Scientific Research Progress | Brain Wisdom Excellence Center has developed a higher -fed and better high -fidelity version of the CAS13 system with higher specificity and better security

Background

CRISPR-CAS13 is a class of gene editing technology that targeted RNA mediated by RNA. CAS13 protein belongs to the 2 VI multi -domain single protein RNA internal cutase. Many in vitro studies have shown that CAS13 protein has the function of cutting target RNA after activation, but also can be non -specific cutting on any RNA (BYSTANDER RNA) around it. This feature is also used to develop RNA detection. Since the report of CAS13A (C2C2) in 2015, a number of CRISPR-CAS13 systems (CAS13B/C/D/X/Y/BT) have been reported at home and abroad, and it can prove that it can perform efficient and efficient and efficient and efficient and efficient and efficient and efficient and efficient target RNA in mammalian cells. Specific to low. Compared with CAS9 -mediated DNA editing technology, the RNA editing tool -based RNA -based RNA -based RNA of CAS13 will not cause permanent changes to the genome, and can control the RNA editing effect through dose adjustment and other methods. More safer.

However, more and more evidence indicates that CAS13A, CAS13B, CAS13D, etc. are generally clied activity in mammalian cells, which can cause serious off -target effects. The latest research also shows that CAS13 (especially CAS13D) will cause great toxic and side effects on animal cells and individuals. Therefore, the edge activity of CAS13 protein itself has become the biggest obstacle to its clinical application. How to reduce or eliminate the edge activity of CAS13 protein is particularly necessary. Developing more special high -fidelity CAS13 protein on RNA -based gene therapy based on RNA editing gene therapy based on RNA editing gene therapy Strategic research and development and subsequent clinical transformation are of great significance.

"Nature BioteChnology"

2022.8.11

On August 11, 2022, the journal "Nature BioteChnology" published an research paper entitled "High-Fidelity Cas13 Variants For Targeted RNA Degradation with Minimal Collateral Effects". Institute of Neuroscience), National Key Laboratory of Neuroscience, Key Laboratory Laboratory of Lingye neurosciences of the Chinese Academy of Sciences, Yang Hui Research Group of Shanghai Brain Science and Brain Research Center and Huida (Shanghai) Biotechnology Co., Ltd. Essence This study comprehensively uses technical means such as protein engineering, streaming cells, in vitro cutting experiments, full transcriptional sequencing, transgenic mice, and safety verification in body gene therapy. Screening and verification have developed high -fidelity CAS13 protein variants with efficient editing but extremely low -edge activity. This study is of great significance for basic scientific research, gene therapy strategy research and development, and subsequent clinical transformation based on RNA editing.

Researchers have developed a dual fluorescent reporting system based on green fluorescent protein (EGFP) and red fluorescent protein (Macherry) to detect the edge effect of CAS13 protein in mammalian cells. By targeting the mRNA of a fluorescent protein (such as Macherry), the expression level of another fluorescent protein (such as EGFP) has found that the activation of CAS13A and CAS13D protein can induce a very significant side -cut effect. In order to reduce or eliminate the edge activity of CAS13D protein, the researchers predicted and analyzed the protein structure of CAS13D. At present, members of the CAS13 family have two aspirational HEPN domains. The RXXXXH conservative base order on it is CAS13 nucleic acid enzyme catalytic activity site. When CAS13 protein and GRNA are activated, the catalytic active site will be exposed to the surface of the protein, and then the surrounding RNA will be cut without different cutting.

Figure 1: The engineering design, screening and off -target effect verification of High Paper CAS13 protein

Researchers use protein engineering methods to conduct a series of mutations and transformations of CAS13D protein to change the interaction between CAS13D protein and non -target RNA, and comprehensively use mammalian cell transfection and flow cell technique to scree Edit activity but cutting activity with a significant decrease in CAS13D protein variants. Among them, the CAS13D-N2v8 variant comprehensively shows the best specificity, which has the highest fidelity (High-Fidelity Cas13D, HFCAS13D). By conducting an experiment of RNA knocking in a large number of endogenous gene sites, HFCAS13D shows the same high activity as the wild type CAS13D. Combined with in vitro cutting experiments, full transcription analysis, stabilizing cell lines, genetically modified mice, and targeting RNA knocking on the body, HFCAS13D has been systematically verified and safety assessment. In summary, this work has developed a high -fidelity CAS13D protein variant with high efficiency editing but extremely low activity. This new tool shows higher specificity and has better safety in gene therapy. Essence Figure 2: Evaluation of the effectiveness and safety of high -fidelity CAS13 protein in mammalian cells and individuals

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It is worth mentioning that in 2021, the Yang Hui research team reported on "Nature Methods" the smallest RNA editing tool CAS13X system (only 775 amino acids). However, the extremely low -edge activity of high -fidelity CAS13X protein variants (HFCAS13X) will show very large application potential in the field of gene therapy edited by RNA.

Huang Jia and Xiao Qingquan, a doctoral student of the Chinese Academy of Sciences, and Dr. He Bingbing, Dong Xue, and Dr. Liu Yuanhua and Hui Da (Shanghai) Biotechnology Co., Ltd., Dr. Tong Huawei, the first author of the first author of the technical development department of Hui Da (Shanghai). Members of Kang, master of graduate students Han Dingyi, and doctoral students Wang Xuchen actively participated in the topic to make important contributions. This work was funded by the National Fund Commission, the Ministry of Science and Technology, the Shanghai Academy of Sciences and the Lingang Laboratory.

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