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Scientific article
Open access
English

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

Published inNature communications, vol. 10, no. 1
Publication date2019-07-04
First online date2019-07-04
Abstract

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.

eng
Affiliation Not a UNIGE publication
Funding
  • Deutsche Forschungsgemeinschaft (German Research Foundation) - [KN498/12-1]
  • Deutsche Forschungsgemeinschaft - [INST 35/1314-1 FUGG]
Citation (ISO format)
BUCHMULLER, Benjamin C. et al. Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast. In: Nature communications, 2019, vol. 10, n° 1. doi: 10.1038/s41467-019-10816-7
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Article (Published version)
Identifiers
ISSN of the journal2041-1723
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