Recent Advances in Genome-Engineering Strategies

Abstract

In October 2020, the chemistry Nobel Prize was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the discovery of a new promising genome-editing tool: the genetic scissors of CRISPR-Cas9. The identification of CRISPR arrays and the subsequent identification of cas genes, which together represent an adaptive immunological system that exists not only in bacteria but also in archaea, led to the development of diverse strategies used for precise DNA editing, providing new insights in basic research and in clinical practice. Due to their advantageous features, the CRISPR-Cas systems are already employed in several biological and medical research fields as the most suitable technique for genome engineering. In this review, we aim to describe the CRISPR-Cas systems that have been identified among prokaryotic organisms and engineered for genome manipulation studies. Furthermore, a comprehensive comparison between the innovative CRISPR-Cas methodology and the previously utilized ZFN and TALEN editing nucleases is also discussed. Ultimately, we highlight the contribution of CRISPR-Cas methodology in modern biomedicine and the current plethora of available applications for gene KO, repression and/or overexpression, as well as their potential implementation in therapeutical strategies that aim to improve patients' quality of life.

Keywords: CAR-T cells; CRISPR-Cas systems; TALENs; ZFNs; dCas9; gene knockout; gene therapy; genome editing; nucleases.

Figure 1

The CRISPR-Cas9 system mechanism on a target gene. The Cas9 protein is guided by a single-stranded RNA that is complementary to the site of interest, in a specific genomic region. The DNA is cleaved by Cas9, and the occurred DSBs can be repaired by two distinct repair pathways: the error-prone non-homologous ending joining (NHEJ), which will induce indels into the repaired DNA (left) and the homologous dependent repair (HDR) that requires a donor repair template that is incorporated in the target DNA sequence (right).

Figure 2

A typical workflow of a CRISPR-Cas9 editing strategy. The basic steps include the selection of the suitable Cas9 endonuclease and the design of the appropriate sgRNA molecule, their cloning into vectors and the subsequent delivery of the construct into eukaryotic cells to mediate changes into the target DNA. The final step corresponds to the experimental verification of the target DNA editing.

Figure 3

Schematic demonstration of the engineered CRISPR-Cas9 systems. (a) The nuclease domains of the Cas9 protein can be mutated independently to generate DNA nickases (Cas9n) that are able to introduce nicks, rather than DBS. DSBs can be introduced through the usage of a pair of sgRNA–Cas9n complexes. (b) The Cas9 protein is engineered into catalytically inactive Cas9 (dCas9). The dCas9 can be fused with specific effector proteins to mediate expression alterations of the gene of interest (GOI).