Guide RNA targeting, PAM recognition, DSB creation, HDR vs NHEJ repair
CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) uses a guide RNA (gRNA = crRNA + tracrRNA) to direct the Cas9 endonuclease to specific genomic sequences. Cas9 scans DNA for PAM sequences (NGG for SpCas9), then unwinds DNA and checks for complementarity to the 20-nt spacer.