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

Nat Commun. 2019 Jul 4;10(1):2960. doi: 10.1038/s41467-019-10816-7.


Clone collections of modified strains ("libraries") 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 present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via 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, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Clone Cells
  • Gene Library
  • Genetic Engineering
  • Genetic Techniques*
  • Genome, Fungal
  • Green Fluorescent Proteins / metabolism
  • Nuclear Proteins / metabolism
  • Saccharomyces cerevisiae / genetics*


  • Nuclear Proteins
  • Green Fluorescent Proteins