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Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches

Authors :
Andrés Felipe Leal
Angelica María Herreno-Pachón
Eliana Benincore-Flórez
Amali Karunathilaka
Shunji Tomatsu
Source :
International Journal of Molecular Sciences, Vol 25, Iss 5, p 2456 (2024)
Publication Year :
2024
Publisher :
MDPI AG, 2024.

Abstract

Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient’s death, respectively. In contrast with classical GT, the CRISPR/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR/Cas9-based GTs.

Details

Language :
English
ISSN :
14220067 and 16616596
Volume :
25
Issue :
5
Database :
Directory of Open Access Journals
Journal :
International Journal of Molecular Sciences
Publication Type :
Academic Journal
Accession number :
edsdoj.1408960c959649a79fda0225ca39635a
Document Type :
article
Full Text :
https://doi.org/10.3390/ijms25052456