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Expeditious evaluation of gRNA constructs driven by a root and callus preferential promoter, MsPRP2, to generate edits of the EcHKT1;1 gene in Eucalyptus camaldulensis using GFP‐tagged hairy roots

Krishnaraj Shamili,M. C. Sandhya,Manoj Kumar Rajendran,Sreeja Sahadevan,V. Chinnusamy,Aiyar Balasubramanian,Sivakumar Veerasamy,Mathish Nambiar-Veetil

Published 2025 in Annals of Applied Biology

ABSTRACT

Clustered regularly interspaced short palindromic repeats/ CRISPR‐associated protein 9 technology involves the use of designed guide RNAs to direct gene editing without any off‐target effects. Rapid evaluation of these guide RNA (gRNAs) for engineering the desired gene edits becomes important in tree species such as Eucalyptus, wherein regeneration of gene‐edited plantlets using Agrobacterium tumefaciens‐mediated transformation takes more than 1 year. In the present study, a synthetic promoter, Medicago sativa proline rich promoter 2 (MsPRP2), reported earlier to drive root‐preferential and salt‐inducible gene expression in Medicago sativa, Arabidopsis thaliana and Glycine max, was evaluated in Eucalyptus camaldulensis for generating the desired deletion in the Eucalyptus camaldulensis high‐affinity potassium transporter (EcHKT)1;1 gene. Two single guide RNA sequences cognate to the EcHKT1;1 promoter and exon 1 region separated by 1411 bp were selected to synthesise the polycistronic gRNA‐tRNA cassette with the MsPRP2 promoter and HSP terminator. After splicing together the MsPRP2 promoter and HSP terminator as flanking sequences, the construct was cloned into cauliflower mosaic virus‐driven Cas9_1 and GFP‐based transformation vectors for Agrobacterium rhizogenes‐mediated transformation of hypocotyl explants. The pooled GFP‐tagged roots generated 36 days after co‐cultivation were used to evaluate the efficiency of the construct by polymerase chain reaction (PCR) analysis and amplicon sequencing. In comparison to the 2375 bp amplicon generated from the IFGTB‐EC‐1 root, the GFP‐tagged roots showed a smaller 964 bp amplicon with the expected 1411 bp deletion between the promoter and exon 1 of the EcHKT1;1 gene. Further, the quantitative reverse transcription polymerase chain reaction (RT‐qPCR) results showed a 3.22‐fold (69%) downregulation when compared to the wild A4RS roots. To further validate these results obtained from GFP‐tagged hairy roots, the EcHKT1;1 gene editing constructs were also evaluated in transgenic events of Eucalyptus generated using A. tumefaciens. These transgenic events showed an expected 1411 bp deletion, albeit in a heterozygous state. Further, RT‐qPCR analysis showed 2.08‐fold (52%) EcHKT1;1 gene downregulation when compared to the control IFGTB‐EC‐1 seedling. However, harvesting sufficient leaves for molecular analysis had to wait for 16 months post‐co‐cultivation. Contrastingly, the higher transformation efficiency of A. rhizogenes‐mediated transformation, at around 20% when compared to 0.59% for A. tumefaciens, made it possible to pool tissue from a larger number of plants for PCR‐based analysis of GFP‐tagged roots in 36 days. Thus, this study demonstrates the feasibility of using rapidly generated GFP‐tagged roots for expeditiously evaluating the efficiency of the MsPRP2 promoter and the selected gRNAs to generate desired gene edits.

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