Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

Luisa Henkel, Benedikt Rauscher, Barbara Schmitt, Jan Winter, Michael Boutros
2020 BMC Biology  
Background In recent years, large-scale genetic screens using the CRISPR/Cas9 system have emerged as scalable approaches able to interrogate gene function with unprecedented efficiency and specificity in various biological contexts. By this means, functional dependencies on both the protein-coding and noncoding genome of numerous cell types in different organisms have been interrogated. However, screening designs vary greatly and criteria for optimal experimental implementation and library
more » ... on and library composition are still emerging. Given their broad utility in functionally annotating genomes, the application and interpretation of genome-scale CRISPR screens would greatly benefit from consistent and optimal design criteria. Results We report advantages of conducting viability screens in selected Cas9 single-cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg) RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a novel genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes. Conclusion We show how empirically designed libraries in combination with an optimized experimental design increase the dynamic range in gene essentiality screens at reduced library coverage.
doi:10.1186/s12915-020-00905-1 pmid:33228647 fatcat:abjcifxu3bg2rkpzqiduk6b72m