Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2

Henri Schmidt; Minsi Zhang; Dimitar Chakarov; Vineet Bansal; Haralambos Mourelatos; Francisco J. Sánchez-Rivera; Scott W. Lowe; Andrea Ventura; Christina S. Leslie; Yuri Pritykin

doi:10.1186/s13059-025-03488-8

Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2

Henri Schmidt
, Minsi Zhang
, Dimitar Chakarov
, Vineet Bansal
, Haralambos Mourelatos
, Francisco J. Sánchez-Rivera
, Scott W. Lowe
, Andrea Ventura
, Christina S. Leslie
, Yuri Pritykin

Research output: Contribution to journal › Article › peer-review

6 Scopus citations

Abstract

We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly design and analysis of individual gRNAs and gRNA libraries for targeting coding and non-coding regions in custom genomes. GuideScan2 analysis identifies widespread confounding effects of low-specificity gRNAs in published CRISPR screens and enables construction of a gRNA library that reduces off-target effects in a gene essentiality screen. GuideScan2 also enables the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome. GuideScan2 will facilitate CRISPR experiments across a wide range of applications.

Original language	English (US)
Article number	41
Journal	Genome biology
Volume	26
Issue number	1
DOIs	https://doi.org/10.1186/s13059-025-03488-8
State	Published - Dec 2025

All Science Journal Classification (ASJC) codes

Ecology, Evolution, Behavior and Systematics
Genetics
Cell Biology

Keywords

Algorithm
Burrows-Wheeler transform
CRISPR
Guide RNA
GuideScan2
Off-targets
Software
Web interface

Access to Document

10.1186/s13059-025-03488-8

Cite this

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abstract = "We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly design and analysis of individual gRNAs and gRNA libraries for targeting coding and non-coding regions in custom genomes. GuideScan2 analysis identifies widespread confounding effects of low-specificity gRNAs in published CRISPR screens and enables construction of a gRNA library that reduces off-target effects in a gene essentiality screen. GuideScan2 also enables the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome. GuideScan2 will facilitate CRISPR experiments across a wide range of applications.",

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AU - Schmidt, Henri

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AU - Chakarov, Dimitar

AU - Bansal, Vineet

AU - Mourelatos, Haralambos

AU - Sánchez-Rivera, Francisco J.

AU - Lowe, Scott W.

AU - Ventura, Andrea

AU - Leslie, Christina S.

AU - Pritykin, Yuri

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KW - GuideScan2

KW - Off-targets

KW - Software

KW - Web interface

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