Recent Updates of the CRISPR/Cas9 Genome Editing System: Novel Approaches to Regulate Its Spatiotemporal Control by Genetic and Physicochemical Strategies

Abstract

Khaled S Allemailem,1 Ahmad Almatroudi,1 Arshad Husain Rahmani,1 Faris Alrumaihi,1 Arwa Essa Alradhi,2 Amal M Alsubaiyel,3 Mohammad Algahtani,4 Rand Mohammad Almousa,5 Ali Mahzari,6 Abdulmajeed AA Sindi,7 Gasim Dobie,8 Amjad Ali Khan9 1Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia; 2General Administration for Infectious Disease Control, Ministry of Health, Riyadh 12382, Saudi Arabia; 3Department of Pharmaceutics, College of Pharmacy, Qassim University, Buraydah 51452, Saudi Arabia; 4Department of Laboratory & Blood Bank, Security Forces Hospital, Mecca 21955, Saudi Arabia; 5Department of Education, General Directorate of Education, Qassim 52361, Saudi Arabia; 6Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Al-Baha University, Al-Baha 65527, Saudi Arabia; 7Department of Basic Medical Sciences, Faculty of Applied Medical Sciences, Al-Baha University, Al-Baha 65527, Saudi Arabia; 8Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Jazan University, Gizan 82911, Saudi Arabia; 9Department of Basic Health Sciences, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi ArabiaCorrespondence: Amjad Ali Khan, Department of Basic Health Sciences, College of Applied Medical Sciences, Qassim University, P.O. Box 6666, Buraydah, 51452, Saudi Arabia, Email [email protected]: The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Streptococcus pyogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9’s undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed.Keywords: CRISPR/Cas9, sgRNA, genome editing, off-target effects, spatiotemporal control, clinical translation

Keywords

• crispr/cas9
• sgrna
• genome editing
• off-target effects
• spatiotemporal control
• clinical translation.