CRISPR genome editing faces four principal limitations, each addressed by specific technological solutions. Specificity: off-target activities of genome editors have been addressed by the development of high-fidelity nuclease variants, chemically modified guide RNAs, and controlled expression of genome editor nucleases. Targeting scope: the NGG PAM sequence requirement of SpCas9 restricts the scope of targetable genomic sites. This is addressed using engineered variants of Cas9 with alternative or relaxed PAM requirements, other naturally derived Cas9 orthologs with alternative PAM requirements, and Cas12a enzymes. Control of editing outcomes: various approaches, including asymmetric or tethered HDR repair templates, cell cycle synchronization, and NHEJ inhibitors, have been developed to enhance the efficiency of HDR and suppress the formation of indels by end-joining pathways. Second-generation technologies such as base or prime editing enable the introduction of precise modifications independently of HDR. Delivery: cellular delivery of genome editor components is facilitated by electroporation/nucleofection, lipid nanoparticles, and viral vectors.
