TPJ article: Twelve genes at one blow: multiplex genome editing with CRISPR/Cas

16 Apr 2021 - By: Leonie Verhage

Twelve genes at one blow: multiplex genome editing with CRISPR/Cas

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

Stuttmann, J., Barthel, K., Martin, P., Ordon, J., Erickson, J.L., Herr, R., Ferik, F., Kretschmer, C., Berner, T., Keilwagen, J. and Marillonnet, S.

TPJ - Twelve genes at one blow- multiplex genome editing with CRISPR:Cas

Next year marks the 10th anniversary of CRISPR/Cas as a tool for targeted gene editing. Like many other life scientists, plant researchers embraced the technique from the start. The long list of plant species in which CRISPR/Cas was applied ranges from liverworts and mosses, to monocots like maize and rice, and dicots like potato, poplar, and banana. The technique has advanced quickly, but there are still challenges left. One of them is the question of how to edit larger numbers of genes by targeting several independent target sites at once (multiplexing). Being able to mutate a series of genes simultaneously allows for the rapid creation of complex higher-order mutants. Such mutants can, for example, be used to reveal the function of redundant genes and understand genetic networks.

In issue 106::1 of The Plant Journal, Johannes Stuttmann and his colleagues explored multiplex genome editing with larger numbers of sgRNAs in two dicot model species: Arabidopsis thaliana and Nicotiana benthamiana. In N. benthamiana, the authors targeted 8 different genes with 9 sgRNAs. They were able to generate a transgene-free mutant with mutations in all targeted genes in one generation. In A. thaliana, the authors targeted 12 genes with 24 different sgRNAs. Isolation of the 12x (duodecuple) mutant proved a bit more difficult. Because the A. thaliana transformation vector contained a high number of sgRNAs, the authors decided to first investigate the effect of the large number of sgRNAs on the gene editing efficiency. The authors produced control constructs that only contained sgRNAs targeting targeting 2 genes with 4 sgRNAs, and found that 80% of the plants showed a double mutant phenotype. However, the vector carrying all 24 sgRNAs resulted in a 30-40% drop in efficiency. This suggests that Cas9 availability might be a limiting factor when introducing a large number of sgRNAs.

Based on these results, the authors realized that isolating the duodecuple mutant in Arabidopsis was going to be more difficult than the octuple mutant in N. benthamiana. Therefore, the functionality of six additional genes, including several genes conferring resistance to plant pathogens or developmental regulators, was first tested in phenotypic assays. Subsequently, they performed an amplicon sequencing approach on PCR samples that were pooled from ten T2 segregants per line. The two lines with the most mutations were selected for individual genotyping. Within these lines, the authors identified a plant with mutations in all of the 12 target genes.

The authors showed that multiplexing is relatively efficient and straightforward in N. benthamiana and A. thaliana. Although they observed reduced efficiencies in A. thaliana when they introduced 24 sgRNAs, they did not observe other issues such as silencing or recombination. Stuttmann thinks there are interesting applications for multiplexing (Figure). Besides classical reverse genetics, he thinks the system can be used for what he describes as ‘targeted forward genetics’, wherein a pool of sgRNAs would be designed for a list of candidate genes, and then assembled into random arrays in a library format. After transformation, genome edits would be analyzed only for plants with a phenotype of interest. Such an approach might identify different combinations of genes that regulate a phenotype of interest. An exciting outlook for CRISPR/Cas on the eve of its 10th anniversary.

Leonie Verhage, Research Highlights Editor


Author: Leonie Verhage
Category: The Plant Journal
Leonie Verhage

Leonie Verhage

Leonie Verhage is Research Highlights Editor at The Plant Journal. For every issue, she highlights one of the key articles with a commentary paper.
On Twitter, Leonie keeps the followers of The Plant Journal (@ThePlantJournal) updated on the latest articles.

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