Abstract
The recent thrust in research has projected the type II clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR-Cas9) system as an avant-garde plant genome editing tool. It facilitates the induction of site-specific double-stranded DNA cleavage by the RNA-guided DNA endonuclease (RGEN), Cas9. Elimination, addition, or alteration of sections in DNA sequence besides the creation of a knockout genotype (CRISPRko) is aided by the CRISPR-Cas9 system in its wild form (wtCas9). The inactivation of the nuclease domain generates a dead Cas9 (dCas9), which is capable of targeting genomic DNA without scissoring it. The dCas9 system can be engineered by fusing it with different effectors to facilitate transcriptional activation (CRISPRa) and transcriptional interference (CRISPRi). CRISPR-Cas thus holds tremendous prospects as a genome-manipulating stratagem for a wide gamut of crops. In this article, we present a brief on the fundamentals and the general workflow of the CRISPR-Cas system followed by an overview of the prospects of bioinformatics in propelling CRISPR-Cas research with a special thrust on the available databases and algorithms/web-accessible applications that have aided in increasing the usage and efficiency of editing. The article also provides an update on the current regulatory landscape in different countries on the CRISPR-Cas edited plants to emphasize the far-reaching impact of the genomic editing technology.
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© 2019, This is the work of a U.S. government)
(Reproduced from Zhao et al. (2021) under provisions of Creative Commons License, http://creativecommons.org/licenses/by/4.0/, © Zhao et al. 2021). b Examples of modifications in the Cas enzyme system. (A) The binding of normal CRISPR-Cas9/sgRNA complex results in DSB, (B) Mutated Cas9 protein, without DNA cutting activity (dCas9), can still bind at the specific sgRNA-guided site on DNA to inhibit the transcription by blocking the progression of the RNA polymerase. (C) The dCas9 fused transcriptional activators can be utilised to switch on or enhance transcription at the target site. (D) The dCas9 fused histone modifiers or DNA methylation enzymes can introduce site-specific epigenetic changes at the target site. (Reproduced from Horodecka & Düchler (2021) under the provisions of Creative Commons License, http://creativecommons.org/licenses/by/4.0/, Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland)
(Reproduced from Chincinska et al. (2022) under the provisions of Creative Commons License, http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2022)
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The first author gratefully acknowledges the help rendered by Ms Rashmi Wanjari, Senior Research Fellow, and Mr Piyush Ghoshe, Project Assistant of DBT—Citrus—NER project at ICAR—National Institute of Biotic Stress Management, Raipur, India in corrections and formatting of the manuscript as per the journal’s instructions. The first author was a recipient of the Indian National Science Academy’s Visiting Scientist Fellowship at ICGEB, New Delhi.
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Kharbikar, L., Konwarh, R., Chakraborty, M. et al. 3Bs of CRISPR-Cas mediated genome editing in plants: exploring the basics, bioinformatics and biosafety landscape. Physiol Mol Biol Plants 29, 1825–1850 (2023). https://doi.org/10.1007/s12298-023-01397-3
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DOI: https://doi.org/10.1007/s12298-023-01397-3