Increasing the performance of pooled CRISPR–Cas9 drop-out screening

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.

Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries

“…One approach is to optimise the gRNA scaffold sequences. Several studies have been conducted thus far and resulted in a similar structure, which showed more robust phenotypic outcomes 38 , 45 , 46 . Another approach is to computationally predict on-target efficiency using nucleotide biases on gRNA KO efficiency.…”

Dual direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance

“…Interestingly, CRISPRi appears to be a more sensitive technique than CRISPRko, indicated by the substantially improved detection for MED12 and MED23 in both screens. Some of this increase in sensitivity is likely to be due to the use of an improved tracrRNA sequence 29 in the CRISPRi vector, which was not used in the CRISPRko screen (Figure S1 ). At the guide level, the increased sensitivity for both MED12 and MED23 might also be attributable to the fact that all 5 guides are active and therefore substantially enriched in the screen, whereas for other potential hits, such as NF1, fewer guides appear to be active (Fig.…”

Combinatorial CRISPR screening reveals functional buffering in autophagy

“…To expand on our 3Cs technology and enable Cas9 gRNA multiplexing, we generated a lentiviral Cas9-gRNA expression plasmid (pLenti-Multiplex) by placing a human 7SK (h7SK) promoter upstream of a previously engineered Cas9-tracrRNA followed by a human U6 (hU6) promoter upstream of a wildtype Cas9-tracrRNA sequence 46,47 . Both gRNA cassettes contain a gRNA placeholder sequence encoding for I-CeuI or I-SceI restriction enzyme sites, respectively ( Figure 1A) 45 .…”