Homo sapiens Raw sequence reads

Prime editing (PE) has its advantages for small insertion, deletion or point mutations without double-stranded DNA breaks. The 3-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity. Here we engineered pegRNA by additional RNA stem loop aptamers at 3-end, or splitting pegRNA into sgRNA and prime RNA containing PBS and RTT. We tethered RNA aptamer binding proteins to Cas9 nickase either directly or in an inducible dimerization manner. We show that additional RNA stem loop increases the PE efficiency. Split pegRNA Prime Editing maintains the PE activity and makes the PE to be chemically inducible