Created a tool that facilitates the genetic modification of plants

(ORDO NEWS) — American bioinformaticians have developed a new open source tool that can significantly simplify and speed up the genetic modification of plants using CRISPR/Cas technology.

The authors hope that the new software will not only speed up research, but also reduce the number of failed experiments.

Created a tool that facilitates the genetic modification of plants 1            The CRISPR/Cas9 complex (white) cuts out a section of DNA (green) guided by a guide RNA (blue)

Today, genetic modification of plants, especially crops, is a widespread practice. It is used to change the characteristics of fruits, increase productivity and plant resistance to stress. CRISPR/Cas technology plays a very important role in the modification process.

In modern genetic engineering, CRISPR/Cas is the workhorse of most targeted gene editing methods. Using it, you can purposefully cut out sections of DNA in the cells of various organisms in order to embed other sections of DNA carrying the desired gene in their place. The target DNA region is recognized by the guide (or guide) RNA (gRNA), after which the Cas protein cuts it out.

In theory, everything sounds simple, but in practice there is a risk of non-specific DNA editing (not only in the place where it was supposed) and violation of the sequence of coding genes.

All due to the fact that gRNA can be complementary to several sections of DNA. The situation is exacerbated in the case of many agricultural crops due to the complexity and polyploidy of their genomes.

The Center for Advanced Innovation in Bioenergy and Bioproducts (CABBI) decided to solve the problem of adapting CRISPR/Cas technology for plant genomes.

Researchers have developed CROPSR, a first-of-its-kind open source software tool for genome-wide design and evaluation of gRNA sequences for CRISPR/Cas experiments.

“CROPSR provides the scientific community with new methods and a new working way to conduct CRISPR/Cas9 knockout experiments,” explains Hans Müller Paul, CROPSR developer, lead author of the study and PhD student in molecular biology . “We hope that the new software will speed up research and reduce the number of failed experiments.”

Created a tool that facilitates the genetic modification of plants 1A — overview of the CRISPR/Cas9 mechanism of action. B is a diagram of a typical CRISPR/Cas9 gene editing experiment on a crop, with the corresponding timeline for each step. It is expected that with the application of the CROPSR software, it is possible to improve the steps highlighted in gray boxes

The fact is that the existing software tools needed to develop and evaluate the effectiveness of CRISPR experiments have so far been based on editing the genomes of bacteria and mammals, which obviously differ from the polyploid genomes of agricultural crops with a multiple set of chromosomes.

In plants, one trait — for example, stress-related, where backup systems are useful — can be regulated by a set of genes.

A scientist can design an experiment with the CRISPR/Cas system to turn off one or more genes and be unaware of others that perform the same function. He also may not be aware of the existence of a similar DNA sequence of another gene with a different function, which may be accidentally cut out.

In either case, the problem may not be identified until the plant has grown and matured, without any trait change, or with some new characteristic (very rarely useful). This problem is especially acute with crops that require special weather conditions to grow, where a missed season can mean a year’s delay in development.

To solve this problem, the team created software (a standalone package written in Python) that revised the approach to designing and evaluating gRNA sequences, taking into account the fact that it is necessary to cut out all copies of the gene and not touch unnecessary sections of DNA.

According to the authors, CROPSR gRNA scoring models provide much more accurate predictions of CRISPR/Cas application even in non-crop genomes.

The developers also included in CROPSR the possibility of creating a gRNA database for the entire crop genome. This process requires significant computational resources and time.But researchers only need to do this once to create a database that can then be reused for many experiments.

Created a tool that facilitates the genetic modification of plants 2Timeline and steps of a typical CRISPR/Cas9 knockout (K/O) experiment in the crop genome using CROPSR. The steps in the gray boxes only need to be done once per genome the first time you use CROPSR (create a database). Sequential use of the same genome only requires a database search, as shown in the figure

As a result, all a scientist needs to do is to find the desired gene in his own database, select gRNA from the proposed list, and CROPSR itself will point to other places in the genome that need to be targeted.

“You can just go into the database, have all the information you need ready to go, and start researching,” concludes Paul. “The less time you spend planning your experiments, the more time you can spend on the experiments themselves.”

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