Clustered Regular Interspaced Short Palindromic Repeats (CRISPR) specific nucleases have quickly become one of the most promising methods of making specifically placed double-stranded breaks in DNA. By expressing a guide RNA with a Cas9 nuclease within an organism, DNA can be completely restricted (amongst other uses), lending itself to the ability to rejoin the DNA with a new piece of DNA inserted or, as in this particular study, simply disrupting a specific gene by rejoining the DNA but with one or more nucleotides missing from the genetic code via non-homologous end joining (see our collaborative piece with PLoS SynBio for a description).
Rice Blast lesion.
A research article in PLoS One recently (have we mentioned we love open science) tackled the issue of increasing resistance to rice blast (Magnaporthe oryzae) using this new tool. Rice, feeding nearly 50% of the world population, is an important staple crop and rice blast, one of the most destructive diseases in rice production, makes the desire to reduce yield loss caused by this fungus one of significant effect should it be realised.
The researchers identified previous studies which had found that a particular gene, OsERF922, is a negative regulator of BLAST resistance. The previous study used a host-expressed RNAi construct to knock-out this gene in rice and, when the rice plants were challenged with the fungus, the transgenic plants showed greater resistance.
The problem with using a RNAi-expressing transgenic plant is that the modification, being an insertion of DNA into the genome, comes under transgenic regulations throughout most of the world, making its adoption for use much more difficult. Traditional breeding to achieve a similar knockout can take nearly a decade to produce. The purpose of this study, therefore, was to use CRISPR to mutate and knockout the gene without leaving any foreign DNA in the genome.
The researchers created a DNA construct that would express the guide RNA and direct the restriction of this gene 7 base pairs away from the initiation codon. To test whether the construct worked it was tested by transforming it into rice protoplasts and then amplifying the target section of DNA. 3 mutant protoplasts were recovered, one with a single base substitution, one with a five base deletion and one with a 30 base insertion, demonstrating that the CRISPR designed worked in the cell.
With the activity of the CRISPR confirmed, rice plants were transformed by using Agrobaterium tumefaciens to deliver the DNA construct which resulted in 50 first generation transgenic plants, of which 21 were sequenced. More than half of the transgenic plants analysed contained base pair deletions (about one third of which were deletions of less than 10 base pairs) while about a quarter had a single base pair insertion and about one-tenth has simultaneous insertions and deletions. Importantly, 16 of the 21 transgenic plants had bi-allelic mutations (mutated on both copies of the gene, although mutated in different ways) and three were homozygous for the mutation.
Having ascertained that mutations could be made in the rice chromosomes, 6 of the analysed rice plants were self pollinated and then genotyped at the target gene. The 120 plants produced all carried the mutations (the progeny of the bi-allelic parents segregated according to the Mendellian ratio of 1:2:1).
Using three of the homozygous, one bi-allelic and one heterozygous plants, a second generation was bred. The homozygous parents produced progeny all of whom had stably transferred the mutation. The bi-allelic and heterozygous plants again followed Mendellian genetic segregation.
Now that the researchers have identified progeny with stably transferred mutations, they searched for the possible transfer of the CRISPR DNA or the transfer DNA from the Agrobacterium tumefacians anywhere in the rice genome of the 120 first generation transgenic rice plants. They generated primers for the Cas9 gene and subjected the primers and the DNA to PCR amplification. The result was the generation of no amplicons for these primers. The story was a little different in the two progeny generations where only 10% of the plants in the second generation didn’t have any detectable remnants of the transfer DNA construct. However, all 30 T2 progeny of one homozygous T1 parent contained no amplified remnant of the CRISPR or transfer DNA, demonstrating that it is possible to make a stably inherited homozygous mutation without incorporating the DNA construct.
Amplified PCR products of T-DNA within mutagenic rice crops. All 30 progeny of T1 plant KS2-45-6 (column 6 of the second row from the top) showed no detectable trace of the T-DNA construct.
So, the story so far…..
We have piece of DNA which can cause mutations in this specific gene in rice protoplasts and rice plants, and the mutation can be passed from generation to generation and can do so without incorporating the CRISPR or vector constructs.
The experiment (cont.)
Now that the ability to mutate the gene has been demonstrated, 6 homozygous T2 transgenic rice plants with differing types of mutation (deletion, addition, substitution) were inoculated with the rice blast fungus at the seedling stage of development. Leaves of the wild type control plant nearly died from the inoculation but the transgenic plants showed significantly decreased lesion areas and lengths, demonstrating that the loss-of-function mutation of OsERF9222 resulted in enhanced resistance to rice blast.
Results from study: Figure A showed sequenced wild-type and the 6 mutated genes of the homozygous mutated crops. Figures B – E show the effects of challenging each of the crops with Magnaporthe oryzae.
Rice blast resistance isn’t worth anything if the mutation also negatively affects the agronomic traits of the crop. Therefore, the 6 homozygous mutants were assessed and compared to the wild type plants in relation to their:
- plant height;
- flag leaf length and width;
- number of productive panicles;
- panicle length;
- number of grains per panicle;
- seed setting rate; and
- thousand seed weight.
All 6 plants (and importantly, the plant which produced all devoid of the CRISPR and vector DNA) showed no significant difference in each of the criteria compared to a health wild type plant.
And a little extra…
Just to check the frequency of producing mutagenic crops from directing mutations to a specific gene, the researchers checked whether the frequency could be be increased by targeting multiple sites in that gene with the one CRISPR construct. They created one construct which targeted two sites in the gene, and another which attacked three sites in the gene. The result was an increased frequency of mutations as the number of target sites increased and a corresponding increase in the percentage of mutants with homozygous sequence changes.
This research demonstrates the ability to induce a specific, advantageous change with this recently discovered, highly adaptable tool of genetic modification, and do so without adding any extraneous DNA to genome. The result is creating a resistant crop in a much shorter amount of time than traditional breeding will allow using a tool that can be adjusted quickly to pivot should evolution of the pest occur. Importantly, the changes in this particular gene didn’t affect the agronomic traits of the rice crop, resulting in a consumer acceptable staple food and decreased yield loss, a significant development in our ability to feed more people with the same amount of land and resources and potentially less fungicides.