Abstract
Genetic screens by CRISPR/Cas9 are increasingly used as a scalable method to introduce targeted gene knockouts (KO) and to interrogate gene function. Within the pharmaceutical industry, the CRISPR-Cas9 system plays a vital role as an unbiased tool for target identification and validation and has a crucial impact on drug discovery. To perform a high quality CRISPR KO screen and to confidently identify target hits, it is important to achieve a high knock-out efficiency. Over the years, numerous sgRNA design algorithms have emerged, each using slightly different approaches but all with a common goal of increasing the efficiency of gene KO. For pooled CRISPR screening the development of a highly efficient gRNA library with fewer guides per gene has the potential to dramatically reduce the cost and scale of screening and enable screening in more complex models. Therefore, we set out to conduct an unbiased benchmarking of 8 publicly available sgRNA libraries in order to empirically assess the performance of the different sgRNA design algorithms. To do so we cloned, into the same backbone, a subset library containing all guides from each sgRNA library, targeting 250 core-essential and 500 non-essential genes and performed an essentiality screen in 4 different cell lines. The results indicated clear and consistent performance differences between the algorithms, with the recently published VBC library (Michlits G. et al 2020) outperforming every other library in terms of log2FC and kinetics. The initial results in a subset screen were further corroborated in a follow up, genome-wide drug-gene interaction screen using the EGFR inhibitor Osimertinib, with two new libraries based on the VBC design. We evaluated the VBCtop3 sgRNAs in a single format and the VBCtop6 sgRNAs in a dual-guide RNA format expressing two gRNAs targeting the same gene, with both libraries clearly outperforming our standard screening library Yusa v3 with 6 gRNAs/gene from the Sanger Institute. These libraries, half the size compared to our previous standard KO library, will now be employed across the diverse portfolio at the Joint AZ-CRH Functional Genomics Centre.
