Massively parallel CRISPR-assisted homologous recombination enables saturation editing of full-length endogenous genes in yeast.

Performing saturation editing of chromosomal genes will enable the study of genetic variants in situ and facilitate protein and cell engineering. However, current in vivo editing of endogenous genes either lacks flexibility or is limited to discrete codons and short gene fragments, preventing a comprehensive exploration of genotype-phenotype relationships. To enable facile saturation editing of full-length genes, we used a protospacer adjacent motif-relaxed Cas9 variant and homology-directed repair to achieve above 60% user-defined codon replacement efficiencies in Saccharomyces cerevisiae genome. Coupled with massively parallel DNA design and synthesis, we developed a saturation gene editing method termed CRISPR-Cas9- and homology-directed repair-assisted saturation editing (CHASE) and achieved highly saturated codon swapping of long genomic regions. By applying CHASE to massively edit a well-studied global transcription factor gene, we found known and unreported genetic variants affecting an industrially relevant microbial trait. The user-defined codon editing capability and wide targeting windows of CHASE substantially expand the scope of saturation gene editing.

Broadening the targetable space: engineering and discovery of PAM-flexible Cas proteins.

The application of CRISPR-Cas systems has been hindered by their requirement for long protospacer-adjacent motifs (PAMs). Recent engineering and discovery of PAM-flexible Cas proteins have substantially broadened the targetable DNA sequence space, thereby facilitating genome editing and improving derivative technologies such as gene regulation, seamless cloning, and large-scale genetic screens.