CRISPR genome editing relies on RNA-guided nucleases such as Cas9 and Cas12a for site-specific target DNA recognition and cleavage. Cas9 utilizes a dual-guide RNA composed of a CRISPR RNA (crRNA)-trans-activating CRISPR RNA (tracrRNA) pair or a single-guide RNA (sgRNA), whereas Cas12a is programmed with a crRNA only. Target DNA recognition is dependent on complementarity with the spacer sequence of the guide RNA as well as the presence of a protospacer adjacent motif (PAM). Cas9 recognizes an NGG PAM, whereas Cas12a requires a TTTV PAM (V = G, C, or A). Upon target binding, the nucleases catalyze DNA cleavage, generating a DNA double-strand break (DSB). DSB repair by cellular DNA repair pathways leads to the introduction of genetic modifications (edits). The end-joining pathways result in short insertions or deletions (indels), whereas homology-directed repair (HDR) using an exogenous DNA repair template can be used to engineer precise modifications.
