CRISPR-Cas9 Mediated Gene Knockout Screening for Drought Tolerance in Cereal Crops
DOI:
https://doi.org/10.5281/zenodo.22687565Keywords:
CRISPR-Cas9, gene knockout screening, drought tolerance, barley, abscisic acid signalling, cereal cropsAbstract
Drought remains the single largest abiotic constraint on cereal yield worldwide, and breeding pipelines built on
natural variation alone are struggling to keep pace with the rate of climatic change. Here we describe an arrayed
CRISPR-Cas9 knockout screen of nine candidate genes drawn from the abscisic acid (ABA) signalling and
biosynthesis pathways of barley (Hordeum vulgare L. cv. Golden Promise), designed to identify loci whose
disruption measurably improves whole-plant performance under progressive soil water deficit. Guide RNAs were
delivered to immature embryo-derived callus by Agrobacterium-mediated transformation, and edited T0
regenerants were advanced to homozygous, transgene-free T2 families before phenotyping. Two knockouts,
hvera1 and hvpp2c1, both encoding negative regulators of ABA signalling, showed a roughly two-fold increase in
post-drought recovery survival relative to Cas9-null segregants (71.6% and 64.8% versus 34.2%, respectively),
together with reduced stomatal conductance and higher leaf relative water content during dry-down. Conversely,
knockouts of the positive regulators hvdreb2a and hvnced1 were markedly more susceptible to drought, consistent
with their established roles in stress-responsive transcription and ABA biosynthesis. Whole-genome resequencing
of the two lead lines detected no confirmed off-target edits at the top ten in-silico predicted sites. These results
reinforce ABA signalling negative regulators as tractable, evolutionarily conserved targets for drought-tolerance
engineering in cereals, and they illustrate how a modestly sized, hypothesis-guided knockout panel can generate
field-relevant leads more quickly than a purely forward-genetic screen.
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