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Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline


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.

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Fig. 1: The Orthrus scoring workflow.
Fig. 2: Key Orthrus functions.
Fig. 3: Schematic demonstrating how Orthrus accounts for guide orientation during combinatorial scoring by scoring guides from different orientations separately.
Fig. 4: Summary plots of mean LFC.
Fig. 5: Total read counts for CHyMErA screens.
Fig. 6: Heatmap of Pearson correlations.
Fig. 7: Summary plots of mean LFC for dual-targeting guides.
Fig. 8: Differential LFC for WT HAP1 guides from the ChyMErA dataset analyzed in Procedure 2.
Fig. 9: Summary plots of mean LFC for combinatorial-targeting guides.
Fig. 10: Differential LFC for WT HAP1 guides comprising two significant hits of the scored combinatorial-targeting guides at T12 from the ChyMErA dataset analyzed in Procedure 2, Steps 15–17.

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Data availability

The example dataset is downloadable with the Orthrus package at The expected output from all procedures is provided under a CC-BY 4.0 license at

Code availability

The Orthrus package is available at, and the version of the code run in the protocol is available at (ref. 20). All code presented from all procedures is also available in separate scripts along with their expected output, and are provided under a CC-BY 4.0 license in the Zenodo repository at Code contained in this repository generated Figs. 410. The code in this protocol has been peer reviewed.


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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).

Author information

Authors and Affiliations



H.N.W. wrote the software and performed all analyses. H.N.W. drafted the protocol, and C.L.M., M.A., T.G.-P., M.B., K.R.B. and T.K.O. provided revisions. H.N.W. and M.B. developed the scoring procedure implemented in Orthrus based on conceptual contributions from M.B. T.K.O. provided feedback to improve the software. M.A., T.G.-P., M.B., K.R.B., J.M., B.J.B. and C.L.M. developed the CHyMErA experimental platform. M.A., T.G.-P. and K.R.B. performed experiments to generate the data analyzed in Procedure 2. K.R.B. contributed data, code and text to Procedure 1. J.M., B.J.B. and C.L.M. acquired funding to support this work and provided supervision throughout the project.

Corresponding author

Correspondence to Chad L. Myers.

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Competing interests

A patent application (no. GB 1907733.8) describing the development and applications of CHyMErA, to the University of Toronto and T.G.-P., M.A., K.R.B., J.M. and B.J.B., is pending. J.M. previously performed sponsored research for Repare Therapeutics.

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Peer review information Nature Protocols thanks Max Horlbeck and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Gonatopoulos–Pournatzis, T. et al. Nat. Biotechnol. 38, 638–648 (2020):

Aregger, M. et al. Nat. Protoc. (2021):

Dede, M. et al. Genome Biol. 21, 262 (2020):

Supplementary information

Reporting Summary

Supplementary Video 1

Tutorial on how to set up input files to run the Orthrus scoring pipeline

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Ward, H.N., Aregger, M., Gonatopoulos-Pournatzis, T. et al. Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline. Nat Protoc 16, 4766–4798 (2021).

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