Genome editing technologies, mechanisms and improved production of therapeutic phytochemicals: Opportunities and prospects

Abstract

Plants produce a large number of secondary metabolites, known as phytometabolites that may be employed as medicines, dyes, poisons, and insecticides in the field of medicine, agriculture, and industrial use, respectively. The rise of genome management approaches has promised a factual revolution in genetic engineering. Targeted genome editing in living entities permits the understanding of the biological systems very clearly, and also sanctions to address a wide-ranging objective in the direction of improving features of plant and their yields. The last few years have introduced a number of unique genome editing systems, including transcription activator-like effector nucleases, zinc finger nucleases, and miRNA-regulated clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). Genome editing systems have helped in the transformation of metabolic engineering, allowing researchers to modify biosynthetic pathways of different secondary metabolites. Given the growing relevance of editing genomes in plant research, the exciting novel methods are briefly reviewed in this chapter. Also, this chapter highlights recent discoveries on the CRISPR-based modification of natural products in different medicinal plants.

Keywords: CRISPR/Cas; biosynthesis pathway; gene encoding; homozygous mutants; knockout; next-generation sequencing.

Figure 1

Various genome‐editing tools. (a) Zinc‐finger nucleases (ZFNs) act as dimer. Each monomer consists of a DNA binding domain and a nuclease domain. Each DNA binding domain consists of an array of 3–6 zinc finger repeats which recognizes 9–18 nucleotides. Nuclease domain consists of type II restriction endonuclease Fok1. (b) Transcription activator‐like nucleases (TALENs): these are dimeric enzymes similar to ZFNs. Each subunit consists of DNA binding domain (highly conserved 33–34 amino acid sequence specific for each nucleotide) and Fok1 nuclease domain. (c) CRISPR/Cas9: Cas9 endonuclease is guided by sgRNA (single guide RNA: crRNA and tracrRNA) for target specific cleavage. Twenty nucleotide recognition site is present upstream of protospacer adjacent motif (PAM) (Adopted from Arora & Narula, 2017). Copyright © 2017 Arora and Narula. This is an open‐access article distributed under the terms of the Creative Commons Attribution License (CC BY).

Figure 2

The principle of CRISPR/Cas9 mediated genome editing and criteria for guide RNA selection. (a) In the CRISPR/Cas9 system, a 20 nt guide RNA (gRNA) is complementary to the target DNA region in the host genome followed by a gRNA scaffold sequence. Each target DNA sequence ends with a protospacer adjacent motif (PAM), which is often the sequence “NGG.” The formation of the gRNA‐DNA complex triggers the binding of the Cas9 endonuclease to the complex and generates a double‐stranded break (DSB) 3 bp in front of the PAM. (b) General rules for choosing a gRNA sequence to improve its effectiveness (Adopted from Y. Zhang & Showalter, 2020). Copyright © 2020 Zhang and Showalter. This is an open‐access article distributed under the terms of the Creative Commons Attribution License (CC BY).