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

Genome Biol. 2025 Feb 26;26(1):41. doi: 10.1186/s13059-025-03488-8.

Authors

Henri Schmidt^#^{1

2}, Minsi Zhang^#³, Dimitar Chakarov⁴, Vineet Bansal⁵, Haralambos Mourelatos^{3

6}, Francisco J Sánchez-Rivera^{3

7}, Scott W Lowe³, Andrea Ventura⁸, Christina S Leslie⁹, Yuri Pritykin^{10

11

12}

Affiliations

¹ Department of Computer Science, Princeton University, Princeton, NJ, USA.
² Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
³ Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
⁴ Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA.
⁵ Center for Statistics and Machine Learning, Princeton University, Princeton, NJ, USA.
⁶ Weill Cornell/Rockefeller/Memorial Sloan Kettering Tri-Institutional MD-PhD Program, New York, NY, USA.
⁷ Present address: David H. Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁸ Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. venturaa@mskcc.org.
⁹ Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. lesliec@mskcc.org.
¹⁰ Department of Computer Science, Princeton University, Princeton, NJ, USA. pritykin@princeton.edu.
¹¹ Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. pritykin@princeton.edu.
¹² Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA. pritykin@princeton.edu.

^# Contributed equally.

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.

Keywords: Algorithm; Burrows-Wheeler transform; CRISPR; Guide RNA; GuideScan2; Off-targets; Software; Web interface.

MeSH terms

Animals
CRISPR-Cas Systems*
Genome
Mice
RNA, Guide, CRISPR-Cas Systems* / genetics
Software*

Substances

RNA, Guide, CRISPR-Cas Systems

Abstract

MeSH terms

Substances

Grants and funding