CRISPR-Cas9 gene-editing technology is one of the most important scientific breakthroughs of recent decades, but there’s always room for improvement. Researchers at the University of Maryland (UMD) have developed a system they call CRISPR-Combo, which can edit multiple genes at once while also changing the expression of others in plants.
The CRISPR system is a powerful tool, allowing scientists to make cut-and-paste edits to specific genes in living organisms. That opened the possibility for new treatments for a range of diseases, including cancers, diabetes, genetic forms of blindness and blood diseases. Edits could also be made to improve crops and farm animals, enable new forms of pest control, and create useful new types of microbes.
Later developments improved on the CRISPR recipe. Some allowed for several different genes to be edited at the same time, while other versions avoided cutting DNA altogether, focusing instead on turning gene expression on or off as needed.
In the new study, the UMD researchers have combined both of these techniques into one tool, fittingly called CRISPR-Combo. Usually, one guide RNA strand performs either the editing or activation functions, meaning they can only do one or the other. For this study the team assigned the two functions to different RNA – the editing to the common Cas9, and the activation to a scaffold sgRNA.
The team tested the concept in several experiments. First, they used tomato and rice plants to demonstrate that they could perform both functions without any crossover – and the tests were successful.
“As a proof of concept, we showed that we could knock out gene A and upregulate, or activate, gene B successfully, without accidentally crossing over and knocking out gene B or upregulating Gene A,” said Yiping Qi, co-author of the study.
Next, they experimented with a common lab plant called rockcress, to investigate how two beneficial changes could be made simultaneously. They edited a gene that makes the plants more resistant to herbicides, and activated a gene that makes the plants flower early. The end result is an engineered rockcress that is both herbicide-resistant and reproduced twice as fast, yielding eight generations in a single year.
In other tests, the team set out to improve the efficiency of breeding new varieties of plants through tissue cultivation. They edited useful genes in poplars and activated three others that promote tissue regeneration in the plants, and found that poplar samples grew new roots much faster, improving the success rate of the engineered crops.
Meanwhile, rice plants edited using CRISPR-Combo were found to grow from cultures without needing the usual growth hormone supplements, and expressed more of the edited gene than those grown with hormones.
The team says that the new technique opens up a wide range of possibilities in editing crops and other plants more efficiently, bestowing them with traits like herbicide resistance and enhanced nutrition.
“The possibilities are really limitless in terms of the traits that can be combined,” said Yiping Qi, an associate professor in the Department of Plant Science and Landscape Architecture and co-author of the study. “But what is really exciting is that CRISPR-Combo introduces a level of sophistication to genetic engineering in plants that we haven’t had before.”
The research was published in the journal Nature Plants.
Source: University of Maryland