Open AccessPINCER: improved CRISPR/Cas9 screening by efficient cleavage at conserved residues
Author(s) -
Brendan Veeneman,
Ying Gao,
Joy Grant,
David Fruhling,
James H. Ahn,
Benedikt Bosbach,
Jadwiga Bienkowska,
Maximillian T. Follettie,
Kim Arndt,
Jeremy S. Myers,
Wenyan Zhong
Publication year - 2020
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gkaa645
Subject(s) - crispr , biology , cas9 , computational biology , genome , genetics , genome editing , gene , gene knockout
CRISPR/Cas9 functional genomic screens have emerged as essential tools in drug target discovery. However, the sensitivity of available genome-wide CRISPR libraries is impaired by guides which inefficiently abrogate gene function. While Cas9 cleavage efficiency optimization and essential domain targeting have been developed as independent guide design rationales, no library has yet combined these into a single cohesive strategy to knock out gene function. Here, in a massive reanalysis of CRISPR tiling data using the most comprehensive feature database assembled, we determine which features of guides and their targets best predict activity and how to best combine them into a single guide design algorithm. We present the ProteIN ConsERvation (PINCER) genome-wide CRISPR library, which for the first time combines enzymatic efficiency optimization with conserved length protein region targeting, and also incorporates domains, coding sequence position, U6 termination (TTT), restriction sites, polymorphisms and specificity. Finally, we demonstrate superior performance of the PINCER library compared to alternative genome-wide CRISPR libraries in head-to-head validation. PINCER is available for individual gene knockout and genome-wide screening for both the human and mouse genomes.