RT Journal Article
SR Electronic
T1 Pooled clone collections by multiplexed CRISPR/Cas12a-assisted gene tagging in yeast
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 476804
DO 10.1101/476804
A1 Buchmuller, Benjamin C.
A1 Herbst, Konrad
A1 Meurer, Matthias
A1 Kirrmaier, Daniel
A1 Sass, Ehud
A1 Levy, Emmanuel D.
A1 Knop, Michael
YR 2018
UL http://biorxiv.org/content/early/2018/11/22/476804.abstract
AB 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 developed 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 new avenues for increased throughput in functional genomics and cell biology research. (max 150 words)