Abstract
Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8′-HYDROXYLASE 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.
Highlights
Genome editing in cereals greatly facilitates genetic improvements that would have been more cumbersome with previously existing mutagenesis tools
We report the evaluation of cas9/gRNA construct performance in planta based on two methods; first, a transient expression assay developed in barley [17], and second, by triticale protoplast transfection
To uncover potential nucleotide polymorphisms between these data and the triticale subgenomes, which may affect targeted mutagenesis, the first two exons of TsABA8 OH1 from the Polish winter triticale variety Bogo, an accession that has been used as experimental model for genetic engineering, were amplified using common primers (Table S1), cloned in plasmids and sequenced (Table S1)
Summary
Genome editing in cereals greatly facilitates genetic improvements that would have been more cumbersome with previously existing mutagenesis tools. A powerful platform for site-specific changes in plant genomes is available for which components of the microbial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) endonuclease immune system have been repurposed [1]. These RNA-guided Cas endonucleases have been employed to generate loss-of-function alleles by targeting single or multiple loci in bread wheat, barley, rice, and other grass species but not yet in triticale. Few reports of successful employment in hexaploid common wheat [4,5,6,7,8,9] suggest that low transformation efficiency for wheat as well as the large genome size makes it exceedingly difficult for the Cas9/gRNA complexes to find their target. It is necessary to improve the specificity and efficiency of Cas on-target mutagenesis and to develop a straightforward in vivo validation system for cas9/gRNA constructs, since the generation of transgenic triticale plants is cost and time-consuming, but has been achieved only by very few laboratories worldwide
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