A New Multiplex CRISPR Method in Plants Can Generate High-Order Mutants in One Shot

Powerful gene-editing technologies, such as CRISPR-Cas9 are needed for efficient gene function analysis. The CRISPR-Cas9 tool has been adapted as an efficient gene-knockout technology in a variety of species. However, in many situations, knocking out just one gene is not enough. In such cases, multiplexing is required to understand the function of a gene.

Multiplexing refers to simultaneous targeting of multiple related or unrelated genes. Commonly, multiplexing for unrelated targets is done by delivering multiple guide RNAs independently or as a single cassette in the cell. However, multiplexing can reduce the efficiency of targeting single guide RNAs (sgRNA). Multiple sgRNAs may compete for Cas proteins or repetitive sequences of sgRNA-coding blocks may undergo recombination.

Why is Multiplexing in Plants Crucial?

Multiplexing is most crucial in plants where more than 50% of genes have more than one copy in the genome. Mutating just one copy may not result in a mutant phenotype as other copies would be still functional. This is seen in the case of Arabidopsis mutants where more than 90% of all single mutant lines had no phenotype. Therefore, targeting multiple genes simultaneously is prudent. However, challenges with multiplexing, including low efficiency and limit to the number of genes targeted simultaneously remain.

The Multiplex Vector Used in the Study

To understand the reason behind these challenges, a group of researchers from Germany explored the efficiency and limits of multiplex editing in two major plant model species, Arabidopsis and Tobacco. The results from the study were published in the Plant Journal.

For their experiments, the authors used the pDGE vector short for Dicot Genome Editing, which has improved efficiency and can target multiple genes with up to 32 sgRNAs. The pDGE vector system contains:

1. Shuttle vector for preparation of sgRNA
2. Pre-assembled recipient vectors

Shuttle vectors are used for cloning sgRNAs and recipient vectors are used for the final assembly of up to 32 sgRNAs. Recipient vectors also contain a highly intron-optimized Cas9 gene called zCas9i. According to the authors, “pDGE system based on pre-assembled recipient vectors containing a range of different zCas9 expression cassettes and selection markers provides simplicity of use.”

Making an Octuple Mutant with High Efficiency in One Shot

The group showed that it is possible to generate multiple mutations in different genes of tobacco plants with high efficiency. Using the pDGE vector, which contained 10 sgRNAs, the group targeted 8 different genes.

All the plants that were analyzed for target editing were chosen in such a way that they do not contain transgene expressing Cas9 protein. According to the authors, the continuous presence of nuclease-encoding transgene may result in mutant genotypes that are unstable or may vary between different parts of the plant. They observed:

1. All plants had mutations in all targeted genes, most of which were on both the copy of chromosomes.
2. They also noted an unexpected overrepresentation of homozygous mutations i.e. mutations on both the copy of chromosomes, which they attribute to high expression of zCas9i protein using RPS5A promoter.
3. 112 out of 116 analyzed sgRNA target sites were scored as edited, which amounted to 96.5% efficiency.
4. They found that the RPS5A promoter used for inducing the expression of sgRNA and zCas9i protein is most suitable for editing in tobacco plants, as it appears to remain active in germline cells throughout development. “This leads to high efficiencies and potential for generation of multiple independent alleles.”

Multiplexing in Arabidopsis at 12 Different Gene Locations

The group attempted targeting 12 different genes by an array of 24 sgRNA in the Arabidopsis plant. The target genes were nucleotide-binding domain-leucine-rich repeat-type resistance genes and developmental regulators. The Arabidopsis genes were targeted using an array of 24-sgRNA assembled in a pDGE vector. The study revealed:

1. Reduced efficiency at individual target sites when 24-sgRNA array was used. 80% targeting efficiency was seen with control sgRNA whereas only 30-40% efficiency was seen with 24-sgRNA construct. This was mainly due to the reduced availability of Cas9 at all the target sites.
2. A prominent problem with repetitive sequences containing multiple sgRNAs is recombination within sgRNA units. However, no such recombination events were observed in these experiments.
3. In second-generation plants, all the targets were found mutated albeit at different frequencies. Rps2 locus was most frequently mutated and high mutation efficiencies were detected for sgRNAs targeting this locus. Rps4 and 5 were least mutated and low efficiencies were detected for the respective sgRNA.
4. Despite the low frequency of editing, the group successfully isolated an Arabidopsis plant within a single generation that was mutated at all the 12 target genes. This was made possible because of the high efficiency achieved by zCas9i endonuclease.

Take-Home Message

According to the authors, it is possible to generate higher-order mutants with this strategy in one generation. The researchers give a workflow that can be used for getting octuples or duodecuples in one shot. They suggest that in the first round of analysis, sequencing of targets should be done to select mutants with low frequency followed by the selection of loci with high mutagenic activity. But the study also points at challenges.

The need for sgRNAs that are highly efficient at targeting specific locations still remains. Moreover, enhancement of nuclease activity by co-expression of TREX2 exonuclease or by using a different nuclease such as SaCas9 will help to generate high-frequency high-order mutations.

“The high multiplex editing efficiencies we report open up new perspectives for the generation of complex genotypes and for functional analysis of numerous candidate genes by RNA-guided nucleases (RGNs).”

Related Article: The Future of CRISPR: Three Areas The Nobel Prize Winning Tech Will Be Most Impactful

Author

Rajaneesh K. Gopinath