Current applications and future perspective of CRISPR/Cas9 gene editing in cancer

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) system provides adaptive immunity against plasmids and phages in prokaryotes. This system inspires the development of a powerful genome engineering tool, the CRISPR/CRISPR-associated nuclease 9 (CRISPR/Cas9) genome editing system. Due to its high efficiency and precision, the CRISPR/Cas9 technique has been employed to explore the functions of cancer-related genes, establish tumor-bearing animal models and probe drug targets, vastly increasing our understanding of cancer genomics. Here, we review current status of CRISPR/Cas9 gene editing technology in oncological research. We first explain the basic principles of CRISPR/Cas9 gene editing and introduce several new CRISPR-based gene editing modes. We next detail the rapid progress of CRISPR screening in revealing tumorigenesis, metastasis, and drug resistance mechanisms. In addition, we introduce CRISPR/Cas9 system delivery vectors and finally demonstrate the potential of CRISPR/Cas9 engineering to enhance the effect of adoptive T cell therapy (ACT) and reduce adverse reactions.

Keywords: CRISPR screen; CRISPR/Cas9; Gene delivery; Gene editing; Immunotherapy.

Fig. 1

Mechanism of type II CRISPR/Cas9 system. a During acquisition, after being infected by the phage, the DNA sequence from the invader is integrated into the host CRIPSPR locus as a spacer and separated by repetitive sequences. b During the transcription stage, pre-crRNA is transcribed, and then pre-crRNA is cleaved to produce mature crRNA. Each crRNA is composed of a repetitive sequence and a spacer sequence against the invader. c In the interference phase, the Cas protein directly cleaves the exogenous nucleic acid at a site complementary to the sequence of the crRNA spacer

Fig. 2

A brief history of CRISPR/Cas9 system development and associated gene editing tools. The CRISPR locus and cas genes were identified in 1987 and 2002 respectively. In 2005, it was discovered by RNA-sequencing that bacterial CRISPR loci contain a number of spacers derived from bacteriophage and other extrachromosomal elements. In 2007, it was confirmed that CRISPR/Cas system mediates the adaptive immunity of prokaryotes to bacteriophages. In 2012, it was confirmed that the double RNA structure formed by tracrRNA and mature crRNA instructed Cas9 to cleave DNA at the target site. In 2013, Type II CRISPR/Cas achieved precise editing of endogenous genome sites in mammalian cells. In the following years, the advent of several CRISPR/Cas9-based gene editing tools has dramatically improved the precision of genome editing and widened its extent of application. In 2016, CRISPR/Cas9 gene editing tools were first applied to clinical treatments, and subsequent clinical trials provided new insights for humans to explore cancer treatments

Fig. 3

The mechanism of base editing and prime editing. a A cytosine base editor (CBE) uses cytidine deaminase to bind to its homologous base to catalyze the deamination reaction and convert the cytosine in the R-loop to uracil. The resulting U•G base mismatch is then converted into T•A pair after DNA replication or repair. b Schematic of the adenine base editor (ABE). ABE-mediated deamination converts adenosine to inosine, which is subsequently read as guanosine during DNA replication. c Schematic diagram of the prime editor structure and prime editing mechanism

Fig. 4

Schematic diagram of in vitro or in vivo CRISPR screening. a CRISPR screening begins by synthesizing oligonucleotide pools containing single guide RNA sequences and cloning them into lentiviral vectors. Lentiviruses then infect cells expressing Cas9 at low multiplicity of infection. After selection, the pool contains cells with different gene knockouts, which can be subsequently used in various screening methods. b In vitro screening is performed by culturing tumor cells under selective pressure such as drug treatment. c In vivo screening transplants the transfected cell population into immunodeficient mice in situ or subcutaneously. d Patient-derived xenotransplantation (PDX) is achieved by transplanting the patient’s tumor into immunodeficient mice. The PDX tumor is harvested, cultured in vitro, and genetically modified to evaluate tumor growth and response to treatment

Fig. 5

Three main approaches to adoptive cell therapy (ACT) and the application of CRISPR in them. a Tumor infiltrating lymphocytes (TILs) are produced by surgical removal of tumors and enrichment and amplification of TILs from tumor samples. b Isolation and purification of primary T cells from cancer patients, followed by CRISPR-mediated targeted insertion of chimeric antigen receptors (CAR) and engineered T cell receptors (TCR). CRISPR can then knock out immune checkpoint genes in T cells to enhance T cell function. c Primary T cells are isolated from healthy donors and purified, and the CRISPR system is used to introduce CAR and engineered TCR. Genes encoding endogenous TCR and human leukocyte antigen are subsequently knocked out with CRISPR/Cas9 to generate “universal” allogeneic CAR-T cells or TCR-T cells

Fig. 6

The structure of CARs and the application of CRISPR/Cas9 in CAR-T cell therapy. a The structure of the first to third generation CARs. b Knock-out of endogenous TCR sites, immune checkpoint protein and major histocompatibility complex class I molecules generates universal CAR-T cells to enhance T cell killing and avoid graft-versus-host disease

Fig. 7

The structure of mixed TCR dimers and the application of CRISPR/Cas9 in TCR-T cell therapy. Introduction of transgenic TCRs can cause formation of new reactive TCR dimers. Knock-out of endogenous TCRs avoids the formation of mixed TCR dimers and increases the expression of transduced TCRs