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Instrumentation and application of the CRISPR-Cas system
- 1 School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China
* Author to whom correspondence should be addressed.
Abstract
The CRISPR-Cas gene editing system has revolutionized molecular biology by providing a versatile and precise method for genetic manipulation. We explore the basic mechanisms of CRISPR-Cas technology, focusing on the role of Cas proteins and guide RNAs in targeting and cleaving specific DNA sequences. Various types of CRISPR-Cas systems are also discussed, focusing on the widely used CRISPR-Cas9, and their applications in genome editing, including gene knockout, gene knock-in, and gene regulation. Additionally, we present advances in CRISPR-Cas technologies, such as base editing and plasmid editing, demonstrating their potential for therapeutic applications. This paper aims to provide a comprehensive overview of the status and applications of CRISPR-Cas technology and its future directions in gene editing.
Keywords
CRISPR-Cas9, Genome Editing, Guide RNA
[1]. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–21.
[2]. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346:1258096.
[3]. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–78.
[4]. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015;523:481–5.
[5]. Li X, Han J, Yang J, Zhang H. The structural biology of type III CRISPR-Cas systems. J Struct Biol. 2024;216:108070.
[6]. Liu Z, Shi M, Ren Y, Xu H, Weng S, Ning W, et al. Recent advances and applications of CRISPR-Cas9 in cancer immunotherapy. Mol Cancer. 2023;22:35.
[7]. Ferrando J, Filluelo O, Zeigler DR, Picart P. Barriers to simultaneous multilocus integration in Bacillus subtilis tumble down: development of a straightforward screening method for the colorimetric detection of one-step multiple gene insertion using the CRISPR-Cas9 system. Microb Cell Fact. 2023;22:21.
[8]. Jiang F, Doudna JA. CRISPR-Cas9 Structures and Mechanisms. Annu Rev Biophys. 2017;46:505–29.
[9]. Goto T, Yogo K, Hochi S, Hirabayashi M. Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease. Mol Biol Rep. 2023;50:1231–9.
[10]. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34:184–91.
[11]. Nuñez JK, Chen J, Pommier GC, Cogan JZ, Replogle JM, Adriaens C, et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell. 2021;184:2503-2519.e17.
[12]. Chuai G, Ma H, Yan J, Chen M, Hong N, Xue D, et al. DeepCRISPR: optimized CRISPR guide RNA design by deep learning. Genome Biology. 2018;19:80.
[13]. Sander JD, Maeder ML, Reyon D, Voytas DF, Joung JK, Dobbs D. ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. Nucleic Acids Res. 2010;38:W462-468.
[14]. Heigwer F, Kerr G, Boutros M. E-CRISP: fast CRISPR target site identification. Nat Methods. 2014;11:122–3.
[15]. Dobson L, Reményi I, Tusnády GE. CCTOP: a Consensus Constrained TOPology prediction web server. Nucleic Acids Res. 2015;43:W408-412.
[16]. Zhang X-H, Tee LY, Wang X-G, Huang Q-S, Yang S-H. Off-target Effects in CRISPR/Cas9-mediated Genome Engineering. Mol Ther Nucleic Acids. 2015;4:e264.
[17]. Rahman MM, Tollefsbol TO. Targeting cancer epigenetics with CRISPR-dCAS9: Principles and prospects. Methods. 2021;187:77–91.
[18]. Concordet J-P, Haeussler M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018;46:W242–5.
[19]. Labun K, Montague TG, Krause M, Torres Cleuren YN, Tjeldnes H, Valen E. CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res. 2019;47:W171–4.
[20]. Manghwar H, Li B, Ding X, Hussain A, Lindsey K, Zhang X, et al. CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off-Target Evaluation, and Strategies to Mitigate Off-Target Effects. Advanced Science. 2020;7:1902312.
[21]. Singh V. Chapter One - An introduction and use of the CRISPR-Cas systems. In: Singh V, editor. Progress in Molecular Biology and Translational Science [Internet]. Academic Press; 2021 [cited 2024 Jul 18]. p. 1–10. Available from: https://www.sciencedirect.com/science/article/pii/S1877117320301812
[22]. Sharpe JJ, Cooper TA. Unexpected consequences: exon skipping caused by CRISPR-generated mutations. Genome Biol. 2017;18:109.
[23]. Hanna RE, Doench JG. Design and analysis of CRISPR–Cas experiments. Nat Biotechnol. 2020;38:813–23.
[24]. Lin Y-Q, Feng K-K, Lu J-Y, Le J-Q, Li W-L, Zhang B-C, et al. CRISPR/Cas9-based application for cancer therapy: Challenges and solutions for non-viral delivery. Journal of Controlled Release. 2023;361:727–49.
[25]. Bernard BE, Landmann E, Jeker LT, Schumann K. CRISPR/Cas-based Human T cell Engineering: Basic Research and Clinical Application. Immunology Letters. 2022;245:18–28.
[26]. Çerçi B, Uzay IA, Kara MK, Dinçer P. Clinical trials and promising preclinical applications of CRISPR/Cas gene editing. Life Sciences. 2023;312:121204.
[27]. Wang X, Xu G, Johnson WA, Qu Y, Yin D, Ramkissoon N, et al. Long sequence insertion via CRISPR/Cas gene-editing with transposase, recombinase, and integrase. Current Opinion in Biomedical Engineering. 2023;28:100491.
Cite this article
Feng,J. (2024). Instrumentation and application of the CRISPR-Cas system. Theoretical and Natural Science,60,128-133.
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The datasets used and/or analyzed during the current study will be available from the authors upon reasonable request.
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Volume title: Proceedings of the 4th International Conference on Biological Engineering and Medical Science
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