second crRNA sequence may independent be from 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotide long.
  • the methods described herein further comprise identifying targetable mutations or SNPs on either side of disease-causing mutation or SNP to silence the disease-causing mutation or SNP.
  • a block of DNA is identified in a phased sequencing experiment.
  • the mutation or SNP of interest is not a suitable substrate for the CRISPR/Cas system, and identifying mutations or SNPs on both side of the disease-causing mutations or SNP that are suitable for CRISPR/Cas cleavage allows removal of a segment of DNA that includes the disease-causing mutations or SNP.
  • the read length may be increased so as to gain longer contiguous reads and a haplotype phased genome by using a technology described in Weisenfeld NI, Kumar V, Shah P, Church DM, Jaffe DB. Direct determination of diploid genome sequences. Genome research. 2017; 27(5):757-767, which is herein incorporated by reference in its entirety.
  • the methods described herein further comprises, prior to administering to the subject the engineered CRISPR/Cas system, obtaining genomic or sequence information of the subject; and selecting the first crRNA sequence and/or the second crRNA sequence based on the genomic or sequence information of the subject.
  • the genomic or sequence information of the subject includes whole or partial genome sequence information of the subject.
  • the human genome is diploid by nature; every chromosome with the exception of the X and Y chromosomes in males is inherited as a pair, one from the male and one from the female parent. When seeking stretches of contiguous DNA sequence larger than a few thousand base pairs, a determination of inheritance is crucial to understand from which parent these blocks of DNA originate.
  • Longer read sequencing technologies have been utilized in attempts to produce a haplotype- resolved genome sequences, i.e. haplotype phasing. Thus, when investigating the genomic sequence of a particular stretch of DNA longer than 50 kbps, a haplotype phased sequence analysis may be utilized to determine which of the paired chromosomes carries the sequence of interest. Longer phased sequencing reads may be employed to determine whether the SNP of interest would be suitable as a target for the CRISPR/Cas gene editing system described herein.
  • the selected first crRNA sequence is configured to cause cleaving at a first cleaving site, within genome of the subject, that is adjacent to the first ancestral variation or snp site; and/or the selected second crRNA sequence is configured to cause cleaving at a second cleaving site, within the genome of the subject, that is adjacent to the second ancestral variation or snp site.
  • the selected first crRNA sequence is configured to cause cleaving only at the first cleaving site; and/or the selected second crRNA sequence is configured to cause cleaving only at the second cleaving site.
  • the selected first crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the first target sequence in trans with the diseasecausing mutation or SNP, said first target sequence not being adjacent to the 5 ’-end of a PAM; and/or the selected second crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence not being adjacent to the 5 ’-end of a PAM.
  • the selected first crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence not being adjacent to the 5 ’-end of a PAM; and the selected second crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the second target sequence in trans with the diseasecausing mutation or SNP, said second target sequence being adjacent to the 5 ’-end of a PAM.
  • the selected first crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence being adjacent to the 5 ’-end of a PAM; and the selected second crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence not being adjacent to the 5 ’-end of a PAM.
  • selecting the first crRNA sequence includes selecting a crRNA sequence that corresponds to the first target sequence in trans, said first target sequence in trans not being adjacent to the 5 ’-end of a PAM
  • selecting the second crRNA sequence includes selecting a crRNA sequence that corresponds to the second target sequence in trans, said second target sequence in trans not being adjacent to the 5 ’-end of a PAM.
  • selecting the first crRNA sequence includes selecting a crRNA sequence that corresponds to the first target sequence in trans, said first target sequence in trans not being adjacent to the 5 ’-end of a PAM
  • selecting the second crRNA sequence includes selecting a crRNA sequence that corresponds to the second target sequence in trans, said second target sequence in trans being adjacent to the 5 ’-end of a PAM.
  • selecting the first crRNA sequence includes selecting a crRNA sequence that corresponds to the first target sequence in trans, said first target sequence in trans being adjacent to the 5 ’-end of a PAM
  • selecting the second crRNA sequence includes selecting a crRNA sequence that corresponds to the second target sequence in trans, said second target sequence in trans not being adjacent to the 5 ’-end of a PAM.
  • the subjects that can be treated with the methods described herein include, but are not limited to, mammalian subjects such as a mouse, rat, dog, baboon, pig or human.
  • the subject is a human.
  • the methods can be used to treat subjects at least 1 year, 2 years, 3 years, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or 100 years of age.
  • the subject is treated for at least one, two, three, or four diseases.
  • a single or multiple crRNA or sgRNA may be designed to alter or delete nucleotides at more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 and/or fewer than 20, 10, 9, 8, 7, 6, 5, 4 or 3 ancestral variation or snp sites.
  • the methods of preventing, ameliorating, or treating the disease in a subject may comprise administering to the subject an effective amount of the engineered CRISPR/Cas system described herein.
  • effective amount or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein.
  • the specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
  • the administering comprises injecting the engineered CRISPR/Cas system into the subject. In additional embodiments, the administering comprises introducing the engineered CRISPR/Cas system into a cell containing and expressing a DNA molecule having the target sequence as described below.
  • the methods of treating the disease provide a positive therapeutic response with respect to a disease or condition.
  • positive therapeutic response is intended an improvement in the disease or condition, and/or an improvement in the symptoms associated with the disease or condition.
  • the therapeutic effects of the subject methods of treatment can be assessed using any suitable method.
  • the subject methods reduce the amount of a diseaseassociate protein deposition in the subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared to the subject prior to undergoing treatment.
  • the present disclosure is related to engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRj/CRISPR associate protein (Cas) systems for preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject.
  • the CRISPR/Cas may comprise at least one vector comprising a nucleotide molecule encoding Cas nuclease and the sgRNAs and/or crRNAs as described herein.
  • the terms “non-naturally occurring” or “engineered” are used interchangeably and indicate the involvement of the hand of man.
  • nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
  • the Cas nuclease and the sgRNA/crRNA do not naturally occur together.
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA- WOocessed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as “crRNA” herein, or a “spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.
  • a tracr trans-activating CRISPR
  • tracr-mate sequence encompassing a “direct repeat” and a tracrRNA- WOocessed partial direct repeat in the context of an endogenous CRISPR system
  • a guide sequence also referred to as “
  • sgRNA is a combination of at least tracrRNA and crRNA.
  • one or more elements of a CRISPR system are derived from a type II CRISPR system.
  • one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes or Staphylococcus aureus.
  • 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 may refer to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex
  • target sequence may refer to a sequence adjacent to a PAM site, which the guide sequence comprises. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
  • target site refers to a site of the target sequence including both the target sequence and its complementary sequence, for example, in double stranded nucleotides.
  • the target site described herein may mean a first target sequence hybridizing to sgRNA or crRNA of CRISPR/Cas system, and/or a second target sequence adjacent to the 5 ’-end of a PAM.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • 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, 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).
  • 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.”
  • Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • “operably 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 types of such vectors can also be selected for targeting particular types of cells.
  • At least one vector of the engineered CRISPR/Cas system described herein further comprises (a) a first regulatory element operably linked to the sgRNA that hybridizes with the target sequence described herein, and (b) a second regulatory element operably linked to the nucleotide molecule encoding Cas nuclease, wherein components (a) and (b) are located on a same vector or different vectors of the system, the sgRNA targets the target sequence, and the Cas nuclease cleaves the DNA molecule.
  • the target sequence may be a nucleotide sequence complementary to from 16 to 25 nucleotides adjacent to the 5’ end of a PAM.
  • the cell is a eukaryotic cell, or a mammalian or human cell, and the regulatory elements are eukaryotic regulators.
  • the cell is a stem cell described herein.
  • the Cas nuclease is codon-optimized for expression in a eukaryotic cell.
  • the first regulatory element is a polymerase III promoter.
  • the second regulatory element is a polymerase II promoter.
  • the term “regulatory element” is intended to include 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). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • 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).
  • 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 I promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pcJ I promoters (e.g., 1, 2, 3, 4, 5, or more pcJ I promoters), or combinations thereof.
  • pol III promoters include, but are not limited to, U6 and Hl promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cy tomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Bosbart et al. Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl a promoter.
  • RSV Rous sarcoma virus
  • CMV cy tomegalovirus
  • PGK phosphoglycerol kinase
  • enhancer elements such as 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 p-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).
  • the Cas nuclease provided herein may be an inducible Cas nuclease that is optimized for expression in a temporal or cell-type dependent manner.
  • the first regulatory element may be an inducible promoter that can be linked to the Cas nuclease including, but are not limited to, tetracycline-inducible promoters, metallothionein promoters; tetracycline-inducible promoters, methionine-inducible promoters (e.g., MET25, MET3 promoters); and galactose-inducible promoters (GALI, GAL7 and GAL 10 promoters).
  • suitable promoters include the ADH1 and ADH2 alcohol dehydrogenase promoters (repressed in glucose, induced when glucose is exhausted and ethanol is made), the CUP1 metallothionein promoter (induced in the presence of Cu 2+ , Zn 2+ ), the PHO5 promoter, the CYC1 promoter, the HIS3 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the LEU2 promoter, the ENO promoter, the TP1 promoter, and the AOX1 promoter.
  • 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., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).
  • CRISPR clustered regularly interspersed short palindromic repeats
  • Exemplary CRISPR/Cas9 systems, sgRNA, crRNA and tracrRNA, and their manufacturing process and use are disclosed in U.S. Patent No. 8697359, U.S. Patent Application Publication Nos. 20150232882, 20150203872, 20150184139, 20150079681, 20150073041, 20150056705, 20150031134, 20150020223, 20140357530, 20140335620, 20140310830, 20140273234, 20140273232, 20140273231, 20140256046, 20140248702, 20140242700, 20140242699, 20140242664, 20140234972, 20140227787, 20140189896, 20140186958, 20140186919, 20140186843, 20140179770, 20140179006, 20140170753, 20140093913, 20140080216, and W02016049024, all of which are incorporated herein by their entirety.
  • the Cas9 nucleases described herein are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2.
  • the Cas9 nuclease may be a Cas9 homolog or ortholog. Mutant Cas9 nucleases that exhibit improved specificity may also be used (see, e.g., Ann Ran et al. Cell 154(6) 1380-89 (2013), which is herein incorporated by reference in its entirety for all purposes and particularly for all teachings relating to mutant Cas9 nucleases with improved specificity for target nucleic acids).
  • the nucleic acid manipulation reagents can also include a deactivated Cas9 nuclease (dCas9).
  • dCas9 deactivated Cas9 binding to nucleic acid elements alone may repress transcription by sterically hindering RNA polymerase machinery.
  • deactivated Cas may be used as a homing device for other proteins (e.g., transcriptional repressor, activators and recruitment domains) that affect gene expression at the target site without introducing irreversible mutations to the target nucleic acid.
  • dCas9 can be fused to transcription repressor domains such as KRAB or SID effectors to promote epigenetic silencing at a target site.
  • Cas9 can also be converted into a synthetic transcriptional activator by fusion to VP16/VP64 or p64 activation domains.
  • a mutant Type II nuclease referred to as an enhanced Cas9 (eCa9) nuclease
  • eCa9 nuclease is used in place of the wild-type Cas9 nuclease.
  • the enhanced Cas9 has been rationally engineered to improve specificity by weakening non-target binding. This has been achieved by neutralizing positively charged residues within the non-target strand groove (Slaymaker et al., 2016).
  • the Cas nucleases direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the Cas nucleases directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • HDR homology directed repair
  • NHEJ non-homologous end joining
  • the first and/or second PAMs and the Cas nuclease described herein are from Streptococcus or Staphylococcus.
  • the Cas nuclease is Cas9 nuclease.
  • the Cas nuclease is Cpfl nuclease.
  • the first and second PAMs are both from Streptococcus or Staphylococcus.
  • the Cas nuclease is from Streptococcus.
  • the Cas nuclease is from Streptococcus pyogenes, Streptococcus dysgalactiae, Streptococcus canis, Streptococcus equi, Streptococcus iniae, Streptococcus phocae, Streptococcus pseudoporcinus, Streptococcus oralis, Streptococcus pseudoporcinus, Streptococcus infantarius, Streptococcus mutans, Streptococcus agalactiae, Streptococcus caballi, Streptococcus equinus, Streptococcus sp.
  • the Cas nuclease is from Staphylococcus.
  • the Cas nuclease is from Staphylococcus aureus, S. simiae, S.
  • the Cas nuclease is Cas9 nuclease.
  • the Cas9 nuclease excludes Cas9 nuclease from Streptococcus pyogenes.
  • N is any of A, T, G, and C
  • R is A or G
  • W is A or T
  • Y is C or T
  • D is any of A, G, and T
  • V is any of A, C, and G.
  • the Cas9 nuclease comprises an amino acid sequence having at least about 60, 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% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or 8.
  • the nucleotide molecule encoding Cas9 nuclease comprises a nucleotide sequence having at least about 60, 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% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 or 7.
  • Cas9 sgRNA sequence may comprises a sequence having at least about 60, 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% sequence identity with SEQ ID NO: 1 or 5.
  • An exemplary tracrRNA or sgRNA scaffold sequence may comprise a sequence having at least about 60, 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% sequence identity with SEQ ID NO: 2 or 6.
  • the Cas9 nuclease is an enhanced Cas9 nuclease that has one or more mutations improving specificity of the Cas9 nuclease.
  • the enhanced Cas9 nuclease is from a Cas9 nuclease from Streptococcus pyogenes having one or more mutations neutralizing a positively charged groove, positioned between the HNH, RuvC, and PAM -interacting domains in the Cas9 nuclease.
  • the Cas9 nuclease comprises an amino acid sequence having at least about 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% sequence identity with a mutant amino acid sequence of a Cas9 nuclease from Streptococcus pyogenes (e.g., SEQ ID NO: 4) with one or more mutations selected from the group consisting of (i) K855A, (ii) K810A, K1003A and R1060A, and (iii) K848A, K1003A and R1060A.
  • the nucleotide molecule encoding Cas nuclease comprises a nucleotide sequence having at least about 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% sequence identity with a nucleotide sequence encoding the mutant amino acid sequence.
  • the CRISPR/Cas system and the methods using the CRISPR/Cas system described herein alter a DNA sequence by the NHEJ.
  • the CRISPR/Cas system or the vector described herein does not include a repair nucleotide molecule.
  • the methods described herein alter a DNA sequence by the HDR.
  • the CRISPR/Cas system or the vector described herein may further comprise a repair nucleotide molecule.
  • the target polynucleotide cleaved by the Cas nuclease may be repaired by homologous recombination with the repair nucleotide molecule, which is an exogenous template polynucleotide.
  • This repair may result in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide.
  • the repair nucleotide molecule introduces a specific allele (e.g., a wild-type allele) into the genome of one or more cells of the plurality of stem cells upon repair of a Type II nuclease induced DSB through the HDR pathway.
  • the repair nucleotide molecule is a single stranded DNA (ssDNA). In other embodiments, the repair nucleotide molecule is introduced into the cell as a plasmid vector. In some embodiments, the repair nucleotide molecule is 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 105, 105 to 110, 110 to 115, 115 to 120, 120 to 125, 125 to 130, 130 to 135, 135 to 140, 140 to 145, 145 to 150, 150 to 155, 155 to 160, 160 to 165, 165 to 170, 170 to 175, 175 to 180, 180 to 185, 185 to 190, 190 to 195, or 195 to 200 nucleotides in length.
  • ssDNA single stranded DNA
  • the repair nucleotide molecule is 200 to 300, 300, to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1,000 nucleotides in length. In other embodiments, the repair nucleotide molecule is 1,000 to 2,000, 2,000 to 3,000, 3,000 to 4,000, 4,000 to 5,000, 5,000 to 6,000, 6,000 to 7,000, 7,000 to 8,000, 8,000 to 9,000, or 9,000 to 10,000 nucleotides in length.
  • the repair nucleotide molecule may further include a label for identification and sorting of cells described herein containing the specific mutation.
  • exemplary labels that can be included with the repair nucleotide molecule include fluorescent labels and nucleic acid barcodes that are identifiable by length or sequence.
  • the CRISPR/Cas system or the vector described herein may include at least one nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • the sgRNA and the Cas nuclease are included on the same vector or on different vectors.
  • crRNA may refer to a guide sequence that may be a part of an sgRNA in an CRISPR/Cas system.
