Differential editing efficiencies in cereal crops: a comparative analysis of tRNA and ribozyme multiplexed guide delivery

Abstract

Cereal transformation and gene editing can be a complex and costly undertaking. It is therefore important to validate and understand the performance of the components to achieve high rates of transformation and gene editing. Here, we have made a direct comparison of different CRISPR/Cas9 guide systems to target the genome in three cereal species. We show that the guide sequences driven by the same pol II promoter in rice, wheat and barley show large differences in editing efficiency. The differences seen were based on the way the guides were presented and factors outside of the guide sequence itself. While both the tRNA system and ribozyme system performed well in rice, their effectiveness varied in wheat and barley. Specifically, the tRNA system outperformed the ribozyme system, achieving higher rates of editing in stable transformed plants. Overall, high levels of editing are observed in all three species when strong expression of the SpCas9 is coupled with the CmYLCV promoter to drive a tRNA array of guide RNAs. Stable inheritance is also achievable in all three species when plants are sampled shortly after the tissue culture concludes. Overall, inheritance rates were above 85% in all three species, particularly when mutations are detected early after plants emerge from tissue culture.

Keywords: CRISPR; CmYLCV; Gsk1; barley; ribozyme; rice; tRNA; wheat.

Figure 1

Schematic diagram of the pMM36 (A) and pMM37 (B) T-DNAs plus the gsk1 genomic sequences from rice (C), barley (D) and wheat (A-homoeologue) (E) with the guide target sites shown. Distances between target guide PAM sites in rice are 698bp (guides 1-2), 330bp (guides 2-3); in barley 796bp (guides 1-2), 336bp (guides 2-3); and in wheat A-genome 806bp (guides 1-2), 341bp (guides 2-3).

Figure 2

Schematic diagram of promoter construct T-DNA structure. The T-DNA structure for the dual reporter binary construct pMS31 containing the CmYLCV promoter is shown. This includes the OsActin promoter-luciferase cassette and the Sc4 promoter-nptII selection gene cassette. Other promoters were substituted for CmYLCV, namely the TaU6 promoter in pMS33 and the Zea mays Ubiquitin promoter in pAK90 and pAK93 (with hygromycin cassette).

Figure 3

Comparison of CRISPR/Cas9-mediated editing efficiency of three guides targeting GSK1 in rice, wheat and barley delivered as a tRNA or ribozyme system in T₀ rice, barley and wheat plants.

Figure 4

Relative GUS Expression Assessed by Semiquantitative PCR. Agarose gels showing OsActin: Luciferase and GUS transcript levels expressed from the pZmUbi, pCmYLCV and pTaU6 promoters in four independent rice (COS) and wheat (CTA) plants (A). M-1Kb gel marker, Wt- non-transformed control plant. Agarose gel bands were quantified using Image J software, and the resulting values were normalized against the luciferase, which served as the transformation control. The fold change was then calculated against the luciferase reference, illustrating the relative expression levels of GUS (B). The error bars depict the standard error of the mean across four independent lines. Utilizing Tukey’s Honest significance test, a notable difference is observed between pZmUbi and pTaU6 in both rice and wheat. In rice alone, a significant variance in promoter activity is evident between pCmYLCV and pTaU6. Significance levels are denoted as **** for p < 0.0001.