PINCER: improved CRISPR/Cas9 screening by efficient cleavage at conserved residues
| Autor: | Maximillian T. Follettie, David Fruhling, Joy Grant, James Ahn, Jeremy S. Myers, Wenyan Zhong, Benedikt Bosbach, Jadwiga Bienkowska, Kim Arndt, Brendan Veeneman, Ying Gao |
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| Rok vydání: | 2020 |
| Předmět: |
AcademicSubjects/SCI00010
Computational biology Biology Genome Conserved sequence Cell Line 03 medical and health sciences Mice 0302 clinical medicine Databases Genetic Genetics Coding region CRISPR Animals Humans Genomic library Amino Acid Sequence Amino Acids Gene Conserved Sequence 030304 developmental biology 0303 health sciences Genomic Library Cas9 Proteins Reproducibility of Results Computational Biology Enzymes Restriction site Narese/24 Narese/29 CRISPR-Cas Systems 030217 neurology & neurosurgery Algorithms RNA Guide Kinetoplastida Thymidine |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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