小柯机器人

德国图宾根大学Stafforst Thorsten团队近日取得一项新成果。他们发现，CLUSTER引导的RNA能够在体内使用内源性腺苷脱氨酶(ADAR)进行精确高效的RNA编辑。该研究于2022年1月3日发表于国际学术期刊《自然-生物技术》。

在本研究中，研究人员利用计算机优化了CLUSTER引导RNA，它们以多价方式结合其目标信使RNA，实现高精度和高效的编辑，并能够靶向之前无法设计gRNA的序列。CLUSTER gRNA可以使用病毒进行基因编码和传递，并且在多种细胞系中都具有活性。在细胞培养中，CLUSTER gRNA实现了内源性转录本的靶向编辑，效率高达45%并且不产生旁观者编辑。在体内，通过流体动力学尾静脉注射将CLUSTER gRNA递送至小鼠肝脏，其编辑报告基因的效率高达10%。CLUSTER方法为以RNA碱基为基础的药物研发开辟了新道路。

据介绍，RNA碱基编辑是一种潜在基因组编辑替代方法。最近研发的RNA编辑方法主要利用内源性RNA编辑酶ADAR对RNA进行编辑，来避免由工程编辑酶异位表达造成的问题。但该方法存在序列限制、效率低和产生旁观者编辑的问题。

附：英文原文

Title: CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo

Author: Reautschnig, Philipp, Wahn, Nicolai, Wettengel, Jacqueline, Schulz, Annika E., Latifi, Ngadhnjim, Vogel, Paul, Kang, Tae-Won, Pfeiffer, Laura S., Zarges, Christine, Naumann, Ulrike, Zender, Lars, Li, Jin Billy, Stafforst, Thorsten

Issue&Volume: 2022-01-03

Abstract: RNA base editing represents a promising alternative to genome editing. Recent approaches harness the endogenous RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) to circumvent problems caused by ectopic expression of engineered editing enzymes, but suffer from sequence restriction, lack of efficiency and bystander editing. Here we present in silico-optimized CLUSTER guide RNAs that bind their target messenger RNAs in a multivalent fashion, achieve editing with high precision and efficiency and enable targeting of sequences that were not accessible using previous gRNA designs. CLUSTER gRNAs can be genetically encoded and delivered using viruses, and are active in a wide range of cell lines. In cell culture, CLUSTER gRNAs achieve on-target editing of endogenous transcripts with yields of up to 45% without bystander editing. In vivo, CLUSTER gRNAs delivered to mouse liver by hydrodynamic tail vein injection edited reporter constructs at rates of up to 10%. The CLUSTER approach opens avenues for drug development in the field of RNA base editing. Optimized guide RNAs improve RNA editing with endogenous enzymes.

DOI: 10.1038/s41587-021-01105-0

Source: https://www.nature.com/articles/s41587-021-01105-0

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