RNA-guided nucleases and DNA binding proteins
Compositions and methods related to Cas proteins, nucleic acids encoding the Cas proteins, and modified host cells comprising the Cas proteins and/or encoding nucleic acids are disclosed. Cas proteins are useful in a variety of applications. Cas proteins bind guide RNAs that in turn provide functional specificity to the Cas proteins, nucleic acids encoding the Cas guide RNAs, and modified host cells comprising the Cas guide RNAs and/or encoding nucleic acids. The Cas polypeptides and corresponding guide RNAs can be used in a variety of applications.
1 . A composition comprising:
a) a CasP6 polypeptide, or a nucleic acid molecule encoding the CasP6 polypeptide, wherein the CasP6 polypeptide has 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3, wherein the CasP6 polypeptide comprises a RuvC-I domain, a RuvC-II domain, and a RuvC-III domain, and
wherein the RuvC-I domain comprises the amino acid sequence of SEQ ID NO: 10, 13, or 16 or one of the sequences comprising a single conservative amino acid substitution, the RuvC-II domain comprises the amino acid sequence of SEQ ID NO: 11, 14, or 17 or one of the sequences comprising a single conservative amino acid substitution, and the RuvC-III domain comprises the amino acid sequence of SEQ ID NO: 12, 15, or 18 or one of the sequences comprising a single conservative amino acid substitution, and
wherein the CasP6 polypeptide comprises catalytic amino acid residues of D434 and E617 when numbered according to SEQ ID NO: 1, D484 and E667 when numbered according to SEQ ID NO: 2, or D503 and E750 when numbered according to SEQ ID NO: 3; and
b) a recombinant CasP6 guide RNA, or one or more recombinant DNA molecules encoding the CasP6 guide RNA, wherein:
(i) the recombinant CasP6 guide RNA is capable of binding to a CasP6 protein, forming a ribonucleoprotein complex (RNP), and targeting the complex to a specific location within a target single stranded RNA (ssRNA); and
(ii) the CasP6 polypeptide and the recombinant CasP6 guide RNA, or one or more recombinant DNA molecules encoding the recombinant CasP6 guide RNA, do not naturally occur together.
2 . The composition of claim 1 , wherein the recombinant CasP6 guide RNA is a single guide RNA.
3 . The composition of claim 2 , wherein the recombinant guide RNA comprises an RNA encoded by residues 17-37 of SEQ ID NO: 4, residues 18-38 of SEQ ID NO: 5, or residues 15-36 of SEQ ID NO: 6.
4 . The composition of claim 1 , comprising one or more of: a lipid, a buffer, a nuclease inhibitor, and a protease inhibitor.
5 . A CasP6 fusion polypeptide comprising: a CasP6 polypeptide fused to a heterologous polypeptide, wherein the CasP6 polypeptide has 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3, wherein the CasP6 polypeptide comprises a RuvC-I domain, a RuvC-II domain, and a RuvC-III domain, and
wherein the RuvC-I domain comprises the amino acid sequence of SEQ ID NO: 10, 13, or 16 or one of the sequences comprising a single conservative amino acid substitution, the RuvC-II domain comprises the amino acid sequence of SEQ ID NO: 11, 14, or 17 or one of the sequences comprising a single conservative amino acid substitution, and the RuvC-III domain comprises the amino acid sequence of SEQ ID NO: 12, 15, or 18 or one of the sequences comprising a single conservative amino acid substitution, and
wherein the CasP6 polypeptide comprises catalytic amino acid residues of D434 and E617 when numbered according to SEQ ID NO: 1, D484 and E667 when numbered according to SEQ ID NO: 2, or D503 and E750 when numbered according to SEQ ID NO: 3.
6 . A nucleic acid molecule encoding the CasP6 fusion polypeptide of claim 5 .
7 . A non-human eukaryotic cell or an isolated human cell comprising:
a) a CasP6 polypeptide, or a nucleic acid molecule encoding the CasP6 polypeptide, wherein the CasP6 polypeptide has 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3, wherein the CasP6 polypeptide comprises a RuvC-I domain, a RuvC-II domain, and a RuvC-III domain, and
wherein the RuvC-I domain comprises the amino acid sequence of SEQ ID NO: 10, 13, or 16 or one of the sequences comprising a single conservative amino acid substitution, the RuvC-II domain comprises the amino acid sequence of SEQ ID NO: 11, 14, or 17 or one of the sequences comprising a single conservative amino acid substitution, and the RuvC-III domain comprises the amino acid sequence of SEQ ID NO: 12, 15, or 18 or one of the sequences comprising a single conservative amino acid substitution, and
wherein the CasP6 polypeptide comprises catalytic amino acid residues of D434 and E617 when numbered according to SEQ ID NO: 1, D484 and E667 when numbered according to SEQ ID NO: 2, or D503 and E750 when numbered according to SEQ ID NO: 3; and
b) a CasP6 guide RNA, or a nucleic acid molecule encoding the CasP6 guide RNA, wherein the recombinant CasP6 guide RNA is capable of binding to a CasP6 protein, forming a ribonucleoprotein complex (RNP), and targeting the complex to a specific location within a target single stranded RNA (ssRNA).