  • at least one of the first and second crRNA sequences described herein comprises a nucleotide sequence selected from the group consisting of sequences listed in Figures 8-23; and/or at least one of the first and second crRNA sequences comprises a nucleotide sequence selected from the group consisting of sequences listed in Table 3.
  • sgRNA refers to a single guide RNA containing a guide sequence (crRNA sequence).
  • the sgRNA also includes a Cas nuclease-recruiting sequence (tracrRNA).
  • the crRNA sequence may be a sequence that is homologous to a region in the gene of interest and may direct Cas nuclease activity.
  • the crRNA sequence and tracrRNA sequence may not naturally occur together.
  • the sgRNA may be delivered as RNA or by transforming with a plasmid with the sgRNA-coding sequence (sgRNA gene) under a promoter.
  • the tracrRNA sequence may be any sequence for tracrRNA for CRISPR/Cas system known in the art. In some embodiments, the sgRNA includes no tracrRNA.
  • the crRNA hybridizes to at least a part of a target sequence (e.g., target genome sequence), and the crRNA may have a complementary sequence to the target sequence.
  • the target sequence herein is a first target sequence that hybridizes to a second target sequence adjacent to a PAM site described herein.
  • the crRNA may comprise the first target sequence or the second target sequence.
  • the first and second target sequences are located in introns of a target gene. “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • “Substantially complementary” as used herein refers to a degree of complementarity that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%.
  • stringent conditions refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme.
  • a sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence.
  • the crRNA or the guide sequence is about 17, 18, 19, 20, 21, 22, 23 or 24 nucleotide long.
  • the term “about” may refer to a range of values that are similar to the stated reference value.
  • the term “about” refers to a range of values that fall within 15, 10, 9, 8,7, 6, 5, 4, 3, 2, 1 percent or less of the stated reference value.
  • the disease is an autosomal dominant disease.
  • the disease is selected from the group consisting of Acropectoral syndrome, Acute intermittent porphyria, Adermatoglyphia, Albright's hereditary osteodystrophy, Arakawa's syndrome II, Aromatase excess syndrome, Autosomal dominant cerebellar ataxia, Axenfeld syndrome, Benign hereditary chorea, Bethlem myopathy, Birt-Hogg-Dube syndrome, Boomerang dysplasia, Branchio- oto-renal syndrome, Buschke-Ollendorff syndrome, Camurati-Engelmann disease, Central core disease, Collagen disease, Collagenopathy, types II and XI, Congenital distal spinal muscular atrophy, Congenital stromal corneal dystrophy, Costello syndrome, Cur
  • the number of the trinucleotide repeat of cytosine-adenine-guanine (CAG) at the end of the huntingtin gene (also called HTT or HD gene) located at 4pl6.3 is associated with the Huntington’s disease. Persons with more than 40 repeats may develop the Huntington’s disease during a normal lifetime, while persons with more than 60 repeats may develop juvenile Huntington’s disease, which begins in childhood or adolescence and has a faster progression.
  • the HTT gene is associated with the expression of huntingtin protein, which plays an important role in neurons. By utilizing the method described herein, in a heterozygote patient, only the HTT gene that contains excessive (e.g., more than 35 repeats, more than 40 repeats, or more than 60 repeats) CAG repeats is cleaved.
  • a mutation in the adenomatous polyposis coli (APC) gene located at 5q21 is associated with the Gardner’s syndrome, which shows an increased risk of colon cancer.
  • a large portion of the mutations occur between amino acid 1061 and amino acid 1513.
  • a “corneal dystrophy” refers to any one of a group of hereditary disorders in the outer layer of the eye (cornea).
  • the corneal dystrophy may be characterized by bilateral abnormal deposition of substances in the cornea.
  • Corneal dystrophies include, but are not limited to the following four IC3D categories of corneal dystrophies (see, e.g., Weiss et al., Cornea 34(2): 117-59 (2015)): epithelial and sub-epithelial dystrophies, epithelial-stromal TGF
  • the corneal dystrophy is selected from the group consisting of Epithelial basement membrane dystrophy (EBMD), Meesmann corneal dystrophy (MECD), Thiel-Behnke corneal dystrophy (TBCD), Lattice corneal dystrophy (LCD), Granular corneal dystrophy (GCD), and Schnyder corneal dystrophy (SCD).
  • EBMD Epithelial basement membrane dystrophy
  • MECD Meesmann corneal dystrophy
  • Thiel-Behnke corneal dystrophy Thiel-Behnke corneal dystrophy
  • LCD Lattice corneal dystrophy
  • GCD Granular corneal dystrophy
  • SCD Schnyder corneal dystrophy
  • the corneal dystrophy is caused by one or more mutations, including SNP, is located in a gene selected from the group consisting of Transforming growth factor, beta-induced (TGFBI), keratin 3 (KRT3), keratin 12 (KRT12), GSN, and UbiA prenyltransferase domain containing 1 (UBIAD1).
  • TGFBI beta-induced
  • KRT3 keratin 3
  • KRT12 keratin 12
  • GSN GSN
  • UbiA prenyltransferase domain containing 1 UbiA prenyltransferase domain containing 1
  • a mutant sequence comprising the mutation or SNP site encodes a mutant protein selected from the group consisting of (i) mutant TGFBI proteins comprising a mutation corresponding to Leu509Arg, Arg666Ser, Gly623Asp, Arg555Gln, Argl24Cys, Val505Asp, Ile522Asn, Leu569Arg, His572Arg, Arg496Trp, Pro501Thr, Arg514Pro, Phe515Leu, Leu518Pro, Leu518Arg, Leu527Arg, Thr538Pro, Thr538Arg, Val539Asp, Phe540del, Phe540Ser, Asn544Ser, Ala546Thr, Ala546Asp, Phe547Ser, Pro551Gln, Leu558Pro, His572del, Gly594Val, Val613del, Val613Gly, Met619Lys, Al
  • mutant KRT3 proteins comprising a mutation corresponding to Glu498Val, Arg503Pro, and/or Glu509Lys in Keratin 3 protein, for example, of Protein Accession No. P12035 or NP 476429.2;
  • mutant KRT12 proteins with Metl29Thr, Metl29Val, Glnl30Pro, Leul32Pro, Leul32Va, Leul32His, Asnl33Lys, Argl35Gly, Argl35Ile, Argl35Thr, Argl35Ser, Alal37Pro, Leul40Arg, Vall43Leu, Vall43Leu, Lle391_Leu399dup, He 426Val, He 426Ser, Tyr429Asp, Tyr429Cys, Arg430Pro, and/or Leu433Arg in KRT12, for example, of Protein Accession No.
  • mutant GSN proteins with Asp214Tyr in GSN for example, of Protein Accession No. P06396
  • mutant UBIAD1 proteins comprising a mutation corresponding to Ala97Thr, Gly98Ser, AsnlO2Ser, Aspl l2Asn, Aspl l2Gly, Aspll8Gly, Argll9Gly, Leul21Val, Leul21Phe, Vall22Glu, Vall22Gly, Serl71Pro, Tyrl74Cys, Thrl75Ile, Glyl77Arg, Lysl81Arg, Glyl86Arg, Leul88His, Asn232Ser, Asn233His, Asp236Glu, and/or Asp240Asn in UBIAD1, for example, of Protein Accession No.
  • a mutant sequence comprising the mutation or SNP site encodes at least a part of mutant TGFBI protein mutated by replacing Leu with Arg at amino acid position corresponding the amino acid position 509 of Protein Accession No. Q15582.
  • a mutation at the mutation or SNP site may be responsible for encoding the mutant amino acid at amino acid position corresponding the amino acid position 509 of Protein Accession No. Q15582.
  • a mutation “corresponding to” a particular mutation in a human protein may include a mutation in a different species that occur at the corresponding site of the particular mutation of the human protein.
  • a mutant protein when a mutant protein is described to include a particular mutant, for example, of Leu509Arg, such a mutant protein may comprise any mutation that occurs at a mutant site corresponding to the particular mutant in a relevant human protein, for example, in TGFBI protein of Protein Accession No. Q15582 as described herein.
  • the corneal dystrophy target nucleic acid is a TGFpi target nucleic acid.
  • the corneal dystrophy target nucleic acid is a COL4A1-4, LOX, SPARC, LRRN1, HGF, AKAP13, ZNF469, ATG12P2, GS1-256O22.5, PLEKHA6, APOL4, SLC44A3, SLC6A18, SLC29A3, RANBP3L, KCNMA1, MUC5AC, CROCC, ATHL1, or PLP1 target nucleic acid.
  • the nucleic acid mutation encodes for an amino acid substitution of arginine 124, arginine 555, or histidine 666 in a TGFpi polypeptide.
  • the nucleic acid mutation encodes for an amino acid substitution selection from R124C, R124H, R124L, R555W, R555Q, and H626P. In some embodiments, the nucleic acid mutation encodes for amino acid substitution Q1334H in COL4A1. In some embodiments, the nucleic acid mutation encodes for amino acid substitution G683A in COL4A2. In some embodiments, the nucleic acid mutation encodes for amino acid substitution P718S in COL4A2. In some embodiments, the nucleic acid mutation encodes for amino acid substitution R517K in COL4A2. In some embodiments, the nucleic acid mutation encodes for amino acid substitution D326Y in COL4A3.
  • the nucleic acid mutation encodes for amino acid substitution H451R in COL4A3. In some embodiments, the nucleic acid mutation encodes for amino acid substitution V1327M in COL4A4. In some embodiments, the nucleic acid mutation encodes for amino acid substitution R158Q in LOX. In some embodiments, the nucleic acid mutation encodes for amino acid substitution A1046T in AKAP13. In some embodiments, the nucleic acid mutation encodes for amino acid substitution G624V in AKAP13. In some embodiments, the nucleic acid mutation encodes for amino acid substitution G2358R in ZNF469. In some embodiments, the nucleic acid mutation encodes for amino acid substitution S158F in SLC29A3. In some embodiments, the nucleic acid mutation encodes for amino acid substitution P4493S in MUC5AC. In some embodiments, the nucleic acid mutation encodes for amino acid substitution P370S in CROCC.
  • the subject has corneal opacity. In some embodiments, the subject is a suitable candidate for LASIK version correction.
  • the present disclosure is also related to methods of altering expression of at least one gene product comprising introducing the engineered CRISPR/Cas system described herein into a cell containing and expressing a DNA molecule having a target sequence and encoding the gene product.
  • the engineered CRISPR/Cas system can be introduced into cells using any suitable method.
  • the introducing may comprise administering the engineered CRISPR/Cas system described herein to cells in culture, or in a host organism.
  • Exemplary methods for introducing the engineered CRISPR/Cas system include, but are not limited to, transfection, electroporation and viral-based methods.
  • the one or more cell uptake reagents are transfection reagents.
  • Transfection reagents include, for example, polymer based (e.g., DEAE dextran) transfection reagents and cationic liposome-mediated transfection reagents. Electroporation methods may also be used to facilitate uptake of the nucleic acid manipulation reagents.
  • the engineered CRISPR/Cas system also may be delivered through viral transduction into the cells. Suitable viral delivery systems include, but are not limited to, adeno-associated virus (AAV), retroviral and lentivirus delivery systems. Such viral delivery systems are useful in instances where the cell is resistant to transfection.
  • AAV adeno-associated virus
  • Methods that use a viral-mediated delivery system may further include a step of preparing viral vectors encoding the nucleic acid manipulation reagents and packaging of the vectors into viral particles.
  • Other method of delivery of nucleic acid reagents include, but are not limited to, lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of nucleic acids. See, also Neiwoehner et al., Nucleic Acids Res. 42:1341-1353 (2014), and U.S. Patent Nos.
  • non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).
  • the cells that have undergone a nucleic acid alteration event can be isolated using any suitable method.
  • the repair nucleotide molecule further comprises a nucleic acid encoding a selectable marker.
  • successful homologous recombination of the repair nucleotide molecule a host stem cell genome is also accompanied by integration of the selectable marker.
  • the positive marker is used to select for altered cells.
  • the selectable marker allows the altered cell to survive in the presence of a drug that otherwise would kill the cell.
  • selectable markers include, but are not limited to, positive selectable markers that confer resistance to neomycin, puromycin or hygromycin B.
  • a selectable marker can be a product that allows an altered cell to be identified visually among a population of cells of the same type, some of which do not contain the selectable marker.
  • selectable markers include, but are not limited to the green fluorescent protein (GFP), which can be visualized by its fluorescence; the luciferase gene, which, when exposed to its substrate luciferin, can be visualized by its luminescence; and P-galactosidase (P-gal), which, when contacted with its substrate, produces a characteristic color.
  • GFP green fluorescent protein
  • P-gal P-galactosidase
  • selectable markers are well known in the art and the nucleic acid sequences encoding these markers are commercially available (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989). Methods that employ selectable markers that can be visualized by fluorescence may further be sorted using Fluorescence Activated Cell Sorting (FACS) techniques. Isolated manipulated cells may be used to establish cell lines for transplantation. The isolated altered cells can be cultured using any suitable method to produce a stable cell line.
  • FACS Fluorescence Activated Cell Sorting
  • the present disclosure is related to methods of treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject in need thereof, comprising: (a) obtaining a plurality of stem cells comprising a nucleic acid mutation in a corneal dystrophy target nucleic acid from the subject; (b) manipulating the nucleic acid mutation in one or more stem cells of the plurality of stem cells to correct the nucleic acid mutation, thereby forming one or more manipulated stem cells; (c) isolating the one or more manipulated stem cells; and (d) transplanting the one or more manipulated stem cells into the subject, wherein manipulating the nucleic acid mutation in the one or more stem cells of the plurality of stem cells includes performing any of the methods of altering expression of a gene product or of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject as described herein.
  • SNP single-n
  • the subject methods may include obtaining a plurality of stem cells. Any suitable stem cells can be used for the subject method, depending on the type of the disease to be treated.
  • the stem cell is obtained from a heterologous donor.
  • the stem cells of the heterologous donor and the subject to be treated are donor-recipient histocompatible.
  • autologous stem cells are obtained from the subject in need of the treatment for the disease. Obtained stem cells carry a mutation in a gene associated with the particular disease to be treated.
  • Suitable stem cells include, but are not limited to, dental pulp stem cells, hair follicle stem cells, mesenchymal stem cells, umbilical cord lining stem cells, embryonic stem cells, oral mucosal epithelial stem cells and limbal epithelial stem cells.
  • Stem cells to be manipulated may include individual isolated stem cells or stem cells from a stem cell line established from the isolated stem cells. Any suitable genetic manipulation method may be used to correct the nucleic acid mutation in the stem cells.
  • kits comprising the CRISPR/Cas system for the treatment of a disease associated with a gene mutation or single-nucleotide polymorphism (SNP).
  • the kit includes one or more sgRNAs described herein, a Cas nuclease and a repair nucleotide molecule that includes a wild-type allele of the mutation to be repaired as described herein.
  • the kit also includes agents that facilitate uptake of the nucleic acid manipulation by cells, for example, a transfection agent or an electroporation buffer.
  • the subject kits provided herein include one or more reagents for the detection or isolation of stem cells, for example, labeled antibodies for one or more positive stem cell markers that can be used in conjunction with FACS.
  • the present disclosure is related to an sgRNA pair, and a kit comprising the sgRNA pair comprising at least two sgRNAs for CRISPR/Cas system to silence a disease-causing mutation or SNP, for example, for preventing, ameliorating or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP).
  • the sgRNA pair comprises an sgRNA comprising a guide sequence for PAM-generating ancestral variation or snp in a target gene, for example, in an intron in cis with a disease-causing mutation or SNP.
  • the sgRNA pair comprises an sgRNA comprising a common guide sequence for PAM generating an ancestral SNP in intronic regions of a target gene.
  • the present disclosure is related to an sgRNA pair designed for CRISPR/Cas system, the sgRNA pair comprising (i) a first sgRNA comprising (a) a first crRNA sequence for a first protospacer adjacent motif (PAM) generating mutation or single-nucleotide polymorphism (SNP) at 3 ’-end side of a disease-causing mutation or SNP in cis, and (b) a tracrRNA sequence, in which the first crRNA sequence and the tracrRNA sequence do not naturally occur together; (ii) a second sgRNA comprising (a) a second crRNA guide sequence for a second PAM generating mutation or SNP at 5 ’-end side of the disease-causing mutation or SNP in cA; (b) a tracrRNA sequence, in which the second crRNA sequence and the tracrRNA sequence do not naturally occur together.
  • a first sgRNA comprising (a) a first crRNA sequence for a first protospacer adjacent
  • the method described herein comprises diagnosing the diseases described herein.
