DOI: 10.1101/476804Nov 22, 2018Paper

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

BioRxiv : the Preprint Server for Biology
Benjamin C BuchmullerMichael Knop

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 new avenues for increased throughput in functional genomics and cell biology research.

Related Concepts

Cloning
Cytology
DNA
Genes
Molecular Probe Techniques
Oligonucleotides
Recombination, Genetic
Research
Saccharomyces cerevisiae
Yeasts

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