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A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast.

Nicholas J McGlincy1, Zuriah A Meacham1, Kendra K Reynaud1,2

  • 1Department of Molecular and Cell Biology, Berkeley, CA, 94720, USA.

BMC Genomics
|March 24, 2021
PubMed
Summary

This study introduces a comprehensive CRISPRi library for yeast, enabling precise gene knock-down and genome-wide screening. The platform effectively identifies essential genes by analyzing fitness defects in pooled screens.

Keywords:
Budding yeastCRISPR interferencePooled screening

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR/Cas9-mediated transcriptional interference (CRISPRi) allows programmable gene knock-down for loss-of-function studies.
  • Effective, inducible CRISPRi systems have been established in budding yeast.
  • Genome-scale guide libraries facilitate systematic, genome-wide genetic analysis.

Purpose of the Study:

  • To develop and validate a comprehensive CRISPRi library for high-precision screening in yeast.
  • To refine CRISPRi design rules using empirical data from pooled screens.
  • To enhance the accuracy and robustness of guide abundance measurements in pooled screens.

Main Methods:

  • Construction of a comprehensive yeast CRISPRi library with multiple guides per gene.
  • Pooled transformation and competitive growth assays to assess gene essentiality and fitness defects.
  • Linking guides with random nucleotide barcodes and using in vitro transcription for improved abundance measurements.

Main Results:

  • The library covers most yeast genes with 10 distinct guides each.
  • Competitive growth screens identified strong fitness defects in most essential genes, confirming library coverage.
  • Analysis of fitness defects refined yeast CRISPRi design rules.

Conclusions:

  • A broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast has been demonstrated.
  • The developed library and methods enable robust genome-wide genetic analysis.