DNA polymerase λ-driven targeted mutagenesis for directed evolution in human cells

Directed evolution is an efficient strategy to steer protein function to either understand specific biological properties or develop new biotechnology tools. Currently available methods for targeted mutagenesis in human cells rely on deaminases which can only modify specific bases, limiting the region of sequence space explored during evolution. By leveraging CRISPR-Cas9 coupled with an error-prone variant of human DNA polymerase λ, here we developed CRISPR-λ, an unbiased mutagenesis tool for directed evolution in human cells. We evaluated CRISPR-λ by reverting the fluorescence of a mutated EGFP and characterized it using ultra-deep sequencing. The mutagenic activity of CRISPR-λ spans 36-46 nucleotides from the target site, with a mutation frequency as high as 1.4e-4 substitutions per base and with no bias for specific nucleotide substitutions. The versatility of CRISPR-λ extends beyond base substitution, enabling modifications of the target gene through insertions and deletions, thereby broadening its potential for genetic diversification. We validated the efficacy of CRISPR-λ in directed evolution approaches by functionally reverting a mutated blasticidin resistance gene. Furthermore, we demonstrated the sequence diversification power of CRISPR-λ by steering the syncytia formation activity of the SARS-CoV-2 Spike envelope protein in cultured cells.