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

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.