Benchmarking the PAM compatibility of Cas12a variants for high-throughput yeast genetic variant engineering

<i>Saccharomyces cerevisiae</i> is an important organism for basic research and applied biotechnology. Genome editing techniques, particularly CRISPR/Cas9 from <i>Streptococcus pyogenes</i>, have greatly facilitated saturation genome editing in yeast to understand mutant functions on a large scale. However, Cas9 is restricted by its targeting preference for G-rich protospacer-adjacent motif (PAM) sequences. To broaden the targeting scope, we established an efficient homology-integrated CRISPR/Cas12a system to install genetic variants through homologous recombination by targeting T-rich PAMs. We benchmarked the PAM compatibility of PAM-relaxed Cas12a variants and identified the improved LbCas12a (impLbCas12a) as the most efficient and PAM-relaxed variant in <i>S. cerevisiae</i>, showing high editing purity and an editing window centering the double-strand break. We show that our system can be used to perform targeted saturation mutagenesis to reveal functional variants not captured previously. By using a homology-integrated CRISPR RNA array, we utilized the multiplexing capability of CRISPR/Cas12a to realize multiplex genetic variant installation. Our system enriches the yeast genetic variant engineering toolbox, complementing the commonly used CRISPR/Cas9 system.IMPORTANCECRISPR/Cas9 has facilitated yeast functional genomics by generating a large number of precise genetic variants in a very short period of time. This enabled the interrogation of reconstituted natural genetic variants across different genetic backgrounds or entirely synthetic mutations to discover novel or improved functions. However, Cas9 only targets a limited genomic sequence space due to its preference for G-rich PAM sequences. In this study, we close this gap by developing a CRISPR/Cas12a-based system to engineer user-defined genetic variants targeting T-rich PAM sequences. Our system adopts a homology-integrated design and the most PAM-relaxed Cas12a characterized in yeast to date. These features collectively enabled the creation of genetic variant libraries and multiplex edited strains. This genome editing tool can be used together with Cas9-based tools to interrogate a greater genomic sequence space.