Elucidating the kinetics of CRISPR-SaCas9 action to obtain effective HIV DNA excision with two gRNAs.

The persistence of integrated human immunodeficiency virus (HIV) proviral DNA poses a major barrier to viral eradication, as the viral reservoir enables rapid rebound upon treatment interruption, despite effective virus inhibition. CRISPR-Cas-based editing strategies, especially those using double-site cleavage, show promise in excising proviral DNA, yet the rate and determinants of excision efficiency remain poorly understood. In this study, we systematically evaluated both single- and dual-SaCas9/gRNA approaches for HIV-1 inactivation. Sequence analysis revealed that SaCas9 can eliminate all wild-type HIV-1 genomes with a single gRNA, unlike other CRISPR-Cas systems. Dual-gRNA strategies improved antiviral efficacy, with the Gag3 + Pol5 combination achieving 97% excision efficiency. Kinetic analysis showed that excision efficiency correlates with the kinetic compatibility of paired gRNAs. Pairs of gRNAs with fast and similar kinetics achieved the highest excision efficiency. In contrast, the Gag3 + Env4 pair exhibited discordant kinetic characteristics (fast and slow), resulting in the failure to induce excision as the cut DNA will be repaired before the second cut is realized. Consequently, no excision but regular editing occurred at the two target sites. These findings provide a mechanistic framework for optimizing CRISPR-Cas-mediated excision, highlighting the critical role of both antiviral activity and kinetic synergy in guiding gRNA selection.