Biology subjects

Lawrenson, T.

Publications and source records attributed to Lawrenson, T..

4 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↗

bioRxiv2022

Improved SpCas9 and LbCas12a genome editing systems in Brassica oleracea and Brassica napus

We report highly efficient genome editing in Brassica species. We compare the efficiency of targeted mutagenesis using four Streptococcus pyogenes Cas9 (SpCas9) systems in Brassica oleracea and Brassica napus over 3 target genes and five guides to identify two which show a striking improvement to our first published system (Lawrenson et al., 2015). Targeted mutagenesis occurred in up to 100% of T0 plants with the improved systems, compared to 20% in the original system. This is the only reported comparison of SpCas9 systems we are aware of in Brassica species. Secondly, we report the first successful use of Lachnospiraceae bacterium Cas12a (LbCas12a) in Brassica oleracea. We test three LbCas12a coding sequences and two guide architectures against one target gene using four guides. From this we identify the best performing combination of our novel, multi-intron coding sequence and a ribozyme flanked guide expression cassette. In this case 68% of T0 plants contained targeted mutations. Heritability of LbCas12a mutations is shown. We show that our two useful and novel LbCas12a coding sequences have utility in Brassica 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↗

bioRxiv2020

CRISPR-Cas9-mediated editing of myb28 impairs glucoraphanin accumulation of Brassica oleracea in the field

We sought to quantify the role of MYB28 in the regulation of aliphatic glucosinolate biosynthesis and associated sulphur metabolism in field-grown B. oleracea with the use of CRISPR-Cas9-mediated gene editing technology. We describe the first characterised myb28 knockout mutant in B. oleracea, and the first UK field trial of CRISPR-Cas9-mediated gene edited plants under the European Court of Justice interpretation of the 2001/18 EU GMO directive. We report that knocking-out myb28 results in downregulation of aliphatic glucosinolate biosynthesis genes and reduction in accumulation of the methionine-derived glucosinolate, glucoraphanin, in leaves and florets of field-grown myb28 mutant broccoli plants. There were no significant changes to the accumulation of sulphate, S-methyl cysteine sulfoxide and indole glucosinolate in leaf and floret tissues.

plant biology↗