In-planta Gene Targeting in Barley Using Cas9 With and Without Geminiviral Replicons

Abstract

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications; however, this remains inefficient especially in key crop species. Here, we describe successful, heritable gene targeting in barley at the target site of Cas9 using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase the copy number of the repair template. Without the replicon, we were able to delete 150 bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared with the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false-positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1, and subsequently, the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

Keywords: homology-dependent recombination; knock-in; precise insertion; repair template; wheat dwarf virus.

Figure 1

(A) Repair template, (B) target locus and (C) successful GT into the target locus. Promoter region of HORVU4Hr1G061310 in light blue, mCherry CDS in purple, HORVU4Hr1G061310 CDS in grey, and 3′ UTR in black. Protospacer sequences (A,B) in dark blue. Associated PAMs in red. Guide efficiency is shown as % of lines in which indels are detected. Complete protospacer/PAM sequences are absent from (A,C) to prevent cleavage by Cas9. A successful event leads to a partial deletion of the HORVU4Hr1G06131 CDS with the remainder being fused in-frame to mCherry. Forward (F) and reverse (R) screening primers are indicated as black horizontal arrows.

Figure 2

Constructs for plant transformation used in this study: RB, right border; LB, left border; HygR, hygromycin resistance cassette; Cas9 Hs, codon-optimised SpCas9 driven by Zm Ubiquitin promoter; sgRNA, guide RNA expression cassette for two guides (A,B); Left arm, left homology arm; mCherry, mCherry reporter CDS; Right arm, right homology arm; LIR, long intergenic region; SIR, short intergenic region; REP, replicase proteins; qPCR probe used for copy number determination. Thick black vertical bars indicate target sites for the Cas9/guides. Construct (A) is the basic GT version, and construct (B) is the same except the repair template is contained between replicon sequences. Construct (C) is the same as (B) but lacks the Cas9/gRNA and ability to introduce DSBs. Construct (D) has no Cas9/gRNA or replicon but has the repair template with extended homology arms. It was transformed into barley and used to establish a sensitive PCR assay. F1, R1, F2, R2, and R3 are PCR primers used for screening.

Figure 3

Gel showing segregation of GT event in 1826-8-1_A T2. Bands are the products of F1/R3 primers. + indicates GT allele; – indicates WT allele.

Figure 4

Key findings comparing in-planta GT with and without geminiviral replicon; 2158 are replicon lines (construct B) and 1826 lines are non-replicon lines (construct A).