Carpet Screening: Leading Editors to Realize Directed Evolution of Rice

Carpet Screening: Leading Editors to Realize Directed Evolution of Rice

Carpet Screening: Guide editors to realize the directed evolution of rice Carpet Screening: guide editors to realize the directed evolution of rice

New herbicide-resistant rice germplasm Photo courtesy of Wei Pengcheng

 New herbicide-resistant rice germplasm Photo courtesy of Wei Pengcheng

More than a year ago, the guided editing technology that can realize the free conversion of all 12 single bases of the genome was born
.

On June 10, Chinese scientists published their latest research results in "Nature-Plants", using guided editing technology to achieve directed evolution in rice for the first time, and created new germplasm for breeding

"Our work proves that artificial evolution has certain advantages over natural selection, which can greatly speed up the process of crop improvement
.

" Wei Pengcheng, the corresponding author of the paper and a researcher at the Anhui Academy of Agricultural Sciences, said in an interview with China Science Daily

Wang Kejian, a researcher at the Chinese Rice Research Institute of the Chinese Academy of Agricultural Sciences, told the Chinese Journal of Science that, compared with CRISPR targeted mutation and single-base editing technology, guided editing technology provides a new method for artificial evolution of key functional sites
.

Exhaust 64 combinations to achieve saturation mutation screening

Exhaust 64 combinations to achieve saturation mutation screening

There are four kinds of bases in the genome
.

The gene editing technology that appeared in 2013 can realize the conversion between bases

How long will it take for a new technology to be applied in the agricultural field?

In one year and eight months, the guided editing technology was successfully developed in mammals, successfully established and optimized in plants, and realized editing of all 20 amino acids at functional sites in rice and creating new germplasm
.

"Early gene editing methods (targeted mutations) can only produce a mutation in a certain segment, but there is no way to control what this mutation looks like
.

That is to say, it can control the location of the mutation, but not the form of the mutation

Moreover, targeted mutations often insert or delete a fragment, causing functional damage
.

"But what is needed in production is not the destruction of this function, but to adjust the function of the corresponding site a little higher or lower, or even create a new function

Scientists have always wanted to try all possible mutations at a gene locus, that is, try what kind of phenotype all amino acids produce
.

According to Xu Rongfang, the first author of the paper, 3 bases make up 1 amino acid, and there are 64 combinations of 4 bases randomly arranged, and these combinations correspond to all 20 amino acids
.

In their research, using guided editing technology, the conversion of all 64 base combinations can be realized at a specific site, that is, the saturation mutation of a single amino acid site can be completed

"In vitro saturation mutation screening is currently an important method for studying protein functions, but there are still great technical difficulties in achieving high-frequency substitution of key amino acids in eukaryotic organisms
.

" Wang Kejian said that this research has successfully established a achievable method in rice.

 

Create new sites that don't exist in nature

Create new sites that don't exist in nature

Genetic variation is the basis of crop breeding
.

Wei Pengcheng introduced that although functional genomics research has provided a large number of major improvement sites/genes for crop molecular breeding in recent years; however, due to technical problems such as insufficient targeting of traditional mutagenesis and large mutation randomness, in most cases, mining and It is still difficult to identify the most suitable alleles for these major genes

In the process of researching rice herbicide resistance genes, many mutations are naturally occurring
.
"But we don't know if these natural mutations are the most suitable mutations
.
" Wei Pengcheng explained.
For example, a mutation can make rice have herbicide resistance, but it may also cause slow growth or even a decline in yield
.

Scientists speculate that natural mutations may not give a gene to its full advantage
.
Wei Pengcheng said that through saturation mutation, that is, to try different amino acid substitution mutations at certain key positions one by one, and then test the effect of different alleles, it is possible to find the most suitable mutation for a certain position of a crop.

.

"How to achieve substitution mutations of saturated amino acids at key sites of important genes in vivo is a difficult problem in this type of research
.
" Wei Pengcheng said
.

Xu Rongfang told the Chinese Journal of Science that the selection of rice herbicide resistance gene OsACC1 as the research object was mainly based on the function of this gene.
OsACC1 gene is a key herbicide response gene, which can distinguish different phenotypes through resistance screening experiments
.

Through a lot of experiments, they found that herbicide resistance genes have great potential
.
Xu Rongfang introduced that through methodological research on 6 reported potential resistance sites, they found that 16 different types of amino acid substitution mutations may be closely related to the acquisition of herbicide resistance
.
Three of these sites were identified for the first time in plants
.

"The guided editing technology has the ability to mutate the target amino acid into the other 19 amino acids, thereby providing a new method of artificial evolution of key functional sites
.
" Wang Kejian said, this actually speeds up the evolution
.

 

Artificial evolution has more advantages

Artificial evolution has more advantages

This is actually a directed evolution
.
Wei Pengcheng explained that directed evolution is a simulation of natural evolution at the molecular level in the laboratory
.
That is to say, according to demand, a large number of mutations are artificially created for the target protein and selective pressure is given to screen out variants with desired characteristics, and then solve the production problem
.

“We have screened out sites that do not exist in nature, even through physical and chemical mutagenesis and gene editing, which are difficult to create
.
” Wei Pengcheng said, which proves that artificial evolution methods do have advantages over natural selection and greatly broadens the creation of germplasm.
Ideas
.

"Researchers have used this technology to successfully screen multiple new herbicide-resistant sites in rice, showing great application value
.
" Wang Kejian said that the research not only established a new method for directed evolution of crop genes, but also helped to fully excavate important genes.
The new allotype, breaking through the limitations of existing germplasm resources, provides new ideas for manual "customization" of excellent traits according to production needs, which has important reference significance
.

Xu Rongfang told the Chinese Journal of Science that the three newly screened sites showed good herbicide resistance in the current experiment.
We will further investigate whether they can maintain good agronomic traits while being resistant to herbicides.

.

"The efficiency of the existing guided editing system is still relatively low.
When selecting gene loci, some other auxiliary means, such as resistance screening, etc.
, are needed to reduce the workload
.
As the efficiency of the guided editing system improves, this method will be available.
For more directed evolution of other functional genes
.
" Xu Rongfang said
.

Compared with other methods, "Through artificial evolution, the endogenous sites of the rice we have screened may soon be directly used in production to help realize the green production model of rice
.
" Wei Pengcheng said
.
They have protected the intellectual property rights of the newly discovered gene locus
.
Wei Pengcheng believes that with the deepening of research and the expansion of applications, it will be possible to apply it to agricultural practice in the future
.

Related paper information: https://doi.
org/10.
1038/s41477-021-00942-w