Abstract

The phosphate-locking loop (PLL), stabilizing Cas9-DNA interactions, is a key target for optimizing efficiency and specificity. Here, we present a protocol for enhancing Cas9 efficiency and fidelity through structure-guided PLL engineering. We describe steps for identifying PLL engineering targets through sequence alignment and structural analysis, constructing variants via inverse PCR, evaluating efficiency using amplicon sequencing, and assessing specificity through Genome-wide Unbiased Identification of DSBs Evaluated by sequencing (GUIDE-seq (GUIDE-seq). This protocol provides a generalizable framework for Cas9 engineering across orthologs. For complete details on the use and execution of this protocol, please refer to Yang et al. 1