Small RNA Toxin-Assisted Evolution of GC-Preferred ErCas12a for Enhanced Genome Targeting Range

CRISPR/Cas12a, a promising gene editing technology, faces limitations due to its requirement for a thymine (T)-rich protospacer adjacent motif (PAM). Despite the development of Cas12a variants with expanded PAM profiles, many genomic loci, especially those with guanine-cytosine (GC)-rich PAMs, have remained inaccessible. This study develops a small RNA toxin-aided strategy to evolve ErCas12a for targeting GC-rich PAMs, resulting in the creation of enhanced ErCas12a (enErCas12a). EnErCas12a demonstrates the ability to recognize GC-rich PAMs and target five times more PAM sequences than the wild-type ErCas12a. Furthermore, enErCas12a achieves efficient gene editing in both bacterial and mammalian cells at various sites with non-canonical PAMs, including GC-rich PAMs such as GCCC, CGCC, and GGCC, which are inaccessible to previous Cas12a variants. Moreover, enErCas12a effectively targets PAM sequences with a GC content exceeding 75% in mammalian cells, providing a valuable alternative to the existing Cas12a toolkit. Importantly, enErCas12a maintains high specificity at targets with canonical PAMs, while also demonstrating enhanced specificity at targets with non-canonical PAMs. Collectively, this work establishes enErCas12a as a promising tool for gene editing in both eukaryotes and prokaryotes.