PAM-flexible SpCas9 variants expand the targeting scope for porcine genome editing and cellular disease modeling

BACKGROUND: CRISPR-Cas-mediated gene editing has revolutionized life sciences, yet the targeting scope of the widely used SpCas9 is limited by its strict requirement for the NGG protospacer adjacent motif (PAM). To overcome this limitation, PAM-flexible SpCas9 variants have been developed and characterized in multiple species; however, their potential in pigs (an important biomedical model for humans) remains unexplored. Here, we systematically evaluated the editing performance of three PAM-flexible SpCas9 variants (SpRY, SpG, and SpCas9-NG) and their derived base editors in porcine fetal fibroblasts (PFFs). RESULTS: Profiling across 228 target sites revealed that SpRY exhibits nearly PAM-less activity, with significantly higher editing efficiency at NRN (15.82%, R = A/G) than at NYN PAMs (5.75%, Y = C/T). SpG and SpCas9-NG preferentially targeted NGN PAMs, achieving mean efficiencies of 14.81% and 16.33%, respectively. PAM‑flexible cytosine base editors (CBEs) mediated efficient C:G‑to‑T:A conversion, with mean efficiencies of 12.01% for SpRY‑BE4max (NNN PAMs), 15.43% for SpG‑BE4max (NGN PAMs), and 18.39% for SpCas9‑NG‑BE4max (NGN PAMs). Similarly, PAM‑flexible adenine base editors (ABEs) mediated efficient A:T‑to‑G:C conversion, with mean efficiencies of 15.66% for SpRY‑ABE8e (NNN PAMs), 24.16% for SpG‑ABE8e (NGN PAMs), and 20.50% for SpCas9‑NG‑ABE8e (NGN PAMs). By exploiting this expanded targeting scope, we successfully introduced 16 pathogenic single‑nucleotide variants (SNVs) at NRN PAM sites in the porcine genome, with editing efficiencies reaching up to 40.68% for CBEs and 61.76% for ABEs. CONCLUSIONS: PAM-flexible SpCas9 variants and their derived base editors greatly expand the targeting scope for porcine genome engineering, thereby substantially broadening the applicability potential of CRISPR-Cas-mediated genome editing tools in porcine genetic improvement and disease model generation.