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. An engineered RNA-guided nuclease protein comprising an amino acid insertion or substitution at least partially encoded by a eukaryotic nucleic acid sequence of at least 17 nucleotides in length,
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,
wherein the insertion relative to SEQ ID NO: 2 or SEQ ID NO: 10 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 RNA-guided nuclease protein of claim 1 , wherein the RNA-guided nuclease has at least about 80% nuclease activity of a wild-type RNA-guided nuclease.
3. The RNA-guided nuclease protein of claim 1 , wherein the eukaryotic nucleic acid sequence is a mammalian sequence.
4. The RNA-guided nuclease protein of claim 1 , wherein the eukaryotic nucleic acid sequence comprises or is adjacent to a protospacer adjacent motif (PAM) that is recognized by the engineered RNA-guided nuclease protein.
5. The RNA-guided nuclease protein of claim 4 , wherein the eukaryotic nucleic acid sequence comprises at least 17 nucleotides adjacent to the PAM.
6. The RNA-guided nuclease protein of claim 1 , wherein the Cas9 protein further comprises an insertion at or near the N-terminus of the Cas9 protein.