  • diagnostic testing is employed to determine one or more genetic conditions by detection of any of a variety of mutations.
  • diagnostic testing is used to confirm a diagnosis when a particular condition is suspected based on for example physical manifestations, signs and/or symptoms as well as family history information.
  • the nucleic acids obtained by the disclosed methods are useful in a variety of diagnostic tests, including tests for detecting mutations such as deletions, insertions, transversions and transitions.
  • diagnostics are useful for identifying unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to be expressed, identifying unaffected individuals who carry one copy of a gene for a disease in which the information could find use in developing a treatment regimen, preimplantation genetic diagnosis, prenatal diagnostic testing, newborn screening, genealogical DNA test (for genetic genealogy purposes), presymptomatic testing for predicting adult-onset disorders such as Huntington's disease, presymptomatic testing for estimating the risk of developing adult-onset cancers and Alzheimer's disease, confirmational diagnosis of a symptomatic individual, and/or forensic/identity testing.
  • the diseases described herein includes corneal dystrophy, for example through detection of Avellino corneal dystrophy-related SNPs, such as those that result in R124 mutations in the TGFBI gene (including for example but not limited to an R124H mutation caused by a G to A transition at nucleotide 418 of TGFBI gene also referred to as a C(G/A)C SNP).
  • Avellino corneal dystrophy-related SNPs such as those that result in R124 mutations in the TGFBI gene (including for example but not limited to an R124H mutation caused by a G to A transition at nucleotide 418 of TGFBI gene also referred to as a C(G/A)C SNP).
  • newborn screening includes any genetic screening employed just after birth in order to identify genetic disorders.
  • newborn screening finds use in the identification of genetic disorders so that a treatment regimen is determined early in life. Such tests include but are not limited to testing infants for phenylketonuria and congenital hypothyroidism.
  • carrier testing is employed to identify people who carry a single copy of a gene mutation.
  • the mutation when present in two copies, the mutation can cause a genetic disorder.
  • one copy is sufficient to cause a genetic disorder.
  • the presence of two copies is contra-indicated for a particular treatment regimen, such as the presence of the Avellino mutation and pre-screening prior to performing surgical procedures in order to ensure the appropriate treatment regiment is pursued for a give patient.
  • such information is also useful for individual contemplating procreation and assists individuals with making informed decisions as well as assisting those skilled in the medical arts in providing important advice to individual patients.
  • predictive and presymptomatic types of testing are used to detect gene mutations associated with a variety of disorders. In some cases, these tests are helpful to people who have a family member with a genetic disorder, but who may exhibit no features of the disorder at the time of testing.
  • predictive testing identifies mutations that increase a person's chances of developing disorders with a genetic basis, including for example but not limited to certain types of cancer.
  • presymptomatic testing is useful in determining whether a person will develop a genetic disorder, before any physical signs or symptoms appear. The results of predictive and presymptomatic testing provide information about a person’s risk of developing a specific disorder and help with making decisions about an appropriate medical treatment regimen described herein.
  • Predictive testing is also employed, in some embodiments, to detect mutations which are contra-indicated with certain treatment regimens, such as the presence of the Avellino mutation being contra-indicated with performing laser eye surgery, such as a refractive surgery (e.g., LASIK, LASEK, PTK, and PRK).
  • a refractive surgery e.g., LASIK, LASEK, PTK, and PRK.
  • LASIK LASIK, LASEK, PTK, and PRK
  • Mutation analysis Mutations associated with various corneal dystrophies were analyzed to determine which were solely caused by missense mutations or in-frame indels. This analysis indicates that for the majority of KI 2 and TGFBI disease, nonsense or frameshifting indel mutations are not associated with disease. Furthermore, an analysis of the exome variant database confirmed that any naturally occurring nonsense, frameshifting indels or splice site mutations found in these genes are not reported to be associated with disease in these individuals.
  • Table 2 Genes and their associated corneal dystrophies that are suitable for a CRISPR/Cas mediated approach.
  • An investigation of the suitable corneal dystrophy genes was conducted to determine the number of mutations targetable by either a PAM-specific approach or a guide allele-specific approach.
  • a PAM-specific approach requires the disease causing SNP to generate a novel PAM, whilst the allele specific approach involves the design of a guide containing the disease causing SNP. All non-disease causing SNPs in TGFBI that generate a novel PAM with a minor allele frequency (MAF) of >10% were identified and analyzed by the Benchling’s online genome-editing design tool.
  • MAF minor allele frequency
  • SNPs with a MAF of >10% may provide a reasonable chance that the SNP resulting in a novel PAM will be found in cis with the disease causing mutation.
  • Being “in cis” with the disease causing mutation refers to being on the same molecule of DNA or chromosome as the disease -causing mutation.
  • the SNP resulting in a novel PAM may be found, for example, in intron or exon in TGFBI gene in cis with the disease-causing mutation. All variants within TGFBI were analyzed to determine whether a novel PAM was created (Table 3).
  • a CRISPR Cas system may target more than one patient or one family with a mutation.
  • One CRISPR/Cas system designed in this way may be used to treat a range of TGFBI mutations.
  • the CRISPR/Cas system may employ an sgRNA adjacent to a PAM site located in the flanking intron that is common to both wild-type and mutant alleles in tandem with a sgRNA adjacent to a PAM site that is specific to the mutant allele ( Figure 16).
  • EBV transformation of lymphocytes A sample of 5ml of whole blood was taken and place in a sterile 50ml Falcon tube. An equal volume of RPMI media containing 20% foetal calf serum was added to the whole blood - mix by gently inverting the tube. 6.25ml of Ficoll-Paque PLUS (GE Healthcare cat no. 17-1440-02) was placed in a separate sterile 50ml Falcon tube. 10 ml of blood/media mix was added to the Ficoll-Paque. The tube was spun at 2000 rpm for 20 min at room temperature. The red blood cells formed at the bottom of the tube above which was the Ficoll layer.
  • the lymphocytes formed a layer on top of the Ficoll layer, while the top layer was the medium.
  • a clean sterile Pastette was inserted to draw off the lymphocytes, which were placed in a sterile 15ml Falcon tube. The lymphocytes were centrifuged and washed. EBV aliquot was thawed and added to resuspended lymphocytes, and the mixture was incubated for 1 hour at 37 degrees C (infection period). RPMI, 20% FCS media and Img/ml phytohaemagglutinin were added to EBV treated lymphocytes, and the lymphocytes were placed on a 24- well plate.
  • EBV Transformed Lymphocytes CRISPR constructs (with either GFP or mCherry co-expressed) were added to suspended EBV transformed lymphocytes cells, and the mixture was transferred to an electroporation cuvette. Electroporation was performed, and 500pl pre-warmed RPMI 1640 media containing 10% FBS was added to the cuvette. The contents of the cuvette was transferred to a 12 well plate containing the remainder of the pre-warmed media, and 6 hours post nucleofection, 1ml of media was removed and was replaced with fresh media.
  • CRISPR constructs with either GFP or mCherry co-expressed

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Abstract

The present disclosure relates to methods of use thereof for gene editing or for preventing, ameliorating or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject, the method including detecting phase of SNPs in cis with the gene mutation or SNP associated with the disease in the subject by droplet digital polymerase chain reaction (PCR).

Description

CRISPR GENE EDITING FOR DISEASES ASSOCIATED WITH A GENE MUTATION OR SINGLE- NUCLEOTIDE POLYMORPHISM (SNP)
RELATED APPLICATIONS
[0001] This application claims the benefit of, and priority to, U.S. Provisional Patent Application Serial No. 63/245,984, filed September 20, 2021, which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The present disclosure relates to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) systems, and methods of use thereof for gene editing or for preventing, ameliorating or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject.
BACKGROUND OF THE INVENTION
[0003] The discovery of a simple endogenous bacterial system for catalytically cleaving doublestranded DNA has revolutionized the field of therapeutic gene editing. The Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) is a programmable RNA guided endonuclease, which has recently been shown to be effective at gene editing in mammalian cells (Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR- Cas9 for genome engineering. Cell 2014; 157: 1262-1278). This highly specific and efficient RNA- guided DNA endonuclease may be of therapeutic importance in a range of genetic diseases. The CRISPR/Cas9 system relies on a single catalytic protein, Cas9 that is guided to a specific DNA sequence by 2 RNA molecules; the tracrRNA and the crRNA (Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 157: 1262- 1278). Combination of the tracrRNA/crRNA into a single guide RNA molecule (sgRNA) (Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 2014; 343: 84-87; Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science 2014; 343: 80-84) has led to the rapid development of gene editing tools potentially specific for any target within the genome. Through the substitution of a nucleotide sequence within the sgRNA, to one complimentary to a chosen target, a highly specific system may be generated in a matter of days. One caveat of this system is that the endonuclease requires a protospacer adjacent motif (PAM), located immediately at the 3' end of the sgRNA binding site. This PAM sequence is an invariant part of the DNA target but not present in the sgRNA, while its absence at the 3' end of the genomic target sequence results in the inability of the Cas9 to cleave the DNA target (Westra ER, Semenova E, Datsenko KA, Jackson RN, Wiedenheft B, Severinov K et al. Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition. PLoS Genet 2013; 9: el003742). This distinction is important as the mutation directly in a PAM-specific approach, or nearby SNPs may be targeted. One SNP allele will represent a PAM site, while the other allele does not. This allows us to discriminate between the two chromosomes.
[0004] The mutation-independent CRISPR method developed (Christie et al., Mol. Ther, 2020) relies on determining the phase of patient-specific SNPs in relation to the disease-causing mutation (Mutation-Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease. Mol Ther 2020;28(8). Doi: 10.1016/j.ymthe.2020.05.002). The allelespecific SNP/SNPs targeted by the gRNA are on the same allele as the disease-causing mutation to remove the mutant allele and leave the wildtype allele untouched. Once phase is determined, the treatment can be tailored to the individual, by selecting two validated guide RNAs (gRNAs) from a pool of guides targeting the SNP/s and where necessary, a common intronic SNP. At present, however, the 10X Genomics method for this analysis is costly and covers the entire genome rather than just the affected genomic region. There is also bioinformatic analysis to be completed after the sequencing. Thus, a more efficient method of identifying the SNPs in cis and selecting sgRNAs is desired.
SUMMARY OF THE INVENTION
[0005] In one aspect, the present disclosure describes the potential of utilizing the PAM-generating mutations in introns of a disease causing gene. For example, the PAM-generating mutations are in adjacent introns of a gene having a disease-causing mutation, and the disease-causing mutation is in exon in between the adjacent introns.
[0006] By utilizing guide sequences that bind adjacent to the PAM sequences in the introns, Cas nuclease may cleave a gene at two intronic sites, between which an exon containing a disease-causing mutation exists, thereby eliminating the disease-causing exon and knocking out the mutated allele. In another aspect, the CRISPR/Cas system utilizing the PAM-generating mutations or SNPs in introns may be used to treat a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject, for example, including an autosomal dominant disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject.
[0007] In one aspect, the present disclosure is related to methods of identifying the PAM-generating mutations or SNPs in introns using droplet digital polymerase chain reaction (PCR).
[0008] In one aspect, the present disclosure is related to methods of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject, the method comprising detecting phase of SNPs in cis with the gene mutation or SNP associated with the disease in the subject by droplet digital polymerase chain reaction (PCR), and administering to the subject an engineered CRISPR/Cas system.
[0009] In some embodiments, the detecting comprises preparing at least 10,000 droplets, each comprising a first labeled probe for the gene mutation or SNP and a second labeled probe for a SNP that is in cis with the gene mutation or SNP. In additional embodiments, the first and second probes are labeled with different fluorescent dyes. [00010] In some embodiments, the methods further comprise detecting the gene mutation or SNP in the subject prior to detecting phase of SNPs. In some embodiments, the methods further comprise diagnosing the disease in the subject prior to detecting phase of SNPs. In some embodiments, the detecting phase of SNPs excludes sequencing a full genome in a sample from the subject. In some embodiments, the methods further comprise obtaining a sample form the subject, and the detecting phase of SNPs from the sample.
[00011] In some embodiments, the administering comprises administering to the subject an engineered CRISPR/Cas system comprising at least one vector comprising at least two different CRISPR targeting RNA (crRNA) sequences or single guide RNA (sgRNA) sequences. In one aspect, the present disclosure is related to methods of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject, comprising administering to the subject an engineered CRISPR/Cas system comprising at least one vector comprising (i) a nucleotide molecule encoding Cas nuclease; (ii) a first sgRNA comprising a first crRNA sequence that hybridizes to a nucleotide sequence complementary to a first target sequence, the first target sequence being adjacent to (e.g., the 5 ’-end of) a first protospacer adjacent motif (PAM) at the 3 ’-end side of a disease-causing mutation or SNP in cis, wherein the first target sequence or the first PAM comprises a first ancestral variation or SNP site; and (iii) a second sgRNA comprising a second crRNA sequence that hybridizes to a nucleotide sequence complementary to a second target sequence, the second target sequence being adjacent to (e.g., the 5 ’-end of) a second PAM at the 5’ side of the disease-causing mutation or SNP in cis. The second target sequence or the second PAM comprises a second ancestral variation or SNP site. The at least one vector does not have a nucleotide molecule encoding Cas nuclease and a sgRNA sequence that naturally occur together. [00012] In some embodiments, the disease is an autosomal dominant disease. In additional embodiments, the disease is selected from the group consisting of Acropectoral syndrome, Acute intermittent porphyria, Adermatoglyphia, Albright's hereditary osteodystrophy, Arakawa's syndrome II, Aromatase excess syndrome, Autosomal dominant cerebellar ataxia, Axenfeld syndrome, Benign hereditary chorea, Bethlem myopathy, Birt-Hogg-Dube syndrome, Boomerang dysplasia, Branchio- oto-renal syndrome, Buschke-Ollendorff syndrome, Camurati-Engelmann disease, Central core disease, Collagen disease, Collagenopathy, types II and XI, Congenital distal spinal muscular atrophy, Congenital stromal corneal dystrophy, Costello syndrome, Currarino syndrome, Darier's disease, Glutl deficiency, Dentatorubral-pallidoluysian atrophy, Dermatopathia pigmentosa reticularis, Dysfibrinogenemia, Transthyretin-related hereditary amyloidosis, Familial atrial fibrillation, Familial hypercholesterolemia, Familial male-limited precocious puberty, Feingold syndrome, Felty's syndrome, Flynn-Aird syndrome, Gardner's syndrome, Gillespie syndrome, Gray platelet syndrome, Greig cephalopoly syndactyly syndrome, Hajdu-Cheney syndrome, Hawkinsinuria, Hay-Wells syndrome, Hereditary elliptocytosis, Hereditary hemorrhagic telangiectasia, Hereditary mucoepithelial dysplasia, Hereditary spherocytosis, Holt-Oram syndrome, Huntington's disease, Huntington's disease-like syndrome, Hypertrophic cardiomyopathy, Hypoalphalipoproteinemia, Hypochondroplasia, Hypodysfibrinogenemia, Jackson-Weiss syndrome, Keratolytic winter erythema, Kniest dysplasia, Kostmann syndrome, Langer-Giedion syndrome, Larsen syndrome, Liddle's syndrome, Marfan syndrome, Marshall syndrome, Medullary cystic kidney disease, Metachondromatosis, Miller-Dieker syndrome, MOMO syndrome, Monilethrix, MonoMAC, Multiple endocrine neoplasia, Multiple endocrine neoplasia type 1, Multiple endocrine neoplasia type 2, Multiple endocrine neoplasia type 2b, Myelokathexis, Myotonic dystrophy, Naegeli-Franceschetti- Jadassohn syndrome, Nail-patella syndrome, Noonan syndrome, Oculopharyngeal muscular dystrophy, Pachyonychia congenital, Pallister-Hall syndrome, PAPA syndrome, Papillorenal syndrome, Parastremmatic dwarfism, Pelger-Huet anomaly, Peutz-Jeghers syndrome, Piebaldism, Platyspondylic lethal skeletal dysplasia, Torrance type, Polydactyly, Popliteal pterygium syndrome, Porphyria cutanea tarda, Pseudoachondroplasia, RASopathy, Reis-Bucklers corneal dystrophy, Romano-Ward syndrome, Rosselli-Gulienetti syndrome, Roussy-Levy syndrome, Rubinstein-Taybi syndrome, Saethre-Chotzen syndrome, Schmitt Gillenwater Kelly syndrome, Short QT syndrome, Singleton Merten syndrome, Spinal muscular atrophy with lower extremity predominance, Spinocerebellar ataxia, Spinocerebellar ataxia type 1, Spinocerebellar ataxia type 6, Spondyloepimetaphyseal dysplasia- Strudwick type, Spondyloepiphyseal dysplasia congenital, Spondyloperipheral dysplasia, Stickler syndrome, Tietz syndrome, Timothy syndrome, Treacher Collins syndrome, Tricho-dento-osseous syndrome, Tuberous sclerosis, Upington disease, Variegate porphyria, Vitelliform macular dystrophy, Von Hippel-Lindau disease, Von Willebrand disease, Wallis-Zieff-Goldblatt syndrome, WHIM syndrome, White sponge nevus, Worth syndrome, Zaspopathy, Zimmermann-Laband syndrome, and Zori-Stalker-Williams syndrome. In yet additional embodiments, the disease is an autosomal dominant disease of an eye. In further embodiments, the disease may include or excludes corneal dystrophy. In some embodiments, the corneal dystrophy is associated with R124H granular corneal dystrophy type 2 mutation.
[00013] In some embodiments, the disease-causing mutation or SNP is in an exon of a gene causing the disease. In further embodiments, the first and second PAMs are in different introns surrounding one or more exons containing the disease-causing mutation or SNP.
[00014] In some embodiments, the first PAM comprises the first ancestral variation or SNP site and/or the second PAM comprises the second ancestral variation or SNP site. In some embodiments, the first crRNA sequence comprises the first target sequence, and the second crRNA sequence comprises the second target sequence. In further embodiments, the first crRNA sequence is from 17 to 24 nucleotide long; and/or the second crRNA sequence is from 17 to 24 nucleotide long.
[00015] In some embodiments, the first and/or second PAMs and the Cas nuclease are from Streptococcus or Staphylococcus. In additional embodiments, the first and second PAMs are both from Streptococcus or Staphylococcus. In some embodiments, the Cas nuclease is Cas9 nuclease. In some embodiments, each of the first and second PAMs independently consists of NGG or NNGRRT, wherein N is any of A, T, G, and C, and R is A or G. In some embodiments, the Cas nuclease is Cpfl nuclease. In some embodiments, the Cas nuclease is selected from the group consisting of: Cas9 nuclease, Cpfl nuclease (also known as Cas 12a nuclease), C2cl nuclease (also known as Cas 12b nuclease), C2c2 nuclease (also known as Casl3al nuclease), C2c3 nuclease (also known as Casl2c nuclease), and Cmsl nuclease. In some embodiments, any other Cas nuclease may be used.
[00016] In some embodiments, the administration comprises injecting the engineered CRISPR/Cas system into the subject. In additional embodiments, the administering comprises introducing the engineered CRISPR/Cas system into a cell containing and expressing a DNA molecule having the target sequence.
[00017] In some embodiments, the disease is associated with the SNP; the first target sequence or the first PAM comprises the first ancestral SNP site; and/or the second target sequence or the second PAM comprises the second ancestral SNP site. In additional embodiments, the target sequence or the PAM comprises a plurality of mutation or SNP sites. In some embodiments, the subject is human. [00018] In some embodiments, the methods described herein further comprises, prior to administering to the subject the engineered CRISPR/Cas system, obtaining genomic or sequence information of the subject; and selecting the first crRNA sequence and/or the second crRNA sequence based on the genomic or sequence information of the subject. In additional embodiments, the genomic or sequence information of the subject includes whole or partial genome sequence information of the subject.
[00019] In some embodiments, the first crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves at a first cleaving site that is adjacent to the first ancestral variation or SNP site; and/or the second crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves at a second cleaving site that is adjacent to the second ancestral variation or SNP site. In additional embodiments, the first crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves only at the first cleaving site that is adjacent to the first ancestral variation or SNP site; and/or the second crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves only at the second cleaving site that is adjacent to the second ancestral variation or SNP site. In further embodiments, the first crRNA sequence is configured to reduce cleaving of the genome of the subject at a site other than a first cleaving site compared to other crRNA sequences hybridizing to the nucleotide sequence complementary to the first target sequences; and/or the second crRNA sequence is configured to reduce cleaving of the genome of the subject at a site other than a second cleaving site compared to other crRNA sequences hybridizing to the nucleotide sequence complementary to the second target sequences. In yet further embodiments, the first crRNA sequence is configured to reduce cleaving of a gene, in trans, that corresponds to a gene causing the disease in cis compared to other crRNA sequences hybridizing to the nucleotide sequence complementary to the first target sequences; and/or the second crRNA sequence is configured to reduce cleaving of a gene, in trans, that corresponds to the gene causing the disease in cis compared to other crRNA sequences hybridizing to the nucleotide sequence complementary to the second target sequences.
[00020] In some embodiments, the selected first crRNA sequence is configured to cause cleaving at a first cleaving site, within genome of the subject, that is adjacent to the first ancestral variation or SNP site; and/or the selected second crRNA sequence is configured to cause cleaving at a second cleaving site, within the genome of the subject, that is adjacent to the second ancestral variation or SNP site. In additional embodiments, the selected first crRNA sequence is configured to cause cleaving only at the first cleaving site; and/or the selected second crRNA sequence is configured to cause cleaving only at the second cleaving site. In further embodiments, the first crRNA sequence hybridizes to the nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence in trans not being adjacent to the 5 ’-end of a PAM; and/or the second crRNA sequence hybridizes to the nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence not being adjacent to the 5 ’-end of a PAM.
[00021] In accordance with some embodiments, a method of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject includes administering to the subject an engineered CRISPR/Cas system comprising: (i) a Cas nuclease; (ii) a first sgRNA comprising a first CRISPR targeting RNA (crRNA) sequence that hybridizes to a nucleotide sequence complementary to a first target sequence, the first target sequence being adjacent to a first protospacer adjacent motif (PAM) at 3 ’-end side of a disease-causing mutation or SNP in cis, wherein the first target sequence or the first PAM comprises a first ancestral variation or SNP site; and (iii) a second sgRNA comprising a second crRNA sequence that hybridizes to a nucleotide sequence complementary to a second target sequence, the second target sequence being adjacent to a second PAM at 5 ’-end side of the disease-causing mutation or SNP in cis, wherein the second target sequence or the second PAM comprises a second ancestral variation or SNP site. The Cas nuclease and a crRNA sequence do not naturally occur together in the subject. In some embodiments, the subject is a vertebrate.
[00022] In accordance with some embodiments, an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) system includes (i) a Cas nuclease; (ii) a first sgRNA comprising a first CRISPR targeting RNA (crRNA) sequence that hybridizes to a nucleotide sequence complementary to a first target sequence, the first target sequence being adjacent to a first protospacer adjacent motif (PAM) at 3 ’-end side of a disease-causing mutation or SNP in cis, wherein the first target sequence or the first PAM comprises a first ancestral variation or SNP site; and (iii) a second sgRNA comprising a second crRNA sequence that hybridizes to a nucleotide sequence complementary to a second target sequence, the second target sequence being adjacent to a second PAM at 5 ’-end side of the disease-causing mutation or SNP in cis. The second target sequence or the second PAM comprises a second ancestral variation or SNP site. The Cas nuclease and a crRNA sequence do not naturally occur together in the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[00023] Figure 1 illustrates an example of a sgRNA sequence, nucleotide and amino acid sequences of Cas9 nuclease from Streptococcus pyogenes (Spy) and Staphylococcus aureus (Sau).
[00024] Figure 2 illustrates an example of a dual-cut approach using intronic PAM sites. Two separate guides are introduced, and Cas9 generates a double stranded break (DSB) at two sites. Repair of this doubly cut region will result in an excision of the region between the two breaks. The deletion encompasses the exonic coding region of the gene shown by the yellow boxes in this figure.
[00025] Figure 3 illustrates an embodiment in which a sgRNA utilizing a flanking SNP within the PAM site is designed in the first intron. Additionally, a sgRNA common to both the wild-type and mutant allele is designed in the second intron. In the wild-type allele the single sgRNA causes NHEJ in the second intron, which may have no functional effect. However, in the mutant allele, the sgRNA utilizing the flanking SNP derived PAM and the common sgRNA result in a large deletion that results in a knockout of the mutant allele.
[00026] Figure 4 illustrates all SNPs in TGFBI with a MAF of >10% that generate a novel PAM. The numbered boxes indicate the exons within TGFBI. The hotspots in TGFBI, where multiple diseasecausing mutations are found, are shown by the red boxes. The blue arrows indicate the position of a SNP that generates a novel PAM. The novel PAM is shown for each arrow, with the required variant highlighted in red.
[00027] Figure 5 depicts experimental results from using an exemplary lymphocyte cell line derived from a patient with a R124H granular corneal dystrophy type 2 mutation that was nucleofected with CRISPR/Cas9 and sgRNA. The guide utilized the novel PAM that is generated by the rs3805700 SNP. This PAM is present on the same chromosome as the patients R124H mutation but does not exist on the wild-type chromosome. Following cell sorting, single clones were isolated to determine whether indels had occurred. Six of the single clones had the unedited wild-type chromosome, indicating stringent allele-specificity of this guide. Four of the isolated clones had the mutant chromosome, and three of these exhibited edits indicating a 75% editing efficiency of the mutant chromosome. Two of the three clones exhibited indels that are frame-shifting. Therefore, at least 66.66% of the edits induced gene disruption.
[00028] Figure 6 shows the results from a dual-guide approach. Two CRISPR plasmids were transfected into the LCLs, one tagged with mCherry the other tagged with GFP. Positive cells were sorted for both mCherry and GFP, collecting 2.6% of the total population. The cells were then allowed to repair and expand, and the genomic DNA was isolated.
[00029] Figure 7, on the right, illustrates that using the original clonal isolation of single alleles, a 565bp deletion encompassing both PAM sites was confirmed. The deletion is shown in red with the PAM sites highlighted in blue. On the left, Figure 7 also illustrates the two guides cutting at their target sites, the region between these cuts being excised upon repair, and the genomic region after repair.
[00030] Figures 8-23 illustrate exemplary common guides in intronic regions of TGFBI gene.
[00031] Figure 24 illustrates a flowchart for exemplary personalized gene editing
DETAILED DESCRIPTION OF THE INVENTION
[00032] As used throughout, ranges are used as shorthand for describing each and every value that is within the range. Any value within the range can be selected as the terminus of the range. In addition, all references cited herein are hereby incorporated by reference in their entireties for all purposes. In the event of a conflict in a definition in the present disclosure and that of a cited reference, the present disclosure controls.
[00033] In one aspect, the present disclosure is related to methods of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject, comprising detecting phase of SNPs in cis with the gene mutation or SNP associated with the disease in the subject by droplet digital polymerase chain reaction (ddPCR), and administering to the subject an engineered CRISPR/Cas system.
[00034] The term ddPCR refers to droplet digital polymerase chain reaction. In ddPCR, one or more PCR amplifications are performed, wherein each reaction is separated into a plurality of water-oil emulsion droplets, so that PCR amplification of the target sequence may occur in each individual droplet. The ddPCR may measure absolute quantities by counting nucleic acid molecules encapsulated in discrete, vohimetrically defined, water-in-oil droplet partitions that support PCR amplification (Hinson et al., 2011, Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem.
84: 1003-1011). A single ddPCR reaction may be comprised of at least 20,000 partitioned droplets per well. A “droplet” or “water-in-oil droplet” refers to an individual partition of the droplet digital PCR assay. A droplet supports PCR amplification of template molecule(s) using homogenous assay chemistries and workflows similar to those widely used for real-time PCR applications (Hinson et al., 2011, Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84:1003-1011). Droplets may be read as either positive or negative for specific fluorescent signals and the fraction of positive droplets (and calculations using Poisson statistics), allowing quantification of the target in the sample.
[00035] The digital droplet system may be useful for determining phase of SNPs. A SNP-specific assay may be run in the same ddPCR reaction as the disease-causing mutation specific assay, each with different fluorescent signals. If the SNP lies in cis with the disease-causing mutation, the droplet will be positive for both assays in a significantly higher portion than if they lie on different alleles (A Rapid Molecular Approach for Chromosomal Phasing. PLoS One 2015;10:e0118270. Doi: 10.1371/joumal.pone.0H8270). This may also be checked via restriction digest between the two sites, which will destroy co-partioning. [00036] Allele-specific probes may also be designed, for example, as shown in Mutation- Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease. Mol Ther 2020;28(8). Doi: 10.1016/j.ymthe.2020.05.002 and adapted to the ddPCR platform.
[00037] In some embodiments, detecting phase of SNPs in cis comprises preparing at least 10,000, 15,000, 20,000, or 25,000 droplets, each comprising a first labeled probe for the gene mutation or SNP and a second labeled probe for a SNP that is in cis with the gene mutation or SNP. In some embodiments, the first and second probes are labeled with different fluorescent dyes. In some embodiments, suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes are described in the 1996 Molecular Probes Handbook by Richard P. Haugland. Suitable fluorescent labels also include, but are not limited to, green fluorescent protein (GFP; Chalfie, et al., Science 263(5148): 802-805, 1994); and EGFP; Clontech — Genbank Accession Number U55762), blue fluorescent protein (BFP; Quantum Biotechnologies, Inc.; Stauber, R. H. Biotechniques 24(3):462-471 (1998); Heim, R. and Tsien, R. Y. Curr. Biol. 6: 178-182 (1996)), enhanced yellow fluorescent protein (EYFP; Clontech Laboratories, Inc.), luciferase (Ichiki, et al., J. Immunol. 150(12):5408-5417 (1993)), P-galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85(8):2603-2607 (April 1988)) and Renilla (WO 92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; U.S. Pat. No. 5,292,658; U.S. Pat. No. 5,418,155; U.S. Pat. No. 5,683,888; U.S. Pat. No. 5,741,668; U.S. Pat. No. 5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304; U.S. Pat. No. 5,876,995; and U.S. Pat. No. 5,925,558). In some embodiments, the labels descried herein include: Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.), Sulfo-Cyanine 3, Sulfo-Cyanine 5, Sulfo-Cyanine 5.5, Sulfo-Cyanine 7, Sulfo- Cyanine 7.5 (Lumiprobe, Hunt Valley, MD.). Tandem conjugate protocols for Cy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, Cy7APC are known. Additional labels are available from commercial sources such as BD Biosciences, Beckman Coulter, AnaSpec, Invitrogen, Cell Signaling Technology, Millipore, eBioscience, Santa Cruz Biotech, Abeam, LiCor, and Sigma-Aldrich.
[00038] In another aspect, the methods described herein comprises administering to the subject an engineered CRISPR/Cas system comprising at least one vector comprising (i) a nucleotide molecule encoding Cas nuclease; (ii) a first sgRNA comprising a first CRISPR targeting RNA (crRNA) sequence that hybridizes to a nucleotide sequence complementary to a first target sequence, the first target sequence being adjacent to the 5 ’-end of a first protospacer adjacent motif (PAM) at 3 ’-end side of a disease-causing mutation or SNP in cis, wherein the first target sequence or the first PAM comprises a first ancestral variation or SNP site; and (iii) a second sgRNA comprising a second crRNA sequence that hybridizes to a nucleotide sequence complementary to a second target sequence, the second target sequence being adjacent to the 5 ’-end of a second PAM at 5 ’-end side of the disease-causing mutation or SNP in cis, wherein the second target sequence or the second PAM comprises a second ancestral variation or SNP site, wherein at least one vector does not have a nucleotide molecule encoding Cas nuclease and a crRNA sequence that naturally occur together. In another aspect, the present disclosure is related to methods of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject comprising altering expression of the gene product of the subject by the methods described above, wherein the gene comprises a mutant or SNP mutant sequence. In some embodiments, the disease is associated with the SNP; the first target sequence or the first PAM comprises the first ancestral SNP site; and/or the second target sequence or the second PAM comprises the second ancestral SNP site. In additional embodiments, the target sequence comprises a plurality of mutation or SNP sites. In some embodiments, the subject is human. In some embodiments, the first crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves at a first cleaving site that is adjacent to the first ancestral variation or SNP site; and/or the second crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves at a second cleaving site that is adjacent to the second ancestral variation or SNP site. In additional embodiments, the first crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves only at the first cleaving site; and/or the second crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves only at the second cleaving site.
[00039] As described herein, being “in cis'” with the disease-causing mutation or SNP refers to being on the same molecule of DNA or chromosome as the disease-causing mutation, and being “in trans” with the disease-causing mutation or SNP refers to being on a different molecule of DNA or chromosome as the disease-causing mutation or SNP. In some embodiments, the first crRNA sequence hybridizes to the nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence not being adjacent to the 5 ’-end of a PAM; and/or the second crRNA sequence hybridizes to the nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence not being adjacent to the 5 ’-end of a PAM. In the absence of the PAM adjacent to the first and/or second target sequences, the first and/or the second target sequences in trans with the diseasecausing mutation or SNP may remain intact without any cleavage (e.g., the Cas nuclease does not cleave the first and/or the second target sequences in trans with the disease-causing mutation or SNP). This approach may permit expression of a gene that is in trans with the disease-causing mutation or SNP and does not include a disease-causing mutation or SNP. This approach may also reduce or eliminate any adverse impacts associated with knocking out both the gene that includes the diseasecausing mutation or SNP and the gene that does not include the disease-causing mutation or SNP in a subject. In additional embodiments, the first crRNA sequence hybridizes to the nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence not being adjacent to the 5 ’-end of a PAM; and the second crRNA sequence hybridizes to the nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence being adjacent to the 5 ’-end of a PAM. In the absence of the PAM adjacent to the first target sequence, the first target sequence in trans with the disease-causing mutation or SNP may remain intact without any cleavage while the second target sequence in trans with the disease-causing mutation or SNP may be cleaved (e.g., the Cas nuclease cleaves the first target sequence in trans with the disease-causing mutation or SNP but does not cleave the second target sequence in trans with the disease-causing mutation or SNP). In further embodiments, the first crRNA sequence hybridizes to the nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence being adjacent to the 5 ’-end of a PAM; and the second crRNA sequence hybridizes to the nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence not being adjacent to the 5 ’-end of a PAM. In the absence of the PAM adjacent to the second target sequence, the second target sequence in trans with the diseasecausing mutation or SNP may remain intact without any cleavage while the first target sequence in trans with the disease-causing mutation or SNP is cleaved (e.g., the Cas nuclease cleaves the second target sequence in trans with the disease -causing mutation or SNP but does not cleave the first target sequence in trans with the disease-causing mutation or SNP). Said “nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP” herein has the identical nucleotide sequence as the nucleotide sequence complementary to the first target sequence in cis with the disease-causing mutation or SNP. Said “nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP” and said “the first target sequence in trans with the disease -causing mutation or SNP,” however, may be located on a different molecule of DNA or chromosome where the same disease-causing mutation or SNP is absent (thus are in trans with the disease-causing mutation or SNP). Similarly, said “nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP” herein has the identical nucleotide sequence as the nucleotide sequence complementary to the second target sequence in cis with the disease-causing mutation or SNP. Said “nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP” and said “the second target sequence in trans with the disease-causing mutation or SNP,” however, may be located on a different molecule of DNA or chromosome where the disease-causing mutation or SNP is absent (thus are in trans with the disease-causing mutation or SNP).
[00040] In some embodiments, the engineered CRISPR/Cas system described herein may comprise at least one vector comprising (i) a nucleotide molecule encoding Cas nuclease described herein, and (ii) a plurality of sgRNA targeting intronic sites surrounding one or more exons containing a disease- associate mutation or SNP of interest as described herein. The sgRNA may comprise a target sequence adjacent to the 5 ’-end of a protospacer adjacent motif (PAM), and/or hybridize to a first target sequence complementary to a second target sequence adjacent to the 5’ end of the PAM. The target sequence or the PAM may comprise the ancestral variation or SNP in an intronic site. In additional embodiments, the ancestral variation or SNP in the intronic site does not cause a disease. In some embodiments, sgRNA may comprise a target sequence adjacent to a PAM site located in the flanking intron that is common to both wild-type and mutant alleles in tandem with a sgRNA adjacent to a PAM site that is specific to the mutant allele. In some embodiments, the Cas nuclease and the sgRNA do not naturally occur together. The sequence of this PAM site is specific to the Cas nuclease being used. In additional embodiments, the PAM comprises the mutation or SNP site. In yet additional embodiments, the PAM consists of a PAM selected from the group consisting of NGG and NNGRRT, wherein N is any of A, T, G, and C, and R is A or G.
[00041] In some embodiments, the disease-causing mutation or SNP is in an exon of a gene associated with the disease, and the first and second PAMs are in different introns surrounding one or more exons containing the disease-causing mutation or SNP. As shown in Figures 2 and 3, first and second CRISPR targeting RNA (crRNA) sequences hybridize to nucleotide sequences complementary to first and second target sequences, the first target sequence being adjacent to the 5 ’-end of a first protospacer adjacent motif (PAM) at 3 ’-end side of a disease-causing mutation or SNP in cis, and the second target sequence being adjacent to the 5 ’-end of a first protospacer adjacent motif (PAM) at 5’- end side of a disease-causing mutation or SNP in cis. Thus, the first and second PAMs are located on opposite sides of one or more exons containing the disease-causing mutation or SNP. As used herein, an “intron” means a section of DNA occurring between two adjacent exons within a gene which is removed during pre-mRNA splicing and does not code for any amino acids constituting the gene product. An “intronic site” is a site within an intron. An “exon” means a section of DNA occurring in a gene which codes for one or more amino acids in the gene product. For example, the constitutively spliced exon known so far has 6 nucleotides or more, and the alternatively spliced exon has 3 nucleotides or more, which is equivalent to 1 or 2 amino acids or more depending on the frame that the mRNA is read in. An “exonic site” is a site within an exon.
[00042] In some embodiments, the first PAM comprises the first mutation or SNP site and/or the second PAM comprises the second mutation or SNP site. In some embodiments, the first crRNA sequence comprises the first target sequence, and the second crRNA sequence comprises the second target sequence. In further embodiments, each of the first crRNA sequence and the second crRNA sequence may independent be from 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotide long.
[00043] In some embodiments, the methods described herein further comprise identifying targetable mutations or SNPs on either side of disease-causing mutation or SNP to silence the disease-causing mutation or SNP. In some embodiments, a block of DNA is identified in a phased sequencing experiment. In some embodiments, the mutation or SNP of interest is not a suitable substrate for the CRISPR/Cas system, and identifying mutations or SNPs on both side of the disease-causing mutations or SNP that are suitable for CRISPR/Cas cleavage allows removal of a segment of DNA that includes the disease-causing mutations or SNP. In some embodiments, the read length may be increased so as to gain longer contiguous reads and a haplotype phased genome by using a technology described in Weisenfeld NI, Kumar V, Shah P, Church DM, Jaffe DB. Direct determination of diploid genome sequences. Genome research. 2017; 27(5):757-767, which is herein incorporated by reference in its entirety.
[00044] In some embodiments, the methods described herein further comprises, prior to administering to the subject the engineered CRISPR/Cas system, obtaining genomic or sequence information of the subject; and selecting the first crRNA sequence and/or the second crRNA sequence based on the genomic or sequence information of the subject. In additional embodiments, the genomic or sequence information of the subject includes whole or partial genome sequence information of the subject.
[00045] The human genome is diploid by nature; every chromosome with the exception of the X and Y chromosomes in males is inherited as a pair, one from the male and one from the female parent. When seeking stretches of contiguous DNA sequence larger than a few thousand base pairs, a determination of inheritance is crucial to understand from which parent these blocks of DNA originate. Longer read sequencing technologies have been utilized in attempts to produce a haplotype- resolved genome sequences, i.e. haplotype phasing. Thus, when investigating the genomic sequence of a particular stretch of DNA longer than 50 kbps, a haplotype phased sequence analysis may be utilized to determine which of the paired chromosomes carries the sequence of interest. Longer phased sequencing reads may be employed to determine whether the SNP of interest would be suitable as a target for the CRISPR/Cas gene editing system described herein.
[00046] In some embodiments, the selected first crRNA sequence is configured to cause cleaving at a first cleaving site, within genome of the subject, that is adjacent to the first ancestral variation or snp site; and/or the selected second crRNA sequence is configured to cause cleaving at a second cleaving site, within the genome of the subject, that is adjacent to the second ancestral variation or snp site. In additional embodiments, the selected first crRNA sequence is configured to cause cleaving only at the first cleaving site; and/or the selected second crRNA sequence is configured to cause cleaving only at the second cleaving site. In some embodiments, the selected first crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the first target sequence in trans with the diseasecausing mutation or SNP, said first target sequence not being adjacent to the 5 ’-end of a PAM; and/or the selected second crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence not being adjacent to the 5 ’-end of a PAM. In additional embodiments, the selected first crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence not being adjacent to the 5 ’-end of a PAM; and the selected second crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the second target sequence in trans with the diseasecausing mutation or SNP, said second target sequence being adjacent to the 5 ’-end of a PAM. In further embodiments, the selected first crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence being adjacent to the 5 ’-end of a PAM; and the selected second crRNA sequence hybridizes to the nucleotide sequence (in trans) complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence not being adjacent to the 5 ’-end of a PAM.
[00047] In some embodiments, selecting the first crRNA sequence includes selecting a crRNA sequence that corresponds to the first target sequence in trans, said first target sequence in trans not being adjacent to the 5 ’-end of a PAM, and/or selecting the second crRNA sequence includes selecting a crRNA sequence that corresponds to the second target sequence in trans, said second target sequence in trans not being adjacent to the 5 ’-end of a PAM. In some embodiments, selecting the first crRNA sequence includes selecting a crRNA sequence that corresponds to the first target sequence in trans, said first target sequence in trans not being adjacent to the 5 ’-end of a PAM, and selecting the second crRNA sequence includes selecting a crRNA sequence that corresponds to the second target sequence in trans, said second target sequence in trans being adjacent to the 5 ’-end of a PAM. In some embodiments, selecting the first crRNA sequence includes selecting a crRNA sequence that corresponds to the first target sequence in trans, said first target sequence in trans being adjacent to the 5 ’-end of a PAM, and selecting the second crRNA sequence includes selecting a crRNA sequence that corresponds to the second target sequence in trans, said second target sequence in trans not being adjacent to the 5 ’-end of a PAM.
[00048] In some embodiments, the subjects that can be treated with the methods described herein include, but are not limited to, mammalian subjects such as a mouse, rat, dog, baboon, pig or human. In some embodiments, the subject is a human. The methods can be used to treat subjects at least 1 year, 2 years, 3 years, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or 100 years of age. In some embodiments, the subject is treated for at least one, two, three, or four diseases. For example, a single or multiple crRNA or sgRNA may be designed to alter or delete nucleotides at more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 and/or fewer than 20, 10, 9, 8, 7, 6, 5, 4 or 3 ancestral variation or snp sites.
[00049] In some embodiments, the methods of preventing, ameliorating, or treating the disease in a subject may comprise administering to the subject an effective amount of the engineered CRISPR/Cas system described herein. The term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
[00050] In some embodiments, the administering comprises injecting the engineered CRISPR/Cas system into the subject. In additional embodiments, the administering comprises introducing the engineered CRISPR/Cas system into a cell containing and expressing a DNA molecule having the target sequence as described below.
[00051] In some embodiments, the methods of treating the disease provide a positive therapeutic response with respect to a disease or condition. By "positive therapeutic response" is intended an improvement in the disease or condition, and/or an improvement in the symptoms associated with the disease or condition. The therapeutic effects of the subject methods of treatment can be assessed using any suitable method. In some embodiments, the subject methods reduce the amount of a diseaseassociate protein deposition in the subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared to the subject prior to undergoing treatment.
[00052] In another aspect, the present disclosure is related to engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRj/CRISPR associate protein (Cas) systems for preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject. The CRISPR/Cas may comprise at least one vector comprising a nucleotide molecule encoding Cas nuclease and the sgRNAs and/or crRNAs as described herein. The terms “non-naturally occurring” or “engineered” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature. In some embodiments, the Cas nuclease and the sgRNA/crRNA do not naturally occur together.
[00053] In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA- WOocessed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as “crRNA” herein, or a “spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus. As described above, sgRNA is a combination of at least tracrRNA and crRNA. In some embodiments, one or more elements of a CRISPR system are derived from a type II CRISPR system. In some embodiments, one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes or Staphylococcus aureus. In general, 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). In the context of formation of a CRISPR complex, “target sequence” may refer to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex, the “target sequence” may refer to a sequence adjacent to a PAM site, which the guide sequence comprises. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. In this disclosure, “target site” refers to a site of the target sequence including both the target sequence and its complementary sequence, for example, in double stranded nucleotides. In some embodiments, the target site described herein may mean a first target sequence hybridizing to sgRNA or crRNA of CRISPR/Cas system, and/or a second target sequence adjacent to the 5 ’-end of a PAM. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast.
[00054] The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. 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, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. 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). 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). Other vectors (e.g., non-episomal mammalian 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. Moreover, 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.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably 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 types of such vectors can also be selected for targeting particular types of cells.
[00055] In some embodiments, at least one vector of the engineered CRISPR/Cas system described herein further comprises (a) a first regulatory element operably linked to the sgRNA that hybridizes with the target sequence described herein, and (b) a second regulatory element operably linked to the nucleotide molecule encoding Cas nuclease, wherein components (a) and (b) are located on a same vector or different vectors of the system, the sgRNA targets the target sequence, and the Cas nuclease cleaves the DNA molecule. The target sequence may be a nucleotide sequence complementary to from 16 to 25 nucleotides adjacent to the 5’ end of a PAM. Being “adjacent” herein means being within 2 or 3 nucleotides of the site of reference, including being “immediately adjacent,” which means that there is no intervening nucleotides between the immediately adjacent nucleotide sequences and the immediate adjacent nucleotide sequences are within 1 nucleotide of each other. In additional embodiments, the cell is a eukaryotic cell, or a mammalian or human cell, and the regulatory elements are eukaryotic regulators. In further embodiments, the cell is a stem cell described herein. In some embodiments, the Cas nuclease is codon-optimized for expression in a eukaryotic cell.
[00056] In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. The term “regulatory element" is intended to include 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). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). 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. In some embodiments, a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol I promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pcJ I promoters (e.g., 1, 2, 3, 4, 5, or more pcJ I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and Hl promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cy tomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Bosbart et al. Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl a promoter. Also encompassed by the term “regulatory1 element” are enhancer elements, such as 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 p-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).
[00057] In some embodiments, the Cas nuclease provided herein may be an inducible Cas nuclease that is optimized for expression in a temporal or cell-type dependent manner. The first regulatory element may be an inducible promoter that can be linked to the Cas nuclease including, but are not limited to, tetracycline-inducible promoters, metallothionein promoters; tetracycline-inducible promoters, methionine-inducible promoters (e.g., MET25, MET3 promoters); and galactose-inducible promoters (GALI, GAL7 and GAL 10 promoters). Other suitable promoters include the ADH1 and ADH2 alcohol dehydrogenase promoters (repressed in glucose, induced when glucose is exhausted and ethanol is made), the CUP1 metallothionein promoter (induced in the presence of Cu2+, Zn2+), the PHO5 promoter, the CYC1 promoter, the HIS3 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the LEU2 promoter, the ENO promoter, the TP1 promoter, and the AOX1 promoter.
[00058] 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. 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., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).
[00059] Exemplary CRISPR/Cas9 systems, sgRNA, crRNA and tracrRNA, and their manufacturing process and use are disclosed in U.S. Patent No. 8697359, U.S. Patent Application Publication Nos. 20150232882, 20150203872, 20150184139, 20150079681, 20150073041, 20150056705, 20150031134, 20150020223, 20140357530, 20140335620, 20140310830, 20140273234, 20140273232, 20140273231, 20140256046, 20140248702, 20140242700, 20140242699, 20140242664, 20140234972, 20140227787, 20140189896, 20140186958, 20140186919, 20140186843, 20140179770, 20140179006, 20140170753, 20140093913, 20140080216, and W02016049024, all of which are incorporated herein by their entirety.
[00060] In some embodiments, the Cas9 nucleases described herein are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. The Cas9 nuclease may be a Cas9 homolog or ortholog. Mutant Cas9 nucleases that exhibit improved specificity may also be used (see, e.g., Ann Ran et al. Cell 154(6) 1380-89 (2013), which is herein incorporated by reference in its entirety for all purposes and particularly for all teachings relating to mutant Cas9 nucleases with improved specificity for target nucleic acids). The nucleic acid manipulation reagents can also include a deactivated Cas9 nuclease (dCas9). Deactivated Cas9 binding to nucleic acid elements alone may repress transcription by sterically hindering RNA polymerase machinery. Further, deactivated Cas may be used as a homing device for other proteins (e.g., transcriptional repressor, activators and recruitment domains) that affect gene expression at the target site without introducing irreversible mutations to the target nucleic acid. For example, dCas9 can be fused to transcription repressor domains such as KRAB or SID effectors to promote epigenetic silencing at a target site. Cas9 can also be converted into a synthetic transcriptional activator by fusion to VP16/VP64 or p64 activation domains. In some instances, a mutant Type II nuclease, referred to as an enhanced Cas9 (eCa9) nuclease, is used in place of the wild-type Cas9 nuclease. The enhanced Cas9 has been rationally engineered to improve specificity by weakening non-target binding. This has been achieved by neutralizing positively charged residues within the non-target strand groove (Slaymaker et al., 2016).
[00061] In some embodiments, the Cas nucleases direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the Cas nucleases directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
[00062] Following directed DNA cleavage by the Cas nuclease, there are two modes of DNA repair available to the cell: homology directed repair (HDR) and non-homologous end joining (NHEJ). While seamless correction of the mutation by HDR following Cas cleavage close to the mutation site is attractive, the efficiency of this method means that it could only be used for in vitro/ex vivo modification of stem cells or induced pluripotent stem cells (iPSC) with an additional step to select those cells in which repair had taken place and purify those modified cells only. HDR does not occur at a high frequency in cells.
[00063] In some embodiments, the first and/or second PAMs and the Cas nuclease described herein are from Streptococcus or Staphylococcus. In some embodiments, additional embodiments, the Cas nuclease is Cas9 nuclease. In some embodiments, the Cas nuclease is Cpfl nuclease. In additional embodiments, the first and second PAMs are both from Streptococcus or Staphylococcus. In additional embodiments, the Cas nuclease is from Streptococcus. In yet additional embodiments, the Cas nuclease is from Streptococcus pyogenes, Streptococcus dysgalactiae, Streptococcus canis, Streptococcus equi, Streptococcus iniae, Streptococcus phocae, Streptococcus pseudoporcinus, Streptococcus oralis, Streptococcus pseudoporcinus, Streptococcus infantarius, Streptococcus mutans, Streptococcus agalactiae, Streptococcus caballi, Streptococcus equinus, Streptococcus sp. oral taxon, Streptococcus mitts, Streptococcus gallolyticus, Streptococcus gordonii, or Streptococcus pasteurianus, or variants thereof. Such variants may include D10A Nickase, Spy Cas9-HF1 as described in Kleinstiver et al, 2016 Nature, 529, 490-495, or Spy eCas9 as described in Slaymaker et al., 2016 Science, 351(6268), 84-88. In additional embodiments, the Cas nuclease is from Staphylococcus. In yet additional embodiments, the Cas nuclease is from Staphylococcus aureus, S. simiae, S. auricularis, S. carnosus, S. condimenti, S. massiliensis, S. piscifermentans, S. simulans, S. capitis, S. caprae, S. epidermidis, S. saccharolyticus, S. devriesei, S. haemolyticus, S. hominis, S. agnetis, S. chromogenes, S. felts, S. delphini, S. hyicus, S. intermedius, S. lutrae, S. microti, S. muscae, S. pseudintermedius, S. rostri, S. schleiferi, S. lugdunensis, S. arlettae, S. cohnii, S. equorum, S. gallinarum, S. kloosii, S. leei, S. nepalensis, S. saprophyticus, S. succinus, S. xylosus, S. fleurettii, S. lentus, S. sciuri, S. Stepanovich, S. vitulinus, S. simulans, S. pasteuri, S. warneri, or variants thereof. [00064] In some embodiments, the Cas nuclease is Cas9 nuclease. In further embodiments, the Cas9 nuclease excludes Cas9 nuclease from Streptococcus pyogenes.
[00065] Examples of Cas nucleases and their PAM sequences are shown in Table 1.
[00066] Table 1: Example Cas nucleases
Figure imgf000022_0001
Figure imgf000023_0001
[00067] In Table 1, N is any of A, T, G, and C; R is A or G; W is A or T; Y is C or T; D is any of A, G, and T; V is any of A, C, and G.
[00068] In additional embodiments, the Cas9 nuclease comprises an amino acid sequence having at least about 60, 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% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or 8. In additional embodiments, the nucleotide molecule encoding Cas9 nuclease comprises a nucleotide sequence having at least about 60, 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% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 or 7. In yet additional embodiments, Cas9 sgRNA sequence may comprises a sequence having at least about 60, 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% sequence identity with SEQ ID NO: 1 or 5. An exemplary tracrRNA or sgRNA scaffold sequence may comprise a sequence having at least about 60, 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% sequence identity with SEQ ID NO: 2 or 6.
[00069] In some embodiments, the Cas9 nuclease is an enhanced Cas9 nuclease that has one or more mutations improving specificity of the Cas9 nuclease. In additional embodiments, the enhanced Cas9 nuclease is from a Cas9 nuclease from Streptococcus pyogenes having one or more mutations neutralizing a positively charged groove, positioned between the HNH, RuvC, and PAM -interacting domains in the Cas9 nuclease. In yet additional embodiments, the Cas9 nuclease comprises an amino acid sequence having at least about 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% sequence identity with a mutant amino acid sequence of a Cas9 nuclease from Streptococcus pyogenes (e.g., SEQ ID NO: 4) with one or more mutations selected from the group consisting of (i) K855A, (ii) K810A, K1003A and R1060A, and (iii) K848A, K1003A and R1060A. In yet further embodiments, the nucleotide molecule encoding Cas nuclease comprises a nucleotide sequence having at least about 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% sequence identity with a nucleotide sequence encoding the mutant amino acid sequence. [00070] In some embodiments, the CRISPR/Cas system and the methods using the CRISPR/Cas system described herein alter a DNA sequence by the NHEJ. In additional embodiments, the CRISPR/Cas system or the vector described herein does not include a repair nucleotide molecule. In some embodiments, the methods described herein alter a DNA sequence by the HDR. In some embodiments, the CRISPR/Cas system or the vector described herein may further comprise a repair nucleotide molecule. The target polynucleotide cleaved by the Cas nuclease may be repaired by homologous recombination with the repair nucleotide molecule, which is an exogenous template polynucleotide. This repair may result in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. The repair nucleotide molecule introduces a specific allele (e.g., a wild-type allele) into the genome of one or more cells of the plurality of stem cells upon repair of a Type II nuclease induced DSB through the HDR pathway. In some embodiments, the repair nucleotide molecule is a single stranded DNA (ssDNA). In other embodiments, the repair nucleotide molecule is introduced into the cell as a plasmid vector. In some embodiments, the repair nucleotide molecule is 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 105, 105 to 110, 110 to 115, 115 to 120, 120 to 125, 125 to 130, 130 to 135, 135 to 140, 140 to 145, 145 to 150, 150 to 155, 155 to 160, 160 to 165, 165 to 170, 170 to 175, 175 to 180, 180 to 185, 185 to 190, 190 to 195, or 195 to 200 nucleotides in length. In some embodiments, the repair nucleotide molecule is 200 to 300, 300, to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1,000 nucleotides in length. In other embodiments, the repair nucleotide molecule is 1,000 to 2,000, 2,000 to 3,000, 3,000 to 4,000, 4,000 to 5,000, 5,000 to 6,000, 6,000 to 7,000, 7,000 to 8,000, 8,000 to 9,000, or 9,000 to 10,000 nucleotides in length.
[00071] The repair nucleotide molecule may further include a label for identification and sorting of cells described herein containing the specific mutation. Exemplary labels that can be included with the repair nucleotide molecule include fluorescent labels and nucleic acid barcodes that are identifiable by length or sequence.
[00072] In additional embodiments, the CRISPR/Cas system or the vector described herein may include at least one nuclear localization signal (NLS). In additional embodiments, the sgRNA and the Cas nuclease are included on the same vector or on different vectors.
[00073] The term “crRNA” may refer to a guide sequence that may be a part of an sgRNA in an CRISPR/Cas system. In some embodiments, at least one of the first and second crRNA sequences described herein comprises a nucleotide sequence selected from the group consisting of sequences listed in Figures 8-23; and/or at least one of the first and second crRNA sequences comprises a nucleotide sequence selected from the group consisting of sequences listed in Table 3. The term, “sgRNA” refers to a single guide RNA containing a guide sequence (crRNA sequence). In some embodiments, the sgRNA also includes a Cas nuclease-recruiting sequence (tracrRNA). The crRNA sequence may be a sequence that is homologous to a region in the gene of interest and may direct Cas nuclease activity. The crRNA sequence and tracrRNA sequence may not naturally occur together. The sgRNA may be delivered as RNA or by transforming with a plasmid with the sgRNA-coding sequence (sgRNA gene) under a promoter. The tracrRNA sequence may be any sequence for tracrRNA for CRISPR/Cas system known in the art. In some embodiments, the sgRNA includes no tracrRNA.
[00074] In some embodiments, the crRNA hybridizes to at least a part of a target sequence (e.g., target genome sequence), and the crRNA may have a complementary sequence to the target sequence. In some embodiments, the target sequence herein is a first target sequence that hybridizes to a second target sequence adjacent to a PAM site described herein. In some embodiments, the crRNA may comprise the first target sequence or the second target sequence. In additional embodiments, the first and second target sequences are located in introns of a target gene. “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. As used herein, “stringent conditions” for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology -Hybridization With Nucleic Acid Probes Part 1 , Second Chapter “Ovendew of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N.Y. “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence. In additional embodiments, the crRNA or the guide sequence is about 17, 18, 19, 20, 21, 22, 23 or 24 nucleotide long. As used herein, the term “about” may refer to a range of values that are similar to the stated reference value. In certain embodiments, the term “about” refers to a range of values that fall within 15, 10, 9, 8,7, 6, 5, 4, 3, 2, 1 percent or less of the stated reference value. [00075] In some embodiments, the disease is an autosomal dominant disease. In additional embodiments, the disease is selected from the group consisting of Acropectoral syndrome, Acute intermittent porphyria, Adermatoglyphia, Albright's hereditary osteodystrophy, Arakawa's syndrome II, Aromatase excess syndrome, Autosomal dominant cerebellar ataxia, Axenfeld syndrome, Benign hereditary chorea, Bethlem myopathy, Birt-Hogg-Dube syndrome, Boomerang dysplasia, Branchio- oto-renal syndrome, Buschke-Ollendorff syndrome, Camurati-Engelmann disease, Central core disease, Collagen disease, Collagenopathy, types II and XI, Congenital distal spinal muscular atrophy, Congenital stromal corneal dystrophy, Costello syndrome, Currarino syndrome, Darier's disease, Glutl deficiency, Dentatorubral-pallidoluysian atrophy, Dermatopathia pigmentosa reticularis, Dysfibrinogenemia, Transthyretin-related hereditary amyloidosis, Familial atrial fibrillation, Familial hypercholesterolemia, Familial male-limited precocious puberty, Feingold syndrome, Felty's syndrome, Flynn-Aird syndrome, Gardner's syndrome, Gillespie syndrome, Gray platelet syndrome, Greig cephalopoly syndactyly syndrome, Hajdu-Cheney syndrome, Hawkinsinuria, Hay-Wells syndrome, Hereditary elliptocytosis, Hereditary hemorrhagic telangiectasia, Hereditary mucoepithelial dysplasia, Hereditary spherocytosis, Holt-Oram syndrome, Huntington's disease, Huntington's disease-like syndrome, Hypertrophic cardiomyopathy, Hypoalphalipoproteinemia, Hypochondroplasia, Hypodysfibrinogenemia, Jackson-Weiss syndrome, Keratolytic winter erythema, Kniest dysplasia, Kostmann syndrome, Langer-Giedion syndrome, Larsen syndrome, Liddle's syndrome, Marfan syndrome, Marshall syndrome, Medullary cystic kidney disease, Metachondromatosis, Miller-Dieker syndrome, MOMO syndrome, Monilethrix, MonoMAC, Multiple endocrine neoplasia, Multiple endocrine neoplasia type 1, Multiple endocrine neoplasia type 2, Multiple endocrine neoplasia type 2b, Myelokathexis, Myotonic dystrophy, Naegeli-Franceschetti- Jadassohn syndrome, Nail-patella syndrome, Noonan syndrome, Oculopharyngeal muscular dystrophy, Pachyonychia congenital, Pallister-Hall syndrome, PAPA syndrome, Papillorenal syndrome, Parastremmatic dwarfism, Pelger-Huet anomaly, Peutz-Jeghers syndrome, Piebaldism, Platyspondylic lethal skeletal dysplasia, Torrance type, Polydactyly, Popliteal pterygium syndrome, Porphyria cutanea tarda, Pseudoachondroplasia, RASopathy, Reis-Bucklers corneal dystrophy, Romano-Ward syndrome, Rosselli-Gulienetti syndrome, Roussy-Levy syndrome, Rubinstein-Taybi syndrome, Saethre-Chotzen syndrome, Schmitt Gillenwater Kelly syndrome, Short QT syndrome, Singleton Merten syndrome, Spinal muscular atrophy with lower extremity predominance, Spinocerebellar ataxia, Spinocerebellar ataxia type 1, Spinocerebellar ataxia type 6, Spondyloepimetaphyseal dysplasia- Strudwick type, Spondyloepiphyseal dysplasia congenital, Spondyloperipheral dysplasia, Stickler syndrome, Tietz syndrome, Timothy syndrome, Treacher Collins syndrome, Tricho-dento-osseous syndrome, Tuberous sclerosis, Upington disease, Variegate porphyria, Vitelliform macular dystrophy, Von Hippel-Lindau disease, Von Willebrand disease, Wallis-Zieff-Goldblatt syndrome, WHIM syndrome, White sponge nevus, Worth syndrome, Zaspopathy, Zimmermann-Laband syndrome, and Zori-Stalker-Williams syndrome. In yet additional embodiments, the disease is an autosomal dominant disease of an eye. In further embodiments, the disease may include or excludes corneal dystrophy.
[00076] For example, the number of the trinucleotide repeat of cytosine-adenine-guanine (CAG) at the end of the huntingtin gene (also called HTT or HD gene) located at 4pl6.3 is associated with the Huntington’s disease. Persons with more than 40 repeats may develop the Huntington’s disease during a normal lifetime, while persons with more than 60 repeats may develop juvenile Huntington’s disease, which begins in childhood or adolescence and has a faster progression. The HTT gene is associated with the expression of huntingtin protein, which plays an important role in neurons. By utilizing the method described herein, in a heterozygote patient, only the HTT gene that contains excessive (e.g., more than 35 repeats, more than 40 repeats, or more than 60 repeats) CAG repeats is cleaved.
[00077] In another example, a mutation in the adenomatous polyposis coli (APC) gene located at 5q21 is associated with the Gardner’s syndrome, which shows an increased risk of colon cancer. A large portion of the mutations occur between amino acid 1061 and amino acid 1513. By utilizing the method described herein, in a heterozygote patient, only the mutant gene is cleaved while maintaining the wild type gene.
[00078] As used herein, a “corneal dystrophy” refers to any one of a group of hereditary disorders in the outer layer of the eye (cornea). For example, the corneal dystrophy may be characterized by bilateral abnormal deposition of substances in the cornea. Corneal dystrophies include, but are not limited to the following four IC3D categories of corneal dystrophies (see, e.g., Weiss et al., Cornea 34(2): 117-59 (2015)): epithelial and sub-epithelial dystrophies, epithelial-stromal TGF|3I dystrophies, stromal dystrophies and endothelial dystrophies. In some embodiments, the corneal dystrophy is selected from the group consisting of Epithelial basement membrane dystrophy (EBMD), Meesmann corneal dystrophy (MECD), Thiel-Behnke corneal dystrophy (TBCD), Lattice corneal dystrophy (LCD), Granular corneal dystrophy (GCD), and Schnyder corneal dystrophy (SCD). In additional embodiments, the corneal dystrophy is caused by one or more mutations, including SNP, is located in a gene selected from the group consisting of Transforming growth factor, beta-induced (TGFBI), keratin 3 (KRT3), keratin 12 (KRT12), GSN, and UbiA prenyltransferase domain containing 1 (UBIAD1). In further embodiments, the mutation or SNP site results in encoding a mutant amino acid in a mutant protein as shown herein. In further embodiments, a mutant sequence comprising the mutation or SNP site encodes a mutant protein selected from the group consisting of (i) mutant TGFBI proteins comprising a mutation corresponding to Leu509Arg, Arg666Ser, Gly623Asp, Arg555Gln, Argl24Cys, Val505Asp, Ile522Asn, Leu569Arg, His572Arg, Arg496Trp, Pro501Thr, Arg514Pro, Phe515Leu, Leu518Pro, Leu518Arg, Leu527Arg, Thr538Pro, Thr538Arg, Val539Asp, Phe540del, Phe540Ser, Asn544Ser, Ala546Thr, Ala546Asp, Phe547Ser, Pro551Gln, Leu558Pro, His572del, Gly594Val, Val613del, Val613Gly, Met619Lys, Ala620Asp, Asn622His, Asn622Lys, Asn622Lys, Gly623Arg, Gly623Asp, Val624_Val625del, Val624Met, Val625Asp, His626Arg, His626Pro, Val627SerfsX44, Thr629_Asn630insAsnValPro, Val631Asp, Arg666Ser, Arg555Trp, Argl24Ser, Aspl23delins, Argl24His, Argl24Leu, Leu509Pro, Leul03_Serl04del, Valll3Ile, Aspl23His, Argl24Leu, and/or Thrl25_Glul26del in TGFBI, for example, of Protein Accession No. Q15582; (ii) mutant KRT3 proteins comprising a mutation corresponding to Glu498Val, Arg503Pro, and/or Glu509Lys in Keratin 3 protein, for example, of Protein Accession No. P12035 or NP 476429.2; (iii) mutant KRT12 proteins with Metl29Thr, Metl29Val, Glnl30Pro, Leul32Pro, Leul32Va, Leul32His, Asnl33Lys, Argl35Gly, Argl35Ile, Argl35Thr, Argl35Ser, Alal37Pro, Leul40Arg, Vall43Leu, Vall43Leu, Lle391_Leu399dup, He 426Val, He 426Ser, Tyr429Asp, Tyr429Cys, Arg430Pro, and/or Leu433Arg in KRT12, for example, of Protein Accession No. Q99456.1 orNP_000214.1; (iv) mutant GSN proteins with Asp214Tyr in GSN, for example, of Protein Accession No. P06396; and (v) mutant UBIAD1 proteins comprising a mutation corresponding to Ala97Thr, Gly98Ser, AsnlO2Ser, Aspl l2Asn, Aspl l2Gly, Aspll8Gly, Argll9Gly, Leul21Val, Leul21Phe, Vall22Glu, Vall22Gly, Serl71Pro, Tyrl74Cys, Thrl75Ile, Glyl77Arg, Lysl81Arg, Glyl86Arg, Leul88His, Asn232Ser, Asn233His, Asp236Glu, and/or Asp240Asn in UBIAD1, for example, of Protein Accession No. Q9Y5Z9. For example, a mutant sequence comprising the mutation or SNP site encodes at least a part of mutant TGFBI protein mutated by replacing Leu with Arg at amino acid position corresponding the amino acid position 509 of Protein Accession No. Q15582. In this case, a mutation at the mutation or SNP site may be responsible for encoding the mutant amino acid at amino acid position corresponding the amino acid position 509 of Protein Accession No. Q15582. As used herein, a mutation “corresponding to” a particular mutation in a human protein may include a mutation in a different species that occur at the corresponding site of the particular mutation of the human protein. Also as used herein, when a mutant protein is described to include a particular mutant, for example, of Leu509Arg, such a mutant protein may comprise any mutation that occurs at a mutant site corresponding to the particular mutant in a relevant human protein, for example, in TGFBI protein of Protein Accession No. Q15582 as described herein. [00079] In some embodiments, the corneal dystrophy target nucleic acid is a TGFpi target nucleic acid. In other embodiments, the corneal dystrophy target nucleic acid is a COL4A1-4, LOX, SPARC, LRRN1, HGF, AKAP13, ZNF469, ATG12P2, GS1-256O22.5, PLEKHA6, APOL4, SLC44A3, SLC6A18, SLC29A3, RANBP3L, KCNMA1, MUC5AC, CROCC, ATHL1, or PLP1 target nucleic acid. In some embodiments, the nucleic acid mutation encodes for an amino acid substitution of arginine 124, arginine 555, or histidine 666 in a TGFpi polypeptide. In some embodiments, the nucleic acid mutation encodes for an amino acid substitution selection from R124C, R124H, R124L, R555W, R555Q, and H626P. In some embodiments, the nucleic acid mutation encodes for amino acid substitution Q1334H in COL4A1. In some embodiments, the nucleic acid mutation encodes for amino acid substitution G683A in COL4A2. In some embodiments, the nucleic acid mutation encodes for amino acid substitution P718S in COL4A2. In some embodiments, the nucleic acid mutation encodes for amino acid substitution R517K in COL4A2. In some embodiments, the nucleic acid mutation encodes for amino acid substitution D326Y in COL4A3. In some embodiments, the nucleic acid mutation encodes for amino acid substitution H451R in COL4A3. In some embodiments, the nucleic acid mutation encodes for amino acid substitution V1327M in COL4A4. In some embodiments, the nucleic acid mutation encodes for amino acid substitution R158Q in LOX. In some embodiments, the nucleic acid mutation encodes for amino acid substitution A1046T in AKAP13. In some embodiments, the nucleic acid mutation encodes for amino acid substitution G624V in AKAP13. In some embodiments, the nucleic acid mutation encodes for amino acid substitution G2358R in ZNF469. In some embodiments, the nucleic acid mutation encodes for amino acid substitution S158F in SLC29A3. In some embodiments, the nucleic acid mutation encodes for amino acid substitution P4493S in MUC5AC. In some embodiments, the nucleic acid mutation encodes for amino acid substitution P370S in CROCC.
[00080] In some embodiments, the subject has corneal opacity. In some embodiments, the subject is a suitable candidate for LASIK version correction.
[00081] In another aspect, the present disclosure is also related to methods of altering expression of at least one gene product comprising introducing the engineered CRISPR/Cas system described herein into a cell containing and expressing a DNA molecule having a target sequence and encoding the gene product. The engineered CRISPR/Cas system can be introduced into cells using any suitable method. In some embodiments, the introducing may comprise administering the engineered CRISPR/Cas system described herein to cells in culture, or in a host organism.
[00082] Exemplary methods for introducing the engineered CRISPR/Cas system include, but are not limited to, transfection, electroporation and viral-based methods. In some cases, the one or more cell uptake reagents are transfection reagents. Transfection reagents include, for example, polymer based (e.g., DEAE dextran) transfection reagents and cationic liposome-mediated transfection reagents. Electroporation methods may also be used to facilitate uptake of the nucleic acid manipulation reagents. By applying an external field, an altered transmembrane potential in a cell is induced, and when the transmembrane potential net value (the sum of the applied and the resting potential difference) is larger than a threshold, transient permeation structures are generated in the membrane and electroporation is achieved. See, e.g., Gehl et a\., Acta Physiol. Scand. 177:437-447 (2003). The engineered CRISPR/Cas system also may be delivered through viral transduction into the cells. Suitable viral delivery systems include, but are not limited to, adeno-associated virus (AAV), retroviral and lentivirus delivery systems. Such viral delivery systems are useful in instances where the cell is resistant to transfection. Methods that use a viral-mediated delivery system may further include a step of preparing viral vectors encoding the nucleic acid manipulation reagents and packaging of the vectors into viral particles. Other method of delivery of nucleic acid reagents include, but are not limited to, lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of nucleic acids. See, also Neiwoehner et al., Nucleic Acids Res. 42:1341-1353 (2014), and U.S. Patent Nos. 5,049,386, 4,946,787; and 4,897,355, which are herein incorporated by reference in its entirety for all purposes, and particularly for all teachings relating to reagent delivery systems. In some embodiments, the introduction is performed by non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). [00083] The cells that have undergone a nucleic acid alteration event (i.e., a “altered” cell) can be isolated using any suitable method. In some embodiments, the repair nucleotide molecule further comprises a nucleic acid encoding a selectable marker. In these embodiments, successful homologous recombination of the repair nucleotide molecule a host stem cell genome is also accompanied by integration of the selectable marker. Thus, in such embodiments, the positive marker is used to select for altered cells. In some embodiments, the selectable marker allows the altered cell to survive in the presence of a drug that otherwise would kill the cell. Such selectable markers include, but are not limited to, positive selectable markers that confer resistance to neomycin, puromycin or hygromycin B. In addition, a selectable marker can be a product that allows an altered cell to be identified visually among a population of cells of the same type, some of which do not contain the selectable marker. Examples of such selectable markers include, but are not limited to the green fluorescent protein (GFP), which can be visualized by its fluorescence; the luciferase gene, which, when exposed to its substrate luciferin, can be visualized by its luminescence; and P-galactosidase (P-gal), which, when contacted with its substrate, produces a characteristic color. Such selectable markers are well known in the art and the nucleic acid sequences encoding these markers are commercially available (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989). Methods that employ selectable markers that can be visualized by fluorescence may further be sorted using Fluorescence Activated Cell Sorting (FACS) techniques. Isolated manipulated cells may be used to establish cell lines for transplantation. The isolated altered cells can be cultured using any suitable method to produce a stable cell line.
[00084] In another aspect, the present disclosure is related to methods of treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject in need thereof, comprising: (a) obtaining a plurality of stem cells comprising a nucleic acid mutation in a corneal dystrophy target nucleic acid from the subject; (b) manipulating the nucleic acid mutation in one or more stem cells of the plurality of stem cells to correct the nucleic acid mutation, thereby forming one or more manipulated stem cells; (c) isolating the one or more manipulated stem cells; and (d) transplanting the one or more manipulated stem cells into the subject, wherein manipulating the nucleic acid mutation in the one or more stem cells of the plurality of stem cells includes performing any of the methods of altering expression of a gene product or of preventing, ameliorating, or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP) in a subject as described herein.
[00085] The subject methods may include obtaining a plurality of stem cells. Any suitable stem cells can be used for the subject method, depending on the type of the disease to be treated. In certain embodiments, the stem cell is obtained from a heterologous donor. In such embodiments, the stem cells of the heterologous donor and the subject to be treated are donor-recipient histocompatible. In certain embodiments, autologous stem cells are obtained from the subject in need of the treatment for the disease. Obtained stem cells carry a mutation in a gene associated with the particular disease to be treated. Suitable stem cells include, but are not limited to, dental pulp stem cells, hair follicle stem cells, mesenchymal stem cells, umbilical cord lining stem cells, embryonic stem cells, oral mucosal epithelial stem cells and limbal epithelial stem cells.
[00086] Stem cells to be manipulated may include individual isolated stem cells or stem cells from a stem cell line established from the isolated stem cells. Any suitable genetic manipulation method may be used to correct the nucleic acid mutation in the stem cells.
[00087] In another aspect, provided herein are kits comprising the CRISPR/Cas system for the treatment of a disease associated with a gene mutation or single-nucleotide polymorphism (SNP). In some embodiments, the kit includes one or more sgRNAs described herein, a Cas nuclease and a repair nucleotide molecule that includes a wild-type allele of the mutation to be repaired as described herein. In some embodiments, the kit also includes agents that facilitate uptake of the nucleic acid manipulation by cells, for example, a transfection agent or an electroporation buffer. In some embodiments, the subject kits provided herein include one or more reagents for the detection or isolation of stem cells, for example, labeled antibodies for one or more positive stem cell markers that can be used in conjunction with FACS.
[00088] In another aspect, the present disclosure is related to an sgRNA pair, and a kit comprising the sgRNA pair comprising at least two sgRNAs for CRISPR/Cas system to silence a disease-causing mutation or SNP, for example, for preventing, ameliorating or treating a disease associated with a gene mutation or single-nucleotide polymorphism (SNP). In some embodiments, the sgRNA pair comprises an sgRNA comprising a guide sequence for PAM-generating ancestral variation or snp in a target gene, for example, in an intron in cis with a disease-causing mutation or SNP. In additional embodiments, the sgRNA pair comprises an sgRNA comprising a common guide sequence for PAM generating an ancestral SNP in intronic regions of a target gene.
[00089] In some embodiments, the present disclosure is related to an sgRNA pair designed for CRISPR/Cas system, the sgRNA pair comprising (i) a first sgRNA comprising (a) a first crRNA sequence for a first protospacer adjacent motif (PAM) generating mutation or single-nucleotide polymorphism (SNP) at 3 ’-end side of a disease-causing mutation or SNP in cis, and (b) a tracrRNA sequence, in which the first crRNA sequence and the tracrRNA sequence do not naturally occur together; (ii) a second sgRNA comprising (a) a second crRNA guide sequence for a second PAM generating mutation or SNP at 5 ’-end side of the disease-causing mutation or SNP in cA; (b) a tracrRNA sequence, in which the second crRNA sequence and the tracrRNA sequence do not naturally occur together.
[00090] In one aspect, the method described herein comprises diagnosing the diseases described herein. In some embodiments, diagnostic testing is employed to determine one or more genetic conditions by detection of any of a variety of mutations. In some embodiments, diagnostic testing is used to confirm a diagnosis when a particular condition is suspected based on for example physical manifestations, signs and/or symptoms as well as family history information.
[00091] The nucleic acids obtained by the disclosed methods are useful in a variety of diagnostic tests, including tests for detecting mutations such as deletions, insertions, transversions and transitions. In some embodiments, such diagnostics are useful for identifying unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to be expressed, identifying unaffected individuals who carry one copy of a gene for a disease in which the information could find use in developing a treatment regimen, preimplantation genetic diagnosis, prenatal diagnostic testing, newborn screening, genealogical DNA test (for genetic genealogy purposes), presymptomatic testing for predicting adult-onset disorders such as Huntington's disease, presymptomatic testing for estimating the risk of developing adult-onset cancers and Alzheimer's disease, confirmational diagnosis of a symptomatic individual, and/or forensic/identity testing. In some embodiments, the diseases described herein includes corneal dystrophy, for example through detection of Avellino corneal dystrophy-related SNPs, such as those that result in R124 mutations in the TGFBI gene (including for example but not limited to an R124H mutation caused by a G to A transition at nucleotide 418 of TGFBI gene also referred to as a C(G/A)C SNP).
[00092] In some embodiments, newborn screening includes any genetic screening employed just after birth in order to identify genetic disorders. In some embodiments, newborn screening finds use in the identification of genetic disorders so that a treatment regimen is determined early in life. Such tests include but are not limited to testing infants for phenylketonuria and congenital hypothyroidism.
[00093] In some embodiments, carrier testing is employed to identify people who carry a single copy of a gene mutation. In some cases, when present in two copies, the mutation can cause a genetic disorder. In some cases, one copy is sufficient to cause a genetic disorder. In some cases, the presence of two copies is contra-indicated for a particular treatment regimen, such as the presence of the Avellino mutation and pre-screening prior to performing surgical procedures in order to ensure the appropriate treatment regiment is pursued for a give patient. In some embodiments, such information is also useful for individual contemplating procreation and assists individuals with making informed decisions as well as assisting those skilled in the medical arts in providing important advice to individual patients.
[00094] In some embodiments, predictive and presymptomatic types of testing are used to detect gene mutations associated with a variety of disorders. In some cases, these tests are helpful to people who have a family member with a genetic disorder, but who may exhibit no features of the disorder at the time of testing. In some embodiments, predictive testing identifies mutations that increase a person's chances of developing disorders with a genetic basis, including for example but not limited to certain types of cancer. In some embodiments, presymptomatic testing is useful in determining whether a person will develop a genetic disorder, before any physical signs or symptoms appear. The results of predictive and presymptomatic testing provide information about a person’s risk of developing a specific disorder and help with making decisions about an appropriate medical treatment regimen described herein. Predictive testing is also employed, in some embodiments, to detect mutations which are contra-indicated with certain treatment regimens, such as the presence of the Avellino mutation being contra-indicated with performing laser eye surgery, such as a refractive surgery (e.g., LASIK, LASEK, PTK, and PRK). For example, patients exhibiting the Avellino mutation should not undergo a refractive surgey (LASIK, LASEK, PTK, and PRK).
[00095] EXAMPLES The following examples are presented to illustrate various embodiments of the invention. It is understood that such examples do not represent and are not intended to represent exclusive embodiments; such examples serve merely to illustrate the practice of this invention.
[00096] Mutation analysis: Mutations associated with various corneal dystrophies were analyzed to determine which were solely caused by missense mutations or in-frame indels. This analysis indicates that for the majority of KI 2 and TGFBI disease, nonsense or frameshifting indel mutations are not associated with disease. Furthermore, an analysis of the exome variant database confirmed that any naturally occurring nonsense, frameshifting indels or splice site mutations found in these genes are not reported to be associated with disease in these individuals.
[00097] Mutation analysis revealed that the following comeal-dystrophy genes are suitable for targeted nuclease gene therapy (Table 2).
[00098] Table 2: Genes and their associated corneal dystrophies that are suitable for a CRISPR/Cas mediated approach.
Figure imgf000033_0001
[00099] An investigation of the suitable corneal dystrophy genes was conducted to determine the number of mutations targetable by either a PAM-specific approach or a guide allele-specific approach. A PAM-specific approach requires the disease causing SNP to generate a novel PAM, whilst the allele specific approach involves the design of a guide containing the disease causing SNP. All non-disease causing SNPs in TGFBI that generate a novel PAM with a minor allele frequency (MAF) of >10% were identified and analyzed by the Benchling’s online genome-editing design tool. The selection of SNPs with a MAF of >10% may provide a reasonable chance that the SNP resulting in a novel PAM will be found in cis with the disease causing mutation. Being “in cis” with the disease causing mutation refers to being on the same molecule of DNA or chromosome as the disease -causing mutation. The SNP resulting in a novel PAM may be found, for example, in intron or exon in TGFBI gene in cis with the disease-causing mutation. All variants within TGFBI were analyzed to determine whether a novel PAM was created (Table 3).
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
[000100] As shown in Figure 4, the positions of the variants within TGFBI, with most of the SNPs clustered in introns. Thus, multiple TGFBI mutations located in the hotspots in exons 11, 12 and 14 may be targeted simultaneously using this approach. Therefore, a CRISPR Cas system may target more than one patient or one family with a mutation. One CRISPR/Cas system designed in this way may be used to treat a range of TGFBI mutations. The CRISPR/Cas system may employ an sgRNA adjacent to a PAM site located in the flanking intron that is common to both wild-type and mutant alleles in tandem with a sgRNA adjacent to a PAM site that is specific to the mutant allele (Figure 16). This would result in NHEJ in the intron of the wild-type allele that should have no functional effect, while in the mutant allele would result in a deletion encompassing the DNA between the two cut sites. This technique is demonstrated in leucocytes isolated from a patient with a suitable SNP profile.
[000101] Confirming allele-specific indels
[000102] EBV transformation of lymphocytes: A sample of 5ml of whole blood was taken and place in a sterile 50ml Falcon tube. An equal volume of RPMI media containing 20% foetal calf serum was added to the whole blood - mix by gently inverting the tube. 6.25ml of Ficoll-Paque PLUS (GE Healthcare cat no. 17-1440-02) was placed in a separate sterile 50ml Falcon tube. 10 ml of blood/media mix was added to the Ficoll-Paque. The tube was spun at 2000 rpm for 20 min at room temperature. The red blood cells formed at the bottom of the tube above which was the Ficoll layer. The lymphocytes formed a layer on top of the Ficoll layer, while the top layer was the medium. A clean sterile Pastettewas inserted to draw off the lymphocytes, which were placed in a sterile 15ml Falcon tube. The lymphocytes were centrifuged and washed. EBV aliquot was thawed and added to resuspended lymphocytes, and the mixture was incubated for 1 hour at 37 degrees C (infection period). RPMI, 20% FCS media and Img/ml phytohaemagglutinin were added to EBV treated lymphocytes, and the lymphocytes were placed on a 24- well plate.
[000103] Electroporation of EBV Transformed Lymphocytes (LCLs): CRISPR constructs (with either GFP or mCherry co-expressed) were added to suspended EBV transformed lymphocytes cells, and the mixture was transferred to an electroporation cuvette. Electroporation was performed, and 500pl pre-warmed RPMI 1640 media containing 10% FBS was added to the cuvette. The contents of the cuvette was transferred to a 12 well plate containing the remainder of the pre-warmed media, and 6 hours post nucleofection, 1ml of media was removed and was replaced with fresh media.
[000104] Cell sorting of GFP+ and/or mCherry+ Live cells: 24 hours post nucleofection, 1ml of media was removed and the remaining media containing cells was collected in a 1.5ml Eppendorf. The cells were centrifuged and resuspended in 200ul PBS add 50ul eFlouro 780 viability stain at 1: 1000 dilution. After another centrifuge, the cells were resuspended in filter sterile FACS buffer containing lx HBSS (Ca/Mg++ free), 5mM EDTA, 25mM HEPES pH 7.0, 5% FCS/FBS (Heat-Inactivated) and lOunits/mL DNase II. Cells were sorted to isolate live GFP+ and/or mCherry+ cells and were collected in RPMI + 20% FBS. Cells were expanded, and DNA was extracted from the cells.
[000105] Isolation of single alleles for sequencing: QIAmp DNA Mini Kit (Qiagen) was used to isolate DNA, and PCR was used across the region targeted by CRISPR/Cas. Specific amplification was confirmed by gel electrophoresis, and the PCR product was purified. The PCR product was blunt ended and ligated into pJET1.2/blunt plasmid from the Clonejet Kit (Thermo Scientific). The ligation mixture was transformed into competent DH5a cells. Single colonies were picked, and Sanger Sequencing was performed to confirm edits. The resulting data is shown in Figure 5.

Claims

Claims
1. A method of preventing, ameliorating, or treating a disease associated with a gene mutation or single -nucleotide polymorphism (SNP) in a subject, comprising detecting phase of SNPs in cis with the gene mutation or SNP associated with the disease in the subject by droplet digital polymerase chain reaction (PCR), and administering to the subject an engineered CRISPR/Cas system.
2. The method according to claim 1, wherein the detecting comprises preparing at least 10,000 droplets, each comprising a first labeled probe for the gene mutation or SNP and a second labeled probe for a SNP that is in cis with the gene mutation or SNP.
3. The method according to claim 2, wherein the first and second probes are labeled with different fluorescent dyes.
4. The method according to any one of the preceding claims, further comprising detecting the gene mutation or SNP in the subject prior to detecting phase of SNPs.
5. The method according to any one of the preceding claims, further comprising diagnosing the disease in the subject prior to detecting phase of SNPs.
6. The method according to any one of the preceding claims, wherein the detecting phase of SNPs excludes sequencing a full genome in a sample from the subject.
7. The method according to any one of the preceding claims, further comprising obtaining a sample form the subject, and the detecting phase of SNPs from the sample.
8. The method according to any one of the preceding claims, wherein the engineered CRISPR/Cas system comprises
(a) an engineered CRISPR/Cas system comprising:
(i) a Cas nuclease;
(ii) a first sgRNA comprising a first CRISPR targeting RNA (crRNA) sequence that hybridizes to a nucleotide sequence complementary to a first target sequence, the first target sequence being adjacent to a first protospacer adjacent motif (PAM) at 3 ’-end side of a disease-causing mutation or SNP in cis, wherein the first target sequence or the first PAM comprises a first ancestral variation or SNP site; and (iii) a second sgRNA comprising a second crRNA sequence that hybridizes to a nucleotide sequence complementary to a second target sequence, the second target sequence being adjacent to a second PAM at 5 ’-end side of the disease-causing mutation or SNP in cis, wherein the second target sequence or the second PAM comprises a second ancestral variation or SNP site, wherein the Cas nuclease and a crRNA sequence do not naturally occur together in the subject; or (b) at least one vector comprising
(i) a nucleotide molecule encoding Cas nuclease;
(ii) a first sgRNA comprising a first CRISPR targeting RNA (crRNA) sequence that hybridizes to a nucleotide sequence complementary to a first target sequence, the first target sequence being adjacent to a first protospacer adjacent motif (PAM) at 3 ’-end side of a disease-causing mutation or SNP in cis, wherein the first target sequence or the first PAM comprises a first ancestral variation or SNP site; and
(iii) a second sgRNA comprising a second crRNA sequence that hybridizes to a nucleotide sequence complementary to a second target sequence, the second target sequence being adjacent to a second PAM at 5 ’-end side of the disease-causing mutation or SNP in cis, wherein the second target sequence or the second PAM comprises a second ancestral variation or SNP site, wherein the at least one vector does not have a nucleotide molecule encoding Cas nuclease and a crRNA sequence that naturally occur together.
9. The method according to any one of the preceding claims, wherein the disease is an autosomal dominant disease.
10. The method according to any one of the preceding claims, wherein the disease is selected from the group consisting of Acropectoral syndrome, Acute intermittent porphyria, Adermatoglyphia, Albright's hereditary osteodystrophy, Arakawa's syndrome II, Aromatase excess syndrome, Autosomal dominant cerebellar ataxia, Axenfeld syndrome, Benign hereditary chorea, Bethlem myopathy, Birt- Hogg-Dube syndrome, Boomerang dysplasia, Branchio-oto-renal syndrome, Buschke-Ollendorff syndrome, Camurati-Engelmann disease, Central core disease, Collagen disease, Collagenopathy, types II and XI, Congenital distal spinal muscular atrophy, Congenital stromal comeal dystrophy, Costello syndrome, Currarino syndrome, Darier's disease, Glutl deficiency, Dentatorubral-pallidoluysian atrophy, Dermatopathia pigmentosa reticularis, Dysfibrinogenemia, Transthyretin-related hereditary amyloidosis, Familial atrial fibrillation, Familial hypercholesterolemia, Familial male-limited precocious puberty, Feingold syndrome, Felty's syndrome, Flynn-Aird syndrome, Gardner's syndrome, Gillespie syndrome, Gray platelet syndrome, Greig cephalopoly syndactyly syndrome, Hajdu-Cheney syndrome, Hawkinsinuria, Hay-Wells syndrome, Hereditary elliptocytosis, Hereditary hemorrhagic telangiectasia, Hereditary mucoepithelial dysplasia, Hereditary spherocytosis, Holt-Oram syndrome, Huntington's disease, Huntington's disease-like syndrome, Hypertrophic cardiomyopathy, Hypoalphalipoproteinemia, Hypochondroplasia, Hypodysfibrinogenemia, Jackson-Weiss syndrome, Keratolytic winter erythema, Kniest dysplasia, Kostmann syndrome, Langer-Giedion syndrome, Larsen syndrome, Liddle's syndrome, Marfan syndrome, Marshall syndrome, Medullary cystic kidney disease, Metachondromatosis, Miller- Dieker syndrome, MOMO syndrome, Monilethrix, MonoMAC, Multiple endocrine neoplasia, Multiple endocrine neoplasia type 1, Multiple endocrine neoplasia type 2, Multiple endocrine neoplasia type 2b, Myelokathexis, Myotonic dystrophy, Naegeli-Franceschetti-Jadassohn syndrome, Nail-patella syndrome, Noonan syndrome, Oculopharyngeal muscular dystrophy, Pachyonychia congenital, Pallister- Hall syndrome, PAPA syndrome, Papillorenal syndrome, Parastremmatic dwarfism, Pelger-Huet anomaly, Peutz-Jeghers syndrome, Piebaldism, Platyspondylic lethal skeletal dysplasia, Torrance type, Polydactyly, Popliteal pterygium syndrome, Porphyria cutanea tarda, Pseudoachondroplasia, RASopathy, Reis-Bucklers comeal dystrophy, Romano-Ward syndrome, Rosselli-Gulienetti syndrome, Roussy-Levy syndrome, Rubinstein-Taybi syndrome, Saethre-Chotzen syndrome, Schmitt Gillenwater Kelly syndrome, Short QT syndrome, Singleton Merten syndrome, Spinal muscular atrophy with lower extremity predominance, Spinocerebellar ataxia, Spinocerebellar ataxia type 1, Spinocerebellar ataxia type 6, Spondyloepimetaphyseal dysplasia-Strudwick type, Spondyloepiphyseal dysplasia congenital, Spondyloperipheral dysplasia, Stickler syndrome, Tietz syndrome, Timothy syndrome, Treacher Collins syndrome, Tricho-dento-osseous syndrome, Tuberous sclerosis, Upington disease, Variegate porphyria, Vitelliform macular dystrophy, Von Hippel-Lindau disease, Von Willebrand disease, Wallis-Zieff- Goldblatt syndrome, WHIM syndrome, White sponge nevus, Worth syndrome, Zaspopathy, Zimmermann-Laband syndrome, and Zori-Stalker-Williams syndrome.
11. The method according to any one of the preceding claims, wherein the disease is an autosomal dominant disease of an eye.
12. The method according to any one of the preceding claims, wherein the disease is corneal dystrophy.
13. The method according to any one of the preceding claims, wherein the disease-causing mutation or SNP is in an exon of a gene causing the disease.
14. The method according to any one of the preceding claims, wherein the first and second PAMs are in different introns surrounding one or more exons containing the disease-causing mutation or SNP.
15. The method according to any one of the preceding claims, wherein the first PAM comprises the first ancestral variation or SNP site and/or the second PAM comprises the second ancestral variation or SNP site.
16. The method according to any one of the preceding claims, wherein the first crRNA sequence comprises the first target sequence; the second crRNA sequence comprises the second target sequence; the first crRNA sequence is from 17 to 24 nucleotide long; and/or the second crRNA sequence is from 17 to 24 nucleotide long.
17. The method according to any one of the preceding claims, wherein the first and/or second PAMs and the Cas nuclease are from Streptococcus or Staphylococcus.
18. The method according to any one of the preceding claims, wherein the first and second PAMs are both from Streptococcus or Staphylococcus.
19. The method according to any one of the preceding claims, wherein the Cas nuclease is Cas9 nuclease.
20. The method according to claim 19, wherein each of the first and second PAMs independently consists of NGG or NNGRRT, wherein N is any of A, T, G, and C, and R is A or G.
21. The method according to any one of the preceding claims, wherein the Cas nuclease is Cpfl nuclease.
22. The method according to any one of the preceding claims, wherein the administering comprises injecting the engineered CRISPR/Cas system into the subject.
23. The method according to any one of the preceding claims, wherein the administering comprises introducing the engineered CRISPR/Cas system into a cell containing and expressing a DNA molecule having the target sequence.
24. The method according to any one of the preceding claims, wherein the disease is associated with the SNP; the first target sequence or the first PAM comprises the first ancestral SNP site; and/or the second target sequence or the second PAM comprises the second ancestral SNP site.
25. The method according to any one of the preceding claims, wherein the target sequence or the PAM comprises a plurality of mutation or SNP sites.
26. The method according to any one of the preceding claims, wherein the subject is human.
27. The method according to any one of the preceding claims, further comprising: prior to administering to the subject the engineered CRISPR/Cas system: obtaining sequence information of the subject; and selecting the first crRNA sequence and/or the second crRNA sequence based on the sequence information of the subject.
28. The method of claim 27, wherein: the sequence information of the subject includes whole-genome sequence information of the subject.
29. The method according to any one of the preceding claims, wherein: the first crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves at a first cleaving site that is adjacent to the first ancestral variation or SNP site; and/or the second crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves at a second cleaving site that is adjacent to the second ancestral variation or SNP site.
30. The method according to claim 29, wherein: the first crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves only at the first cleaving site that is adjacent to the first ancestral variation or SNP site; and/or the second crRNA sequence hybridizes to the nucleotide sequence so that the Cas nuclease cleaves only at the second cleaving site that is adjacent to the second ancestral variation or SNP site.
31. The method according to any one of the preceding claims, wherein: the first crRNA sequence hybridizes to the nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence in trans not being adjacent to the 5 ’-end of a PAM; and/or the second crRNA sequence hybridizes to the nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence in trans not being adjacent to the 5 ’-end of a PAM.
32. The method according to any one of the preceding claims, wherein: the first crRNA sequence hybridizes to the nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence in trans not being adjacent to the 5 ’-end of a PAM; and the second crRNA sequence hybridizes to the nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence in trans being adjacent to the 5 ’-end of a PAM.
33. The method according to any one of the preceding claims, wherein: the first crRNA sequence hybridizes to the nucleotide sequence complementary to the first target sequence in trans with the disease-causing mutation or SNP, said first target sequence in trans being adjacent to the 5 ’-end of a PAM; and the second crRNA sequence hybridizes to the nucleotide sequence complementary to the second target sequence in trans with the disease-causing mutation or SNP, said second target sequence in trans not being adjacent to the 5 ’-end of a PAM.
34. The method according to any one of the preceding claims, wherein the disease-causing mutation or SNP is not located in TGFBI.
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