Pooled clone collections by multiplexed CRISPR/Cas12a-assisted gene tagging in yeast

Clone collections of modified strains (9libraries9) are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we developed CRISPR/Cas12a (Cpf1)-assisted tag library engineering ( CASTLING) for multiplexed strain construction. CASTLINGuses microarray-synthesized oligonucleotide pools and in vitro recombineeringto program the genomic insertion of long DNA constructsvia homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLINGunlocks new avenues for increased throughput in functional genomicsand cell biology research.