New Hope for Genome Editing in Cultivated Grasses: CRISPR Variants and Application

Abstract

With the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) mediated genome editing, crop improvement has progressed significantly in recent years. In this genome editing tool, CRISPR-associated Cas nucleases are restricted to their target of DNA by their preferred protospacer adjacent motifs (PAMs). A number of CRISPR-Cas variants have been developed e.g. CRISPR-Cas9, -Cas12a and -Cas12b, with different PAM requirements. In this mini-review, we briefly explain the components of the CRISPR-based genome editing tool for crop improvement. Moreover, we intend to highlight the information on the latest development and breakthrough in CRISPR technology, with a focus on a comparison of major variants (CRISPR-Cas9, -Cas12a, and -Cas12b) to the newly developed CRISPR-SpRY that have nearly PAM-less genome editing ability. Additionally, we briefly explain the application of CRISPR technology in the improvement of cultivated grasses with regard to biotic and abiotic stress tolerance as well as improving the quality and yield.

Keywords: CRISPR; Cas12; Cas9; SPRY; cultivated-grasses; plant genome editing; stress tolerance.

FIGURE 1

A schematic model explaining the plant genome editing using CRISPR-associated enzymes with limitations and extra feature of SpRY as compared to others. Cas9 targets a G enriched site with PAM = NGG (Black arrow), Cas12 targets T enriched site with PAM = TTTV (Red arrow), whereas SpRY with no PAM restriction (Green arrow).

FIGURE 2

A schematic illustration of the steps involved in CRISPR/Cas9 Genetic Transformation; 1; (A) Specific gene-targeted; (B) Designing sgRNA for the desired gene; (C) Vector; (D) Transformation of the CRISPR/Cas9 system; (E) Callus formation; (F) Regeneration of shoots from callus; (G) T0- Mutated plants; (H) Transgenic plants testing by PCR; (I) Identification of mutated plants by T7E1; (J) Screening of mutants by sequencing; (K) Various techniques to detect edited plants; (L) Self-pollination of T0 transgenic plants; (M) Mutated T0 seeds; (N) T1 progeny; (O) Phenotypic analysis of T2 plants. (2) The scale mentions the year in which each grass was employed for CRISPR-based genome editing. (3) The scale mentions the year in which each (major) CRISPR tool was developed and used in agriculture.