8 . The non-human eukaryotic cell or isolated human cell of claim 7 , comprising the nucleic acid molecule encoding the CasP6 polypeptide, wherein said nucleic acid molecule is integrated into the genomic DNA of the cell.
9 . The eukaryotic cell of claim 7 , wherein the eukaryotic cell is a plant cell, a non-human mammalian cell, an insect cell, an arachnid cell, a fungal cell, a bird cell, a reptile cell, an amphibian cell, an invertebrate cell, a mouse cell, a rat cell, an isolated primate cell, or a non-human primate cell.
10 . A method of modifying a target single stranded RNA (ssRNA), the method comprising contacting the target ssRNA with:
a) a CasP6 polypeptide, wherein the CasP6 polypeptide has 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3, wherein the CasP6 polypeptide comprises a RuvC-I domain, a RuvC-II domain, and a RuvC-III domain, and
wherein the RuvC-I domain comprises the amino acid sequence of SEQ ID NO: 10, 13, or 16 or one of the sequences comprising a single conservative amino acid substitution, the RuvC-II domain comprises the amino acid sequence of SEQ ID NO: 11, 14, or 17 or one of the sequences comprising a single conservative amino acid substitution, and the RuvC-III domain comprises the amino acid sequence of SEQ ID NO: 12, 15, or 18 or one of the sequences comprising a single conservative amino acid substitution, and
wherein the CasP6 polypeptide comprises catalytic amino acid residues of D434 and E617 when numbered according to SEQ ID NO: 1, D484 and E667 when numbered according to SEQ ID NO: 2, or D503 and E750 when numbered according to SEQ ID NO: 3; and
b) a recombinant CasP6 guide RNA comprising a guide sequence that hybridizes to a target sequence of the target ssRNA, wherein:
(i) the recombinant CasP6 guide RNA is capable of binding to a CasP6 protein, forming a ribonucleoprotein complex (RNP), and targeting the complex to a specific location within the target ssRNA; and
(ii) said contacting results in modification of the target ssRNA by the CasP6 polypeptide/CasP6 guide RNA RNP complex; and
(iii) the CasP6 polypeptide and the recombinant CasP6 guide RNA do not naturally occur together.
11 . The method of claim 10 , wherein said contacting takes place inside of a cell in culture or inside of a cell in vivo and wherein the cell is a non-human eukaryotic cell or an isolated human cell, optionally wherein the non-human eukaryotic cell is a plant cell, a fungal cell, a non-human mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, or a non-human primate cell.
12 . The method of claim 10 , wherein said contacting results in cleavage of the target ssRNA.
13 . The method of claim 10 , wherein said contacting comprises: introducing into a cell: (a) the CasP6 polypeptide, or a nucleic acid molecule encoding the CasP6 polypeptide, and (b) the CasP6 guide RNA, or a nucleic acid molecule encoding the CasP6 guide RNA.
14 . The method of claim 10 , wherein the recombinant CasP6 guide RNA is a single guide RNA.
15 . The method of claim 14 , wherein the guide RNA comprises an RNA encoded by residues 17-37 of SEQ ID NO: 4, residues 18-38 of SEQ ID NO: 5, or residues 15-36 of SEQ ID NO: 6.
16 . A composition comprising:
a) a CasP6 fusion polypeptide comprising a CasP6 polypeptide fused to a heterologous polypeptide, or a nucleic acid molecule encoding the CasP6 fusion polypeptide, wherein the CasP6 fusion polypeptide has 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3, wherein the CasP6 polypeptide comprises a RuvC-I domain, a RuvC-II domain, and a RuvC-III domain, and
wherein the RuvC-I domain comprises the amino acid sequence of SEQ ID NO: 10, 13, or 16 or one of the sequences comprising a single conservative amino acid substitution, the RuvC-II domain comprises the amino acid sequence of SEQ ID NO: 11, 14, or 17 or one of the sequences comprising a single conservative amino acid substitution, and the RuvC-III domain comprises the amino acid sequence of SEQ ID NO: 12, 15, or 18 or one of the sequences comprising a single conservative amino acid substitution, and
wherein the CasP6 polypeptide comprises catalytic amino acid residues of D434 and E617 when numbered according to SEQ ID NO: 1, D484 and E667 when numbered according to SEQ ID NO: 2, or D503 and E750 when numbered according to SEQ ID NO: 3; and
b) a recombinant CasP6 guide RNA, or one or more DNA molecules encoding the recombinant CasP6 guide RNA, wherein:
(i) the recombinant CasP6 guide RNA is capable of binding to the CasP6 fusion protein, forming a ribonucleoprotein complex (RNP), and targeting the complex to a specific location within a target single stranded RNA (ssRNA); and
(ii) the CasP6 fusion polypeptide and the recombinant CasP6 guide RNA, or one or more DNA molecules encoding the recombinant CasP6 guide RNA, do not naturally occur together.