Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline

Henry N. Ward, Michael Aregger, Thomas Gonatopoulos-Pournatzis, Maximilian Billmann, Toshiro K. Ohsumi, Kevin R. Brown, Benjamin J. Blencowe, Jason Moffat, Chad L. Myers

Research output: Contribution to journalArticle

5 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)4766-4798
Number of pages33
JournalNature Protocols
Issue number10
StatePublished - Oct 2021

Bibliographical note

Funding Information:
We are grateful to K. Lin for his help testing the Orthrus package. H.N.W, M.B. and C.L.M. were partially supported by grants from the National Science Foundation (1818293) and the National Institutes of Health (R01HG005084, R01HG005853). T.G.-P. was supported by the NIH Earl Stadtman Investigator Program and the NIH Distinguished Scholars Program. M.A. was supported by a Swiss National Science Foundation fellowship (P300PA_164667). M.B. was supported by the German Research Foundation DFG (Bi 2086/1-1). B.J.B was supported by a Canadian Institutes for Health Research Foundation grant (FDN-148434) and by an Ontario Institute of Regenerative Medicine grant. J.M. was supported by grants from the Canadian Institutes for Health Research (MOP-142375) and by Genome Canada (OGI-157).

Publisher Copyright:
© 2021, The Author(s), under exclusive licence to Springer Nature Limited.


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