Development of an Agrobacterium-delivered codon-optimized CRISPR/Cas9 system for chickpea genome editing

Abstract

Chickpea is considered recalcitrant to in vitro tissue culture. The Clustered, Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) based genome editing in chickpea can remove the bottleneck of limited genetic variation in this cash crop rich in nutrients and protein. However, the generation of stable mutant lines using CRISPR/Cas9 requires efficient and highly reproducible transformation approaches. We modified a binary vector pPZP200 by introducing a codon-optimized Cas9 gene for chickpea and the promoters of Medicago truncatula U6 snRNA for expressing guide RNA targeted to the Phytoene Desaturase (PDS) gene. The dissected single cotyledons with half embryo of chickpea were used as explants for genetic transformation. A single gRNA was found sufficient to achieve high efficiency (42%) editing with the generation of PDS mutants with albino phenotypes. A simple, rapid, highly reproducible, stable transformation and CRISPR/Cas9-based genome editing system for chickpea was established. For the first time, this study aimed to demonstrate this system's applicability by performing a gene knockout of the chickpea phytoene desaturase gene (CaPDS) in stable shoots using an improved chickpea transformation protocol. Keywords: Chickpea transformation, CRISPR/Cas9, PDS, Codon optimization, Inference of CRISPR Edits (ICE)

CRISPR/Cas9 based genome editing of Phytoene desaturase (PDS) gene in chilli pepper (Capsicum annuum L.)

“…Several previously published studies indicated that CRISPR/Cas9-mediated PDS gene-edited mutants were photosensitive and failed to survive. 23 , 32 …”

“… 26 It also allows for simple screening of mutant lines. 27 , 28 Recent studies have shown the establishment of efficient genome editing using the PDS gene through the CRISPR/Cas9 system in several crops, including potato, 24 pea, 29 papaya, 30 coffee, 31 chickpea, 32 hop, 23 onion 25 and squash. 33 Nevertheless, the development of a biolistic-mediated CRISPR/Cas9-reagent delivery system using the PDS gene in chilli pepper has not yet been reported.…”

Optimized protoplast isolation and transfection with a breakpoint: Accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot

“…8C, D, and F). A handful of reports are available that show editing experiments in chickpeas (Gupta et al 2023). This protocol will facilitate researchers to undertake genome editing in chickpeas.…”