Abstract
Components of the type II CRISPR–Cas complex in bacteria have been used successfully in eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation and functional genomic screens. Such developments are providing new opportunities for drug target identification and validation, particularly with the application of pooled genetic screening. As CRISPR–Cas is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen. To examine critical aspects of screening quality, we constructed ultra-complex libraries containing sgRNA sequences targeting a collection of essential genes. We examined the performance of screening in both haploid and hypotriploid cell lines, using two alternative guide design algorithms and two tracrRNA variants in a time-resolved analysis. Our data indicate that a simple adaptation of the tracrRNA substantially improves the robustness of guide loss during a screen. This modification minimises the requirement for high numbers of sgRNAs targeting each gene, increasing hit scoring and creating a powerful new platform for successful screening.
Highlights
Clustered regularly interspaced short palindromic repeats (CRISPRs) form part of an adaptive immune response in bacteria that is activated by the presence of foreign DNA elements from virus or plasmids[1,2]
As the CRISPR–CRISPR associated (Cas) technology is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen
16 sgRNAs against each essential gene were captured in the library, including 10 guides per gene from a third generation guide design and 6 guides per gene from the algorithm used by Feng Zhang and colleagues[10]
Summary
Clustered regularly interspaced short palindromic repeats (CRISPRs) form part of an adaptive immune response in bacteria that is activated by the presence of foreign DNA elements from virus or plasmids[1,2]. Recognition of these genetic elements triggers the assembly of a protein–RNA complex, similar to RNA interference complexes in eukaryotes, which cleaves the invading DNA Components of this complex — CRISPR associated (Cas) nuclease, CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) — have been modified and used in eukaryotic cells to facilitate rapid and accurate cell line engineering and animal model generation, as well as functional genomic screens[3]. Such developments provide new opportunities for drug target identification and validation, in the context of a pooled genetic screening format. Alterations to the tracrRNA that increase the residency time of Cas[9] and remove a potential pol III stop site[12], further improve the sensitivity of drop-out screens and essentially negate the effect of numbers of sgRNAs targeting each target gene
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