Genome-editing approaches and applications: a brief review on CRISPR technology and its role in cancer

Abstract

The development of genome-editing technologies in 1970s has discerned a new beginning in the field of science. Out of different genome-editing approaches such as Zing-finger nucleases, TALENs, and meganucleases, clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR/Cas9) is a recent and versatile technology that has the ability of making changes to the genome of different organisms with high specificity. Cancer is a complex process that is characterized by multiple genetic and epigenetic changes resulting in abnormal cell growth and proliferation. As cancer is one of the leading causes of deaths worldwide, a large number of studies are done to understand the molecular mechanisms underlying the development of cancer. Because of its high efficiency and specificity, CRISPR/Cas9 has emerged as a novel and powerful tool in the field of cancer research. CRISPR/Cas9 has the potential to accelerate cancer research by dissecting tumorigenesis process, generating animal and cellular models, and identify drug targets for chemotherapeutic approaches. However, despite having tremendous potential, there are certain challenges associated with CRISPR/Cas9 such as safe delivery to the target, potential off-target effects and its efficacy which needs to be addressed prior to its clinical application. In this review, we give a gist of different genome-editing technologies with a special focus on CRISPR/Cas9 development, its mechanism of action and its applications, especially in different type of cancers. We also highlight the importance of CRISPR/Cas9 in generating animal models of different cancers. Finally, we present an overview of the clinical trials and discuss the challenges associated with translating CRISPR/Cas9 in clinical use.

Keywords: Animal models; CRISPR/Cas9; Cancer; Genetics; Genome editing; Nucleic acids.

Fig. 1

Schematic representation of timelines of CRISPR development which highlights the important discoveries related to CRISPR technology from 1987 to current year. CRISPR technology was first reported in 1987 in Osaka University, whereas the term CRISPR–Cas9 was first coined in 2002. In 2012, first patent for CRISPR–Cas9 technology was submitted, and in 2015, first report of human genes edited by CRISPR came out which fueled the controversy about ethical issues related to gene-editing technologies. In the same year, US scientists used CRISPR/Cas9 for making genetically modified mosquitoes, to prevent them carrying malaria parasite. In 2018, first CRISPR–Cas9 clinical trial was launched. In 2020, first patient received gene therapy where CRISPR was administered directly into the body and in the same year Emmanuelle Charpentier and Jennifer Doudna won the Nobel Prize in chemistry for CRISPR technology

Fig. 2

A simple schematic representation of CRISPR system and associated nucleases. The CRISPR/Cas system can be divided into two classes as Class1 and Class2. Class1 CRISPR/Cas system utilize multi-Cas protein complex, whereas Class2 CRISPR/Cas system employ single Cas protein. Furthermore, these two classes are subdivided into six types based on the presence of specific genes. Class1 includes types I, III, and IV, whereas Class2 includes types II, V, and VI

Fig. 3

Simplified mechanism of the CRISPR technology (modified from “Delivery Strategies of the CRISPR–Cas9 Gene-Editing System for Therapeutic Applications” (Liu et al. 2017a). a Single guide RNA (sgRNA structure) consists of CRISPR-RNA (crRNA) and trans-activating CRISPR-RNA (tracrRNA). The crRNA and tracrRNA forms a complex and acts as a guide RNA for the Cas9 enzyme. b Cas9 is a dual RNA-guided DNA endonuclease enzyme in Streptococcus pyrogenes. There are two nuclease domains, RuvC (which cleaves the non-target DNA strand) and HNH nuclease domain (that cleaves the target strand of DNA). Target DNA must contain a PAM-motif which is recognized by PAM-interacting domain (PI) of Cas9. Cas9 also have a recognition lobe (REC). Both REC and Nuclease lobe folds to give a positive charge that can accommodate the negative charged sgRNA:target DNA heteroduplex. c CRISPR/Cas9 induces double-stranded breaks which can be repaired either by the non-homologous end-joining DNA repair pathway (NHEJ) or the homology-directed repair (HDR) pathway. In NHEJ process, the two broken ends of DNA are ligated without a template donor which causes insertion and deletion (indel) mutations. This repair process is error-prone and can result in frameshift or loss-of-function and finally gene disruption. In case of HDR, it requires almost identical DNA template to repair the breaks that result in to precise insertion or edition ultimately leading to full correction of the DNA cleavage. d RNA targeting by CRISPR–FnCas9. Cas9 from Francisella novicida (Fn) can target and degrade mRNA. FnCas9 forms a complex with its tracrRNA and a novel and small CRISPR/Cas-associated RNA (termed a scaRNA) instead of the crRNA. The exact mechanism is not very clear yet

Fig. 4

A pictorial representation of different types of CRISPR delivery systems. The effective delivery of CRISPR is one of the most challenging steps in the genome-editing process. The main mode of delivery is either by viral (adenoassociated, adenoviral, or lentiviral) or by non-viral methods (lipid nanoparticles, gold nanoparticles, cell penetrating peptides, etc.). Non-viral methods have lesser advantage over viral vectors especially in case of gene knock-ins. Viral vectors are the most prominent one, but it induces off-targets and immune response which needs to be improved