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 CasJ fusion polypeptide comprising a heterologous polypeptide fused to an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 1, or a nucleic acid molecule encoding the CasJ fusion polypeptide; and
b) a CasJ guide RNA, or one or more DNA molecules encoding the CasJ guide RNA.
2. The composition of claim 1 , wherein the CasJ guide RNA is a single guide RNA.
3. The composition of claim 1 , wherein the CasJ guide RNA comprises an RNA encoded by SEQ ID NO:15.
4. The composition of claim 1 , wherein the composition comprises a lipid or is within a liposome.
5. The composition of claim 1 , wherein the CasJ fusion polypeptide and the CasJ guide RNA are within and/or at least partially coat a particle.
6. The composition of claim 1 , wherein the composition further comprises one or more of: a buffer, a nuclease inhibitor, and a protease inhibitor.
7. A composition comprising:
a) a CasJ polypeptide comprising an amino acid sequence having at least 80% but less than 100% identity to the amino acid sequence of SEQ ID NO: 1, or a nucleic acid molecule encoding the CasJ polypeptide, wherein the CasJ polypeptide is a catalytically inactive CasJ Polypeptide (dCasJ) and
b) a CasJ guide RNA, or one or more DNA molecules encoding the CasJ guide RNA.
8. The composition of claim 7 , wherein the CasJ polypeptide comprises one or more mutations at a position corresponding to those selected from D901, E1128, and/or D1298 of SEQ ID NO: 1.
9. The composition of claim 1 , further comprising a DNA donor template.
10. A CasJ fusion polypeptide comprising a heterologous polypeptide fused to a CasJ polypeptide comprising an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 1.
11. The CasJ fusion polypeptide of claim 10 , wherein the CasJ polypeptide is a catalytically inactive CasJ Polypeptide (dCasJ).
12. The CasJ fusion polypeptide of claim 11 , wherein the CasJ polypeptide comprises one or more mutations selected from D901A, E1128A, and/or D1298A by reference to SEQ ID NO: 1.
13. The CasJ fusion polypeptide of claim 10 , wherein the heterologous polypeptide is operably linked to the N-terminus and/or the C-terminus of the CasJ polypeptide.
14. The CasJ fusion polypeptide of claim 10 , wherein the heterologous polypeptide comprises a comprising a nuclear localization signal (NLS), and endosomal escape peptide, and/or a chloroplast transit peptide.
15. The CasJ fusion polypeptide of claim 10 , wherein the heterologous polypeptide exhibits an enzymatic activity that modifies target DNA.
16. The CasJ fusion polypeptide of claim 15 , wherein the heterologous polypeptide exhibits one or more enzymatic activities selected from: nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, and/or glycosylase activity.
17. The CasJ fusion polypeptide of claim 14 , wherein the chloroplast transit peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3-12, and 13.
18. The CasJ fusion polypeptide of claim 11 , wherein the heterologous polypeptide protein increases or decreases transcription of a gene when the CasJ fusion polypeptide is bound to the gene and a guide RNA in comparison to when the CasJ fusion polypeptide is not bound to the gene and a guide RNA.
19. The CasJ fusion polypeptide of claim 18 , wherein the heterologous polypeptide is a transcriptional repressor domain.
20. The CasJ fusion polypeptide of claim 18 , wherein the heterologous polypeptide is a transcriptional activation domain.