The ability of CRISPR, more specifically RNA-guided CRISPR activation (CRISPRa) systems, to activate genes in plants has been previously demonstrated. However, the simultaneous activation of multiple genes remains challenging. It was this aspect of the CRISPR toolbox that researchers at the University of Maryland (UMD) sought to expand. The ability to activate genes to gain functionality is essential to creating better plants and crops for the future.

To achieve this goal, the researchers introduced a new and improved CRISPR system into  plants named CRISPR–Act 3.0. This third-generation CRISPR system focuses on multiplexed gene activation, boosting the function of multiple genes simultaneously. According to the researchers, led by Yiping Qi, PhD, associate professor of plant science at UMD, the system boasts four to six times the activation capacity of current state-of-the-art CRISPR technology, and demonstrates high accuracy and efficiency in up to seven genes at once.

This work is published in Nature Plants, in the paper, “CRISPR–Act3.0 for highly efficient multiplexed gene activation in plants.

“While my lab has produced systems for simultaneous gene editing [multiplexed editing] before, editing is mostly about generating loss of function to improve the crop,” explained Qi. “But if you think about it, that strategy is finite, because there aren’t endless genes that you can turn off and actually still gain something valuable. Logically, it is a very limited way to engineer and breed better traits, whereas the plant may have already evolved to have different pathways, defense mechanisms, and traits that just need a boost. Through activation, you can really uplift pathways or enhance existing capacity, even achieve a novel function. Instead of shutting things down, you can take advantage of the functionality already there in the genome and enhance what you know is useful.”

The researchers validated the CRISPR–Act 3.0 system in rice, tomatoes, and Arabidopsis. The team showed that you can simultaneously activate many kinds of genes, including faster flowering to speed up the breeding process. But this is just one of the many advantages of multiplexed activation, said Qi.

“Having a much more streamlined process for multiplexed activation can provide significant breakthroughs. For example, we look forward to using this technology to screen the genome more effectively and efficiently for genes that can help in the fight against climate change and global hunger. We can design, tailor, and track gene activation with this new system on a larger scale to screen for genes of importance, and that will be very enabling for discovery and translational science in plants.”

The highly robust CRISPRa system, the authors wrote, works through “systematically exploring different effector recruitment strategies and various transcription activators based on deactivated Streptococcus pyogenes Cas9 (dSpCas9).” Deactivated CRISPR-Cas9 can bind, but not cut, DNA. Therefore, the system recruits activation proteins for specific genes of interest by binding to certain segments of DNA.

Qi also tested the SpRY variant of CRISPR-Cas9 that greatly broadens the scope of what can be targeted for activation, as well as a deactivated form of the CRISPR-Cas12b system to show versatility across CRISPR systems. This shows the great potential of expanding for multiplexed activation, which can change the way genome engineering works.

“People always talk about how individuals have potential if you can nurture and promote their natural talents,” said Qi. “This technology is exciting to me because we are promoting the same thing in plants—how can you promote their potential to help plants do more with their natural capabilities? That is what multiplexed gene activation can do, and it gives us so many new opportunities for crop breeding and enhancement.”

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