CRISPR/CAS9 ribonucleoprotein complex mediated editing of TaASN2 to develop low acrylamide wheat
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Date
2022
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Punjab Agricultural University, Ludhiana
Abstract
Processed food products of wheat have been found to possess acrylamide, a potent neurotoxin and a potential carcinogenic compound. It is formed non-enzymatically when free reducingsugars and free asparagine present in wheat flour react at high temperature cooking via Maillard reaction. Asparagine synthetase 2 (TaASN2) has been identified to be responsible for free asparagine accumulation in grain, the concentration of which is directly related to its acrylamide forming potential. In the present study, CRISPR/Cas9 based genome editing approach was used to generate TaASN2 specific knock-outs to develop low acrylamide genetic stocks of wheat. BWL 5429, a high yielding, disease resistant and a high grain protein wheat line known for its superior chapatti quality was used as the target genotype. To achieve DNA-free editing, vectorless direct delivery approach that uses pre-assembled ribonucleoproteins was employed. Target specific gRNAs were designed using CRISPOR and Wheat CRISPR online available softwares. D-genome specific gRNA [having MM (MorMateos) = 83, CFD (Cutting Frequency Determination) = 81, MIT Specificty Score = 63)] was designed from exon 4 of TaASN2, which showed cleavage of the amplified TaASN2 gene segment upon in vitro cleavage, so was chosen for particle gene gun mediated genetict ransformation. Ready-to-use Cas9 was procured commercially. Pre-assembled RNP complex was used to genetically transform three explants- ‗mature embryo‘, ‗mature embryo with endosperm intact‘ and ‗immature embryo‘. 323 TaASN2-specific and 185 TaPDS-specific T1 spikes were harvested from T0 regenerated embryo/seeds transplanted in pots. ∼1500 spikes of TaASN2 specific knock-outs were harvested in the following crop season. DNA extractedfrom T0 and T1 putative genome edited plants was subjected to amplification of exon 4 of TaASN2 specific to D-genome copy. Upon restriction digestion of exon 4 specific amplicon with HaeIII, the appearance of an intact 267 bp band in a few plants enabled the detection of positive genome edited events. These positive identified TaASN2 knockouts may be further confirmed via amplicon sequencing.
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Prabhjot Kaur (2022). CRISPR/CAS9 ribonucleoprotein complex mediated editing of TaASN2 to develop low acrylamide wheat (Unpublished M.Sc. thesis). Punjab Agricultural University, Ludhiana, Punjab, India.