Biology subjects

Hinchliffe, A.

Publications and source records attributed to Hinchliffe, A..

3 recordsLinked to original sources

bioRxiv2022

Highly efficient genome editing in barley using novel LbCas12a variants and impact of sgRNA architecture

We report the first successful, high efficiency use of LbCas12a in barley and describe the development and application of two novel Cas12a variants. In total we compared five coding sequence (CDS) variants including the two novel ones and two guide architectures over 5 different target genes using twenty different guides. We found large differences in editing efficiencies between the different CDS versions (0-87%) and guide architectures (0-70%) and show our two novel CDS versions massively outperform the others on test in this species. We show heritability of mutations generated. Our findings highlight the importance of optimising CRISPR systems for individual species and are likely to aid the use of LbCas12a in other monocot species.

genomics↗

bioRxiv2021

In-planta Gene Targeting in Barley using Cas9, with and without Geminiviral Replicons

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 using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase copy number of the repair template. Without the replicon, we were able to delete 150bp 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 to 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.

plant biology↗

bioRxiv2017

Speed breeding: a powerful tool to accelerate crop research and breeding

The growing human population and a changing environment have raised significant concern for global food security, with the current improvement rate of several important crops inadequate to meet future demand [1]. This slow improvement rate is attributed partly to the long generation times of crop plants. Here we present a method called speed breeding, which greatly shortens generation time and accelerates breeding and research programs. Speed breeding can be used to achieve up to 6 generations per year for spring wheat (Triticum aestivum), durum wheat (T. durum), barley (Hordeum vulgare), chickpea (Cicer arietinum), and pea (Pisum sativum) and 4 generations for canola (Brassica napus), instead of 2-3 under normal glasshouse conditions. We demonstrate that speed breeding in fully-enclosed controlled-environment growth chambers can accelerate plant development for research purposes, including phenotyping of adult plant traits, mutant studies, and transformation. The use of supplemental lighting in a glasshouse environment allows rapid generation cycling through single seed descent and potential for adaptation to larger-scale crop improvement programs. Cost-saving through LED supplemental lighting is also outlined. We envisage great potential for integrating speed breeding with other modern crop breeding technologies, including high-throughput genotyping, genome editing, and genomic selection, accelerating the rate of crop improvement.

plant biology↗