CRISPR 3.0 is coming. It will activate the expression of multiple genes at one time and will play a great role in breeding

Cutting edge scientific progress lies in the biological world

Since the birth of crispr-cas9 gene editing technology, people have been so impressed with its nickname "gene magic scissors" that they have neglected that it can also be used as a tool for gene expression enhancement and gene expression reduction.
Qi Lei of Stanford University was the first to develop crispr-dcas9 and apply it to gene expression enhancement (crispra) and gene expression reduction (crispri). This technology can accurately regulate gene expression without causing DNA double strand breaks, which also greatly expands the application scope and potential of CRISPR technology.
On June 24, 2021, Qi Yiping's team from the University of Maryland published a research paper entitled CRISPR – act3.0 for highly efficient multiplex gene activation in plants in nature plants.
The research developed a gene editing technology called CRISPR – act3.0, which can realize multiple gene activation in plants. The activation ability of this system is four to six times that of the most advanced CRISPR activation technology, and it can activate as many as seven genes at a time. Activating genes to obtain functional gain is of great significance for creating better plants, especially crops.

Although CRISPR activation (crispra) has been successfully used in the activation of plant genes in previous studies, there are still challenges in activating multiple genes at one time, and activating gene expression to obtain functional gain is of great significance for creating better plants, especially crops.
In order to achieve this goal, the research team introduced a new and improved CRISPR system in plants and named it CRISPR – act 3.0. This third generation CRISPR system focuses on multiple gene activation and can enhance the function of multiple genes at the same time.
Qi Yiping's team has previously developed multiple gene editing technology in plants to improve crops by knocking out multiple genes, but the potential of this strategy is limited because there are not many genes that can achieve crop improvement by reducing expression.
Enhancement is different. We can improve the function we want by enhancing gene expression or even adding new genes.
Crispra is based on the loss of cleavage activity of dcas9 binding to a variety of transcription activators, which is achieved in the guide RNA (gRNA), which guides dcas9 and transcription factors to bind to specific DNA sequences, thereby activating gene expression. Due to the loss of DNA cleavage activity of dcas9, it will not cause DNA double strand breaks, so it has higher safety.
The first generation crispra system is based on dcas9-vp64, and the second generation crispra system includes dcas9 suntag. The second generation system can connect multiple vp64 in series to achieve a higher level of gene activation.

In this study, CRISPR – act 3.0 developed by the research team, dspcas9 fused with vp64, and its coupled gr2.0 contained two MS2 RNA aptamers, which were used to recruit more MS2 phage coat proteins (MCPs) fused with suntag.

Researchers have verified the effect of CRISPR act 3.0 system in rice, tomato and Arabidopsis. The results showed that CRISPR act 3.0 can activate many genes at the same time, including genes that accelerate the flowering speed to speed up the breeding process.
In addition, in order to target PAM sequences rich in T, the research team also applied CRISPR act 3.0 to cas12b, and developed crispr-act3.0 system independent of PAM sequence based on spry variant, which is superior to dcas9 ng system in activation efficacy and targeting range.

In general, this research has developed a simple strategy of multiple gene activation, which is far superior to the current CRISPR activation technology in both effect and efficiency. This technology has important application value for designing, customizing and creating better crops, and also helps to cope with climate change and global hunger.

Paper link:
https://www.nature.com/articles/s41477-021-00953-7