Systems and methods for one-shot guide RNA (ogRNA) targeting of endogenous and source DNA

A method of altering a target site in a cell comprising delivering to the cell a transiently active genome editing system comprising a guide RNA (gRNA) and an engineered Cas9 protein, or a nucleic acid composition encoding the gRNA and the Cas9 protein, wherein

• a) the gRNA comprises a targeting domain that is complementary to a eukaryotic nucleic acid sequence;
• and
• b) the Cas9 protein is encoded by a nucleic acid comprising the eukaryotic nucleotide sequence and a protospacer adjacent motif (PAM), wherein the PAM is recognized by the Cas9 protein and is within or adjacent to the eukaryotic nucleotide sequence, and wherein the Cas9 protein comprises:
• i) an amino acid insertion relative to SEQ ID NO:
• 2 selected from the group consisting of:
• E271_N272insGX6-10G, L371_N372insGX6-10G, and Q737_A738insGX6-10G;
• or
• ii) an amino acid insertion S12_M13insGX6-10G relative to SEQ ID NO:
• 10,
• wherein the amino acid 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 the sequence set forth in SEQ ID NO:
• 3, SEQ ID NO:
• 4, SEQ ID NO:
• 5, or SEQ ID NO:
• 10, and having an RNA-guided nuclease activity.