CRISPR-cas9: a powerful tool towards precision medicine in cancer treatment

Abstract

Cancer is a highly heterogeneous disease in term of molecular signature even though it is originated from the same tissue type. Cancer heterogeneity may occur during its development or treatment, which is the main cause resulting in drug resistance and recurrence. Precision medicine refers to matching the right medicine to the right patients based on their molecular signatures. Therefore, a thorough understanding of the mechanism of tumorigenesis and drug resistance is essential to precision medicine. CRISPR-cas9 system is a powerful tool for gene editing and CRISPR-based high-throughput screening has been widely applied especially in searching for tumor-driven or synergistic lethal genes aiming to overcome drug resistance. In this review, we describe the progress of CRISPR-cas9-based unbiased screening in precision medicine including identification of new drug targets, biomarkers and elucidation of mechanisms leading to drug resistance. The existing challenges as well as the future directions are also discussed.

Keywords: CRISPR-cas9; biomarker; drug resistance; drug target; precision medicine; synergistic lethality.

Fig. 1. Mechanism of CRISPR-cas9-mediated gene editing.

The synthesized 20 nt sgRNA is complementary to the target sequence near the protospacer adjacent motif (PAM). The cas9 protein introduces double-strand break (DSB) through the endonuclease domains HNH and Ruvc and then mediates gene repair by nonhomologous end joining (NHEJ) and homology directed repair (HDR), which may lead to an indel mutation or gene knockout. Moreover, cas9 can also be transformed into dcas9 by inactivating the endonuclease domain, which could cause gene transcriptional repression or activation via fusion to a transcriptional activator or repressor.

Fig. 2. CRISPR-cas9 screening process in vitro and in vivo.

A large number of sgRNAs were synthesized and cloned into a lentiviral vector to form pooled libraries. Meanwhile, cells stably expressing cas9/dcas9 were constructed. The viruses were then transduced at a low MOI, and the cells with stable perturbation remained after antibiotic selection. Positive or negative screening in vitro or in vivo was carried out according to different experimental purposes, and bioinformatics analysis was conducted according to the results of next-generation sequencing to identify the candidate genes.

Fig. 3. Screening methods used for CRISPR-cas9.

A CRISPR-cas9 screen can utilize either positive or negative screening according to the screening purpose. A positive screen focuses on surviving cells, whereas a negative screen focuses on dead cells. Screening can be further divided into loss-of-function (LOF knockout) or gain-of-function (GOF overexpression) screening according to the different functions of gene editing. Drug-resistance or suppressor genes are subsequently identified by the corresponding approach.