Systems and methods for one-shot guide RNA (ogRNA) targeting of endogenous and source DNA
Engineered nucleic acids encoding genome editing system components are provided, as are engineered RNA-guided nucleases that include inserts encoded in part by cellular genomic or other sequences recognized by guide RNAs.
1. A transiently active genome editing system, comprising: a guide RNA (gRNA) comprising a targeting domain that is complementary to a eukaryotic nucleic acid sequence; and an engineered RNA-guided nuclease encoded by a nucleic acid comprising the eukaryotic nucleotide sequence and a protospacer adjacent motif (PAM), wherein the PAM is recognized by the RNA-guided nuclease and is within or adjacent to the eukaryotic nucleotide sequence,
wherein the engineered RNA-guided nuclease is a Cas9 protein,
wherein the Cas9 protein comprises an amino acid insertion relative to SEQ ID NO: 2 selected from the group consisting of:
E271_N272insGX 6-10 G,
L371_N372insGX 6-10 G, and
Q737_A738insGX 6-10 G; or
wherein the Cas9 protein comprises an amino acid insertion S12_M13insGX 6-10 G relative to SEQ ID NO: 10,
which results in a polypeptide comprising an amino acid sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 3-5 and 10 and having an RNA-guided nuclease activity.
2. The transiently active genome editing system of claim 1 , wherein the engineered Cas9 protein and the gRNA form a Cas9/gRNA complex.
3. The transiently active genome editing system of claim 2 , wherein the Cas9/gRNA complex is adapted to cleave the nucleic acid encoding the engineered RNA-guided nuclease.
4. The transiently active genome editing system of claim 1 , wherein the engineered Cas9 protein has at least about 80% nuclease activity of a wild-type Cas9 protein.
5. The transiently active genome editing system of claim 1 , wherein the engineered Cas9 protein is encoded by a nucleic acid comprising an insertion, relative to SEQ ID NO: 6, selected from the group consisting of:
c.813_814insN 24-36 ,
c.1113_1114insN 24-36 , and
c.2211_2212insN 24-36 ; or
comprising an insertion c.36_37insN 24-36 relative to SEQ ID NO: 11.
6. The transiently active genome editing system of claim 5 , wherein the engineered Cas9 protein is encoded by a nucleic acid comprising a sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 7-9 and 11.
7. The transiently active genome editing system of claim 1 , wherein the engineered Cas9 protein is an engineered S. aureus Cas9.
8. The transiently active genome editing system of claim 1 , wherein the system alters both a cellular endogenous target gene and the RNA-guided nuclease expression.
9. The transiently active genome editing system of claim 1 , wherein the eukaryotic nucleic acid sequence is at least 17 nucleotides in length.
10. The transiently active genome editing system of claim 1 , wherein the targeting domain of the gRNA is 16-24 nucleotides in length.
11. The transiently active genome editing system of claim 1 , wherein the engineered Cas9 protein comprises an amino acid insertion relative to SEQ ID NO: 2, which is E271_N272insGX 6-10 G.
12. The transiently active genome editing system of claim 1 , wherein the engineered Cas9 protein comprises an amino acid insertion relative to SEQ ID NO: 2, which is L371_N372insGX 6-10 G.
13. The transiently active genome editing system of claim 1 , wherein the engineered Cas9 protein comprises an amino acid insertion relative to SEQ ID NO: 2, which is Q737_A738insGX 6-10 G.
14. The transiently active genome editing system of claim 1 , wherein the engineered Cas9 protein comprises an amino acid insertion relative to SEQ ID NO: 10, which is S12_M13insGX 6-10 G.
15. The transiently active genome editing system of claim 1 , wherein the nucleic acid encoding the engineered Cas9 protein comprises an insertion relative to SEQ ID NO: 6, which is c.813_814insN 24-36 .
16. The transiently active genome editing system of claim 1 , wherein the nucleic acid encoding the engineered Cas9 protein comprises an insertion relative to SEQ ID NO: 6, which is c.1113_1114insN 24-36 .
17. The transiently active genome editing system of claim 1 , wherein the nucleic acid encoding the engineered Cas9 protein comprises an insertion relative to SEQ ID NO: 6, which is c.2211_2212insN 24-36 .
18. The transiently active genome editing system of claim 1 , wherein the nucleic acid encoding the engineered Cas9 protein comprises an insertion relative to SEQ ID NO: 11, which is c.36_37insN 24-36 .