Anti-CRISPR nucleic acid inhibitors of CRISPR-Cas effector enzymes
A CRISPR inhibitor molecule is provided, comprising an artificial nucleic acid construct having a first polynucleotide, the inhibitor molecule capable of establishing several points of contact with a CRISPR protein and high binding affinity thereto, is provided. The first polynucleotide may comprise a sequence selected from the group consisting of: a polynucleotide that interacts with a protospacer adjacent motif (PAM)-interaction (PI) domain of a CRISPR-associated (Cas) protein, a polynucleotide that interacts with a guide sequence of a crRNA or an equivalent position of a single-guide RNA, and a polynucleotide that interacts with a repeat region of a tracrRNA or an equivalent position of a single-guide RNA. The CRISPR inhibitor molecule may also comprise a second polynucleotide and a linker. Methods of using the CRISPR inhibitor molecule in therapeutic agent selection and creation, as well as part of a therapeutic treatment, are also provided.
1 . A CRISPR inhibitor molecule comprising an artificial nucleic acid construct having a first polynucleotide, a second polynucleotide, and a linker molecule, wherein:
the first polynucleotide is a polynucleotide that interacts with a protospacer adjacent motif-interaction domain of a CRISPR-associated protein and comprises a sequence selected from the group consisting of:
SEQ ID NO: 1 or a sequence at least 95% identical thereto; and
SEQ ID NO: 2 or a sequence at least 95% identical thereto;
the second polynucleotide is a polynucleotide that interacts with a repeat region of a tracrRNA or an equivalent position of a single-guide RNA and comprises a sequence selected from the group consisting of:
SEQ ID NO: 4 or a sequence at least 95% identical thereto;
SEQ ID NO: 5 or a sequence at least 95% identical thereto; and
SEQ ID NO: 6 or a sequence at least 95% identical thereto;
and wherein the linker molecule is operably connected to said first polynucleotide and operably connected to said second polynucleotide.
2 . The CRISPR inhibitor molecule of claim 1 , wherein said second polynucleotide further comprises a polynucleotide that interacts with a guide sequence of a crRNA or an equivalent position of a single-guide RNA.
3 . The CRISPR inhibitor molecule of claim 2 , wherein said polynucleotide that interacts with said guide sequence of said crRNA or said equivalent position of said single-guide RNA comprises:
a 2′-deoxyribonucleotide;
a 2′-deoxyribonucleotide analog;
a ribonucleotide;
a ribonucleotide analog; or
a combination thereof.
4 . The CRISPR inhibitor molecule of claim 2 , wherein said polynucleotide that interacts with said guide sequence of said crRNA or said equivalent position of said single-guide RNA comprises at least one internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, phosphotriester, phosphorodithioate, boranophosphate, Rp- and/or Sp-phosphorothioate, 3′ thioformacetal, methylene, amide, methylphosphonate, phosphoramidate and any combination thereof.
5 . The CRISPR inhibitor molecule of claim 2 , wherein said polynucleotide that interacts with said guide sequence of said crRNA or said equivalent position of said single-guide RNA comprises SEQ ID NO: Z or a sequence at least 95% identical thereto.
6 . The CRISPR inhibitor molecule of claim 1 , wherein said polynucleotide that interacts with said repeat region of said tracrRNA or said equivalent position of said single-guide RNA comprises:
a 2′-deoxyribonucleotide;
a 2′-deoxyribonucleotide analog;
a ribonucleotide;
a ribonucleotide analog; or
a combination thereof.
7 . The CRISPR inhibitor molecule of claim 1 , wherein said polynucleotide that interacts with said repeat region of said tracrRNA or said equivalent position of said single-guide RNA comprises at least one internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, phosphotriester, phosphorodithioate, boranophosphate, Rp- and/or Sp-phosphorothioate, 3′ thioformacetal, methylene, amide, methylphosphonate, phosphoramidate and any combination thereof.
8 . The CRISPR inhibitor molecule of claim 1 , wherein said linker molecule comprises polyethylene glycol.
9 . The CRISPR inhibitor molecule of claim 1 , wherein said linker molecule comprises one or more nucleic acids, wherein a linker molecule having more than one nucleic acid may comprise a single stranded oligonucleotide having a spacer functionality within the artificial nucleic acid construct.
10 . The CRISPR inhibitor molecule of claim 1 , wherein the first polynucleotide comprises:
a 2′-deoxyribonucleotides;
a 2′-deoxyribonucleotide analog;
a ribonucleotide;
a ribonucleotide analog; or
any combination thereof.
11 . The CRISPR inhibitor molecule of claim 1 , wherein the first polynucleotide comprises at least one internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, phosphotriester, phosphorodithioate, boranophosphate, Rp- and/or Sp-phosphorothioate, 3′ thioformacetal, methylene, amide, methylphosphonate, phosphoramidate and any combination thereof.