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.

CRISPR-Mediated In Situ Introduction or Integration of F9-Padua in Human iPSCs for Gene Therapy of Hemophilia B

“…Researchers constantly attempt to refine nuclease systems that bind to specific DNA sequences and precisely achieve genomic modification. There are three genomic editing tools commonly used: zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and CRISPR/Cas systems [36]. ZFN and TALEN have played an important role, but their use is limited by several factors, such as the complex and costly preparation process [37].…”

Protocol for efficient CRISPR-Cas9-mediated fluorescent tag knockin in hard-to-transfect erythroid cell lines

“…Targeted genome editing by CRISPR-Cas9 technology has revolutionized life sciences and has become an invaluable tool for a myriad of biological applications. 2 , 3 , 4 , 5 , 6 , 7 , 8 It relies on the use of an RNA-guided endonuclease (Cas9) capable of producing double strand DNA cleavage at chosen genomic regions based on sequence complementarity with a co-expressed guide RNA (gRNA) molecule. Cas9-induced double strand breaks are repaired by the error-prone non-homologous end joining (NHEJ) which introduces small insertions or deletions creating frame-shifts (gene KO), or which can be repaired by homologous recombination (HR) upon the introduction of an HR template to precisely engineer genomic sequences (gene KI).…”

“…Cas9-induced double strand breaks are repaired by the error-prone non-homologous end joining (NHEJ) which introduces small insertions or deletions creating frame-shifts (gene KO), or which can be repaired by homologous recombination (HR) upon the introduction of an HR template to precisely engineer genomic sequences (gene KI). 8 Whereas CRISPR-Cas9 appears, in principle, rather simple i.e., relying on the introduction into a cell of the Cas9 endonuclease and a guide RNA (gRNA) to target the enzyme to a defined genomic site, its successful deployment can be cumbersome in a wide range of situations. Some of the major limitations and common bottlenecks relate to the following.…”

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

“…While NHEJ is an error-prone repair process that often leads to the introduction of mutations such as minor insertions and deletions (Indels), HDR leads to the precise repair of DSBs. A common result of DSBs in the genome is the generation of random insertions or deletions through NHEJ, which is the predominant DSB repair pathway in plants [ 26 , 27 , 28 , 29 ].…”