Researchers allegedly create a new 'controllable, reversible' gene-editing method in China

Scientists from the Chinese Academy of Sciences (CAS) have allegedly developed a new "controllable, reversible and safer" gene-editing approach using CRISPR technology.

The system, named Cas13d-N2V8, showed a significant reduction in the number of off-target genes and no detectable collateral damage in cell lines and somatic cells, which indicated its future potential, according to a report published in South China Morning Post newspaper on Wednesday.

The new approach using the Cas13 enzyme, targeting RNA, is safer because RNAs are transient molecules that only exist in the cell for a short period of time and are not integrated into the genome, the researchers claimed.

Compared to other DNA editing techniques, "the Cas13 gene editing system is safer, and the effects are more controllable and short-lived," said Yang Hui, the corresponding author of the study and a researcher at the CAS Centre for Excellence in Brain Science and Intelligence Technology, China.

The technique involves CRISPR

The technique involves the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology – a natural defense mechanism that allows bacterial cells to detect and destroy the viruses that attack them and has become one of the most commonly used gene-editing techniques in recent years.

CRISPR-associated protein-9 (Cas9) is an enzyme that can cut the two strands of DNA in the genome to add or remove material in the most widely used system.

"CRISPR-Cas9 and Cas9-based gene editing tools are well protected by patents. Other companies don't have a chance [to develop them]," Hui told Bioworld, a WeChat official account focusing on research.

"The CRISPR-Cas13 systems are more specific and precise, so they have a broader scope of application."

However, the enzyme's ability to cleave non-target RNAs, or collateral cleavage, limits its clinical application.

"Cas13 can degrade both target and non-target RNAs at random," making it difficult to design experiments and interpret results when using Cas13, the researchers explained on the CAS website.

But "the CRISPR-based gene editing tool does not permanently change the genome, and the effects of editing are controllable, reversible, and safer," he added.

System to detect the collateral effects

Yang and colleagues described how they created a system to detect the collateral effects of Cas13 in mammalian cells, which they then used to create a large number of variants.

"In short, Cas13 variants with minimal collateral effect we developed are expected to be more competitive for in vivo RNA editing and future therapeutic applications," the authors concluded in the study.

Earlier, Feng Zhang, a gene-editing researcher at the Broad Institute of MIT and Harvard and one of the CRISPR pioneers, created the Rescue platform, which used Cas13 to edit RNA.

"By developing this new enzyme and combining it with the programmability and precision of CRISPR, we were able to fill a critical gap in the toolbox," Zhang said in 2019.

The study was originally published in the peer-reviewed journal Nature Biotechnology.

Abstract:

CRISPR–Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications.