Methods and compositions for modifying a single stranded target nucleic acid

Abstract

The present disclosure provides compositions and methods for binding and/or cleaving a single stranded target nucleic acid. Subject compositions include a Cas9 polypeptide, a guide nucleic acid, and a PAMmer. A subject PAMmer is a single stranded oligonucleotide having a proto spacer adjacent motif (PAM) sequence and at least one of: a specificity segment positioned 5′ of the PAM sequence, and an orientation segment positioned 3′ of the PAM sequence. In some embodiments, the Cas9 polypeptide is a variant Cas9 polypeptide having reduced nuclease activity relative to a corresponding wild type Cas9 polypeptide. In some cases, methods of binding are for visualizing single stranded target nucleic acids using a detectable label. In some cases, methods of binding are for isolating, collecting, and/or analyzing at least one of: (i) bound single stranded target nucleic acids; and (ii) polypeptides associated with bound single stranded target nucleic acids.

What is claimed is: 
     
         1 . A method of cleaving a single stranded target nucleic acid, the method comprising:
 contacting the single stranded target nucleic acid with:
 (i) a Cas9 polypeptide; 
 (ii) a guide nucleic acid comprising: 
   (a) a targeting segment comprising a nucleotide sequence that is complementary to a first target nucleotide sequence in the single stranded target nucleic acid, and   (b) a protein-binding segment comprising two stretches of nucleotides that hybridize with one another to form a double-stranded RNA duplex that interacts with the Cas9 polypeptide; and
 (iii) a PAMmer, wherein the PAMmer is a single stranded oligonucleotide comprising:
 (a) a protospacer adjacent motif (PAM) sequence, and 
 (b) at least one of: 
 
   (i) an orientation segment, positioned 3′ of the PAM sequence, comprising a nucleotide sequence that is complementary to a second target nucleotide sequence in the target nucleic acid; and   (ii) a specificity segment, positioned 5′ of the PAM sequence, comprising a nucleotide sequence that is complementary to said first target nucleotide sequence.   
     
     
         2 . The method according to  claim 1 , wherein the single stranded target nucleic acid is a single stranded RNA (ssRNA). 
     
     
         3 . The method according to  claim 2 , wherein the target ssRNA is selected from: mRNA, rRNA, tRNA, non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and microRNA (miRNA). 
     
     
         4 . The method according to  claim 3 , wherein the target ssRNA is mRNA and the method results in reduced production of a protein encoded by the mRNA. 
     
     
         5 . The method according to  claim 1 , wherein the single stranded target nucleic acid is single stranded DNA (ssDNA). 
     
     
         6 . The method according to any of  claims 1  to  5 , wherein the single stranded target nucleic acid is from a virus. 
     
     
         7 . The method according to any of  claims 1  to  6 , wherein the PAMmer is covalently linked to the guide nucleic acid. 
     
     
         8 . The method according to any of  claims 1  to  7 , wherein the PAMmer is covalently linked to the Cas9 polypeptide. 
     
     
         9 . The method according to any of  claims 1  to  8 , wherein the PAMmer comprises an orientation segment. 
     
     
         10 . The method according to any of  claims 1  to  9 , wherein the PAMmer does not comprise a specificity segment. 
     
     
         11 . The method according to  claim 10 , wherein the PAM sequence is the 5′ end of the PAMmer. 
     
     
         12 . The method according to any of  claims 1  to  9 , wherein the PAMmer comprises a specificity segment. 
     
     
         13 . The method according to any of  claims 1  to  8  or  claim 12 , wherein the PAMmer does not comprise an orientation segment. 
     
     
         14 . The method according to  claim 13 , wherein the PAM sequence is the 3′ end of the PAMmer. 
     
     
         15 . The method according to any of  claims 1  to  8 , wherein the PAMmer comprises an orientation segment and a specificity segment. 
     
     
         16 . The method according to any of  claims 1  to  15 , wherein the PAMmer comprises a detectable label. 
     
     
         17 . The method according to  claim 16 , wherein the detectable label is a fluorescent label. 
     
     
         18 . The method according to any of  claims 1  to  17 , wherein the PAM sequence is GG. 
     
     
         19 . The method according to any of  claims 1  to  17 , wherein the PAM sequence is 5′-NGG-3′ and N can be any nucleotide. 
     
     
         20 . The method of any of  claims 1  to  19 , wherein said contacting is outside of a cell in vitro. 
     
     
         21 . The method according to any of  claims 1  to  19 , wherein said contacting is in a cell in vitro or ex vivo. 
     
     
         22 . The method according to any of  claims 1  to  19 , wherein said contacting is in a cell in vivo. 
     
     
         23 . The method according to any of  claims 1  to  22 , wherein the guide nucleic acid is a DNA/RNA hybrid nucleic acid and the first segment of the guide nucleic acid comprises DNA. 
     
     
         24 . The method according to any of  claims 1  to  22 , wherein the guide nucleic acid is a guide RNA. 
     
     
         25 . The method according to any of  claims 1  to  24 , wherein the guide nucleic acid is a dual guide nucleic acid, wherein the two stretches of nucleotides that hybridize with one another are present on separate nucleic acid molecules. 
     
     
         26 . The method according to any of  claims 1  to  24 , wherein the guide nucleic acid is a single guide nucleic acid, wherein the two stretches of nucleotides that hybridize with one another are present on the same nucleic acid molecule and are covalently linked by intervening nucleotides. 
     
     
         27 . The method according to any of  claims 1  to  26 , wherein the target nucleic acid:
 (i) does not have a nucleotide sequence that is complementary to the PAM sequence at a position within 10 nucleotides of the 3′ end of the specificity segment of the PAMmer; or 
 (ii) does not have a nucleotide sequence that is complementary to the PAM sequence at a position within 10 nucleotides of the 5′ end of the orientation segment of the PAMmer. 
 
     
     
         28 . The method according to any of  claims 1  to  27 , wherein 10 or less nucleotides are present in the target nucleic acid between the first and second target nucleotide sequences. 
     
     
         29 . The method according to any of  claims 1  to  28 , wherein 2 or 3 nucleotides are present in the target nucleic acid between the first and second target nucleotide sequences. 
     
     
         30 . A method of binding a single stranded target nucleic acid, the method comprising:
 contacting the single stranded target nucleic acid with:
 (i) a variant Cas9 polypeptide having an amino acid mutation that results in reduced nuclease activity relative to a corresponding wild type Cas9 polypeptide; and 
 (ii) a guide nucleic acid comprising: 
   (a) a targeting segment comprising a nucleotide sequence that is complementary to a first target nucleotide sequence in the single stranded target nucleic acid, and   (b) a protein-binding segment comprising two stretches of nucleotides that hybridize with one another to form a double-stranded RNA duplex that interacts with the Cas9 polypeptide,
 wherein said contacting produces a variant-Cas9/target complex. 
   
     
     
         31 . The method according to  claim 30 , further comprising contacting the single stranded target nucleic acid with a PAMmer, wherein the PAMmer is a single stranded oligonucleotide comprising:
 (a) a protospacer adjacent motif (PAM) sequence, and   (b) at least one of:   (i) an orientation segment, positioned 3′ of the PAM sequence, comprising a nucleotide sequence that is complementary to a second target nucleotide sequence in the target nucleic acid; and   (ii) a specificity segment, positioned 5′ of the PAM sequence, comprising a nucleotide sequence that is complementary to said first target nucleotide sequence.   
     
     
         32 . The method according to  claim 31 , wherein the PAMmer comprises a detectable label. 
     
     
         33 . The method according to  claim 31  or  claim 32 , wherein the PAMmer comprises an orientation segment and a specificity segment. 
     
     
         34 . The method according to any of  claims 30  to  33 , wherein the variant Cas9 polypeptide comprises at least one of:
 (i) an H840A mutation of the  S. pyogenes  Cas9 amino acid sequence (SEQ ID NO:8) or the corresponding mutation in the amino acid sequence set forth in any of SEQ ID NOs: 1-7, 9-256, and 795-1346; and 
 (ii) W476A and W1126A mutations of the  S. pyogenes  Cas9 amino acid sequence (SEQ ID NO:8) or the corresponding mutation in the amino acid sequence set forth in any of SEQ ID NOs: 1-7, 9-256, and 795-1346. 
 
     
     
         35 . The method according to any of  claims 30  to  33 , wherein the variant Cas9 polypeptide comprises at least one of:
 (i) D10A and H840A mutations of the  S. pyogenes  Cas9 amino acid sequence (SEQ ID NO:8) or the corresponding mutations in the amino acid sequence set forth in any of SEQ ID NOs: 1-7, 9-256, and 795-1346; and 
 (ii) W476A and W1126A mutations of the  S. pyogenes  Cas9 amino acid sequence (SEQ ID NO:8) or the corresponding mutation in the amino acid sequence set forth in any of SEQ ID NOs: 1-7, 9-256, and 795-1346. 
 
     
     
         36 . The method according to any of  claims 30  to  35 , wherein the variant Cas9 polypeptide comprises a detectable label. 
     
     
         37 . The method according to  claim 36 , wherein the detectable label of the Cas9 polypeptide is a fluorescent protein. 
     
     
         38 . The method according to any of  claims 30  to  37 , wherein the guide nucleic acid comprises a detectable label. 
     
     
         39 . The method according to  claim 38 , wherein the detectable label of the guide nucleic acid is a directly detectable label. 
     
     
         40 . The method according to  claim 38 , wherein the detectable label of the guide nucleic acid is an indirectly detectable label. 
     
     
         41 . The method according to  claim 40 , wherein the indirectly detectable label of the guide nucleic acid is a nucleotide sequence that specifically binds a labeling protein. 
     
     
         42 . The method according to any of  claims 30  to  41 , wherein the variant Cas9 polypeptide comprises a fusion partner with an enzymatic activity, and the single stranded target nucleic acid is modified as a result of the method. 
     
     
         43 . The method according to any of  claims 30  to  42 , further comprising:
 isolating the variant-Cas9/target complex; 
 releasing the single stranded target nucleic acid from the variant-Cas9/target complex; and 
 collecting and/or analyzing the released single stranded target nucleic acid and/or a polypeptide associated with the single stranded target nucleic acid. 
 
     
     
         44 . A hybrid guide nucleic acid, comprising:
 (i) a targeting segment comprising a DNA nucleotide sequence that is complementary to a first target nucleotide sequence in a target nucleic acid; and   (ii) a protein-binding segment that comprises RNA, forms a double-stranded RNA duplex, and interacts with a Cas9 polypeptide.   
     
     
         45 . The hybrid guide nucleic acid of  claim 44 , wherein the protein-binding segment of the hybrid guide nucleic acid comprises a nucleotide sequence with 60% or more identity over a stretch of 8 or more contiguous nucleotides to a nucleotide sequence set forth in any of SEQ ID NOs: 431-682, or a complement thereof. 
     
     
         46 . The hybrid guide nucleic acid of  claim 44  or  claim 45 , wherein the hybrid guide nucleic acid is a dual guide nucleic acid. 
     
     
         47 . The hybrid guide nucleic acid of  claim 44  or  claim 45 , wherein the hybrid guide nucleic acid is a single guide nucleic acid. 
     
     
         48 . The hybrid guide nucleic acid of any of  claims 44  to  47 , wherein the guide nucleic acid is covalently linked to a PAMmer, wherein the PAMmer is a single stranded oligonucleotide comprising:
 a protospacer adjacent motif (PAM) sequence, and at least one of: 
 (i) an orientation segment, positioned 3′ of the PAM sequence, comprising a nucleotide sequence that is complementary to a second target nucleotide sequence in the target nucleic acid; and 
 (ii) a specificity segment, positioned 5′ of the PAM sequence, comprising a nucleotide sequence that is complementary to said first target nucleotide sequence. 
 
     
     
         49 . A PAM-modified Cas9 polypeptide, comprising a Cas9 polypeptide that is conjugated to a PAMmer, wherein the PAMmer is a single stranded oligonucleotide comprising:
 (a) a protospacer adjacent motif (PAM) sequence, and   (b) at least one of:   (i) an orientation segment, positioned 3′ of the PAM sequence, comprising a nucleotide sequence that is complementary to a second target nucleotide sequence in the target nucleic acid, and   (ii) a specificity segment, positioned 5′ of the PAM sequence, comprising a nucleotide sequence that is complementary to said rust target nucleotide sequence.   
     
     
         50 . A composition comprising:
 (i) a PAMmer, wherein the PAMmer is a single stranded oligonucleotide comprising:
 (a) a protospacer adjacent motif (PAM) sequence, and 
 (b) at least one of: 
   (b.i) an orientation segment, positioned 3′ of the PAM sequence, comprising a nucleotide sequence that is complementary to a first target nucleotide sequence in a target nucleic acid, and   (b.ii) a specificity segment, positioned 5′ of the PAM sequence, comprising a nucleotide sequence that is complementary to a second target nucleotide sequence in a target nucleic acid; and   at least one of:
 (ii) a Cas9 polypeptide, or a nucleic acid encoding the same; and 
 (iii) a guide nucleic acid, or a nucleic acid encoding the same, wherein the guide nucleic acid comprises: 
   (a) a targeting segment comprising a nucleotide sequence that is complementary to said first target nucleotide sequence in said single stranded target nucleic acid, and   (b) a protein-binding segment comprising two stretches of nucleotides that hybridize with one another to form a double-stranded RNA duplex that interacts with a Cas9 polypeptide.   
     
     
         51 . The composition of  claim 50 , wherein the guide nucleic acid is a guide RNA. 
     
     
         52 . The composition of  claim 50 , wherein the guide nucleic acid is a DNA/RNA hybrid guide nucleic acid, wherein the targeting segment of the guide nucleic acid comprises DNA. 
     
     
         53 . The composition of any of  claims 50  to  52 , wherein the guide nucleic acid is a dual guide nucleic acid, wherein the two stretches of nucleotides that hybridize with one another are present on separate nucleic acid molecules. 
     
     
         54 . The composition of any of  claims 50  to  52 , wherein the guide nucleic acid is a single guide nucleic acid, wherein the two stretches of nucleotides that hybridize with one another are present on the same nucleic acid molecule and are covalently linked by intervening nucleotides. 
     
     
         55 . The composition of any of  claims 50  to  54 , wherein the PAM sequence is the 5′ end of the PAMmer. 
     
     
         56 . The composition of any of  claims 50  to  55 , wherein the PAM sequence is GG. 
     
     
         57 . The composition of any of  claims 50  to  55 , wherein the PAM sequence is 5′-NGG-3′ and N can be any nucleotide. 
     
     
         58 . The composition of any of  claims 50  to  57 , wherein 10 or less nucleotides are present in the target nucleic acid between the first and second target sequences. 
     
     
         59 . The composition of any of  claims 50  to  58 , wherein 2 or 3 nucleotides are present in the target nucleic acid between the first and second target sequences. 
     
     
         60 . The composition of any of  claims 50  to  59 , wherein the Cas9 polypeptide is a variant Cas9 polypeptide having reduced nuclease activity relative to a corresponding wild type Cas9 polypeptide. 
     
     
         61 . A kit comprising:
 (i) a guide nucleic acid, or a nucleic acid encoding the same, wherein the guide nucleic acid comprises:   (a) a targeting segment comprising a nucleotide sequence that is complementary to a first target sequence in a target nucleic acid, and   (b) a protein-binding segment comprising two stretches of nucleotides that hybridize with one another to form a double-stranded RNA duplex that interacts with a Cas9 polypeptide; and   (ii) a PAMmer, or a nucleic acid encoding the same, wherein the PAMmer is a single stranded oligonucleotide comprising:
 (a) a protospacer adjacent motif (PAM) sequence, and 
 (b) at least one of: 
   (b.i) an orientation segment, positioned 3′ of the PAM sequence, comprising a nucleotide sequence that is complementary to a second target sequence in the target nucleic acid, and   (b.ii) a specificity segment, positioned 5′ of the PAM sequence, comprising a nucleotide sequence that is complementary to said first target sequence.   
     
     
         64 . A library comprising:
 two or more targeting nucleic acid pairs, wherein each targeting pair comprises:   (i) a guide nucleic acid, or a nucleic acid encoding the same, wherein the guide nucleic acid comprises:   (a) a targeting segment comprising a nucleotide sequence that is complementary to a first target sequence in a target nucleic acid, and   (b) a protein-binding segment comprising two stretches of nucleotides that hybridize with one another to form a double-stranded RNA duplex that interacts with a Cas9 polypeptide; and   (ii) a PAMmer, or a nucleic acid encoding the same, wherein the PAMmer is a single stranded oligonucleotide comprising:
 (a) a protospacer adjacent motif (PAM) sequence, and 
 (b) at least one of: 
   (b.i) an orientation segment, positioned 3′ of the PAM sequence, comprising a nucleotide sequence that is complementary to a second target sequence in the target nucleic acid, and   (b.ii) a specificity segment, positioned 5′ of the PAM sequence, comprising a nucleotide sequence that is complementary to said first target sequence in the target nucleic acid.