An in-library ligation strategy and its application in CRISPR/Cas9 screening of high-order gRNA combinations

Abstract

Simultaneous targeting multiple genes is a big advantage of CRISPR (clustered regularly interspaced short palindromic repeats) genome editing but challenging to achieve in CRISPR screening. The crosstalk among genes or gene products is a common and fundamental mechanism to ensure cellular stability and functional diversity. However, the screening approach to map high-order gene combinations to the interesting phenotype is still lacking. Here, we developed a universal in-library ligation strategy and applied it to generate multiplexed CRISPR library, which could perturb four pre-designed targets in a cell. We conducted in vivo CRISPR screening for potential guide RNA (gRNA) combinations inducing anti-tumor immune responses. Simultaneously disturbing a combination of three checkpoints in CD8+ T cells was demonstrated to be more effective than disturbing Pdcd1 only for T cell activation in the tumor environment. This study developed a novel in-library ligation strategy to facilitate the multiplexed CRISPR screening, which could extend our ability to explore the combinatorial outcomes from coordinated gene behaviors.

Optimized minimal genome-wide human sgRNA library

“…CRISPR-Cas9 K562/Jurkat screens. K562 and Jurkat cell lines expressing Cas9 stably and lentivirus pools carrying sgRNA libraries were produced as previously described 14 . K562-Cas9 cells were cultured in 1640 medium with 10% FBS and 1 μg/ml blasticidin on confluency of 0.5 million/ml in shaking incubators at 120 rpm, 37 °C, and with 5% CO2.…”

“…These CRISPR-based knockout screening systems have been applied to hundreds of cell types in different organisms 3 , 4 and made significant advancements in unraveling biological processes in many aspects, for example, functional genomics 5 , cancer, and immunotherapy 6 – 9 . Accordingly, many studies have been performed to improve the performance of CRISPR-based knockout screening systems, including CRISPR/Cas enzyme optimization 10 sgRNA library design 11 – 13 , and sgRNA delivery 14 . However, the sgRNA library size remains a stringent barrier to the application of CRISPR-based knockout screening systems.…”

CRISPR–Cas9 applications in T cells and adoptive T cell therapies

“…In addition, TCR-driven kinases, critical for evaluating T cell responses, can be pinpointed through these screens. In a study targeting 25 TCR-driven kinases within primary T cells, perturbing these kinases revealed that only p38 kinase functioned as a central regulator, influencing T cell phenotypic attributes such as cell expansion, differentiation, response to oxidative stress, and genomic stability [135,136]. In engineered human T cells, the application of CRISPR-Cas9 library screening serves a pivotal role in the identification of primary regulators governing cell fate.…”