Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline.

Autor: Ward HN; Bioinformatics and Computational Biology Graduate Program, University of Minnesota-Twin Cities, Minneapolis, MN, USA., Aregger M; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.; RNA Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD, USA., Gonatopoulos-Pournatzis T; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.; RNA Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD, USA., Billmann M; Department of Computer Science and Engineering, University of Minnesota-Twin Cities, Minneapolis, MN, USA., Ohsumi TK; Repare Therapeutics, Cambridge, MA, USA., Brown KR; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada., Blencowe BJ; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada., Moffat J; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.; Institute for Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada., Myers CL; Bioinformatics and Computational Biology Graduate Program, University of Minnesota-Twin Cities, Minneapolis, MN, USA. chadm@umn.edu.; Department of Computer Science and Engineering, University of Minnesota-Twin Cities, Minneapolis, MN, USA. chadm@umn.edu.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2021 Oct; Vol. 16 (10), pp. 4766-4798. Date of Electronic Publication: 2021 Sep 10.
DOI: 10.1038/s41596-021-00596-0
Abstrakt: The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.
(© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
Databáze: MEDLINE
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