CRISPR/Cas: Advances, Limitations, and Applications for Precision Cancer Research

Abstract

Cancer is one of the most leading causes of mortalities worldwide. It is caused by the accumulation of genetic and epigenetic alterations in 2 types of genes: tumor suppressor genes (TSGs) and proto-oncogenes. In recent years, development of the clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized genome engineering for different cancer research ranging for research ranging from fundamental science to translational medicine and precise cancer treatment. The CRISPR/CRISPR associated proteins (CRISPR/Cas) are prokaryote-derived genome editing systems that have enabled researchers to detect, image, manipulate and annotate specific DNA and RNA sequences in various types of living cells. The CRISPR/Cas systems have significant contributions to discovery of proto-oncogenes and TSGs, tumor cell epigenome normalization, targeted delivery, identification of drug resistance mechanisms, development of high-throughput genetic screening, tumor models establishment, and cancer immunotherapy and gene therapy in clinics. Robust technical improvements in CRISPR/Cas systems have shown a considerable degree of efficacy, specificity, and flexibility to target the specific locus in the genome for the desired applications. Recent developments in CRISPRs technology offers a significant hope of medical cure against cancer and other deadly diseases. Despite significant improvements in this field, several technical challenges need to be addressed, such as off-target activity, insufficient indel or low homology-directed repair (HDR) efficiency, in vivo delivery of the Cas system components, and immune responses. This study aims to overview the recent technological advancements, preclinical and perspectives on clinical applications of CRISPR along with their advantages and limitations. Moreover, the potential applications of CRISPR/Cas in precise cancer tumor research, genetic, and other precise cancer treatments discussed.

Targeting Cancer with CRISPR/Cas9-Based Therapy

“…Alternatively, Cas9 orthologs encoded by shorter genes have been found in different bacterial species which can replace the SpCas9 in AAV payloads. The sequence of Staphylococcus aureus Cas9 (SaCas9) gene is 1 kbp smaller in size than Cas9 found in Streptococcus pyogenes and Campylobacter jejuni Cas9 protein (CjCas9) gene is shorter by 2.95 kbp than SpCas9 [ 48 , 49 , 50 ]. When considering the use of SpCas9 ortholog it is worth remembering, that alternative Cas9 proteins require different PAM sequences for successful gene manipulation.…”

“…Liposomal transporters offer a broad variety of available modifications, allowing for precise control over the physico-chemical properties of their surface and the introduction of targeting ligands [ 55 ]. Antibody-guided system emerged in combination with Lipid nanoparticles (LNPs), that comprise a well-known delivery system for genetic material, initially developed and standardized for treatment with small interfering RNA (siRNA) [ 49 , 56 ]. Recently, LNPs were used in a proof-of-concept study to target disseminated ovarian cancer in mice with CRISPR/Cas9.…”

CRISPR/Cas9 in Gastrointestinal Malignancies

“…However, several challenges need to be overcome before CRISPR/Cas9 can be translated for clinical routine management of GI cancers. For instance, off-target effects of CRISPR-based technologies are still a major concern, especially in the context of in vivo applications ( Yang et al, 2021 ). Furthermore, current approaches only provide low efficiency in some cases and some genes cannot be targeted with standard approaches due to missing PAM sequences ( Yang, et al, 2021 ; You et al, 2019 ).…”

“…For instance, off-target effects of CRISPR-based technologies are still a major concern, especially in the context of in vivo applications ( Yang et al, 2021 ). Furthermore, current approaches only provide low efficiency in some cases and some genes cannot be targeted with standard approaches due to missing PAM sequences ( Yang, et al, 2021 ; You et al, 2019 ). Intensive research is being performed to overcome these challenges and thus, CRISPR/Cas9 is still among the most promising candidates to become an indispensable tool for personalized medicine of GI cancers in the future.…”

Performance of large scale pooled CRISPR screens is dependent on Cas9 expression levels

“…In particular, large scale pooled CRISPR screening technologies have yielded many new discoveries in a variety of research fields (1)(2)(3)(4). Since its establishment, the technology has undergone many improvements, and the CRISPR toolbox has been significantly expanded (5,6). sgRNA design has improved to a level where efficient screening is possible with whole-genome (WG) libraries containing only two independent sgRNAs per gene (7).…”