Comparative optimization of combinatorial CRISPR screens

Ruitong Li, Olaf Klingbeil, Davide Monducci, Michael J. Young, Diego J. Rodriguez, Zaid Bayyat, Joshua M. Dempster, Devishi Kesar, Xiaoping Yang, Mahdi Zamanighomi, Christopher R. Vakoc, Takahiro Ito () and William R. Sellers ()
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Ruitong Li: Broad Institute of Harvard and MIT
Olaf Klingbeil: Cold Spring Harbor Laboratory
Davide Monducci: Broad Institute of Harvard and MIT
Michael J. Young: Broad Institute of Harvard and MIT
Diego J. Rodriguez: Broad Institute of Harvard and MIT
Zaid Bayyat: Broad Institute of Harvard and MIT
Joshua M. Dempster: Broad Institute of Harvard and MIT
Devishi Kesar: Broad Institute of Harvard and MIT
Xiaoping Yang: Broad Institute of Harvard and MIT
Mahdi Zamanighomi: Broad Institute of Harvard and MIT
Christopher R. Vakoc: Cold Spring Harbor Laboratory
Takahiro Ito: Broad Institute of Harvard and MIT
William R. Sellers: Broad Institute of Harvard and MIT

Nature Communications, 2022, vol. 13, issue 1, 1-10

Abstract: Abstract Combinatorial CRISPR technologies have emerged as a transformative approach to systematically probe genetic interactions and dependencies of redundant gene pairs. However, the performance of different functional genomic tools for multiplexing sgRNAs vary widely. Here, we generate and benchmark ten distinct pooled combinatorial CRISPR libraries targeting paralog pairs to optimize digenic knockout screens. Libraries composed of dual Streptococcus pyogenes Cas9 (spCas9), orthogonal spCas9 and Staphylococcus aureus (saCas9), and enhanced Cas12a from Acidaminococcus were evaluated. We demonstrate a combination of alternative tracrRNA sequences from spCas9 consistently show superior effect size and positional balance between the sgRNAs as a robust combinatorial approach to profile genetic interactions of multiple genes.

Date: 2022
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DOI: 10.1038/s41467-022-30196-9

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