Advancements and Obstacles of CRISPR-Cas9 Technology in Translational Research

Abstract

The expanding CRISPR-Cas9 technology is an easily accessible, programmable, and precise gene-editing tool with numerous applications, most notably in biomedical research. Together with advancements in genome and transcriptome sequencing in the era of metadata, genomic engineering with CRISPR-Cas9 meets the developmental requirements of precision medicine, and clinical tests using CRISPR-Cas9 are now possible. This review summarizes developments and established preclinical applications of CRISPR-Cas9 technology, along with its current challenges, and highlights future applications in translational research.

Keywords: CRISPR-Cas9; gene editing; gene therapy; off-target; tumor immunotherapy.

Figure 1

Timeline and Key Studies of the CRISPR-Cas System Major developments during the past three decades are shown. Research on CRISPR exploded after 2013 (represented by ever-increasing number of bubbles), when the technology was used to modify genes in human cells and many other eukaryotes.

Figure 2

Mechanisms of CRISPR-Cas9 as a Genome Engineering Platform (A) Cas9 nuclease cleaves double-stranded DNA via RuvC and HNH domains to introduce double-strand breaks (DSBs) that are then repaired by NHEJ or HDR. Error-prone NHEJ repair pathways always introduce random insertions or deletions (indels) (left), but with the use an exogenous DNA donor, the HDR pathway can introduce precise insertions (right). (B) Cas9 nickase (nCas9) can cleave a single strand of double-stranded DNA when inactivating either HNH or RuvC domains. The use of two nCas9 complexes can reduce off-target effects. (C) Dead Cas9 (dCas9) contains inactivating domains in HNH or RuvC. It can be tethered with transcriptional factors to mediate downregulation or activation of target genes. In addition, dCas9 can be fused to labeling proteins (e.g., GFP), for nucleic acid imaging. (D) dCas9 can be fused to deaminase for catalytic conversion of C to U, thus achieving single-base editing during DNA replication. Similarly, dCas9 can be tethered with epigenetic modification enzymes to obtain desired edits. (E) dgRNA can guide Cas9 to regulate gene expression. (F) Workflow schematic of CRISPR genome-scale functional screening.

Figure 3

Applications of CRISPR-Cas9 Genomic Engineering As a genetic and epigenetic engineering technology, CRISPR-Cas9 has a range of applications. This study focuses on biomedical and clinical applications.