小柯机器人

韩国科学技术院Yong-Sam Kim团队利用超小型Cas12f1和经设计的引导RNA通过腺相关病毒传递实现高效的CRISPR编辑。相关论文于2021年9月2日在线发表于国际学术期刊《自然—生物技术》。

研究人员重新设计了Un1Cas12f1的天然引导RNA的五个部位：反式激活CRISPR RNA（tracrRNA）的5′末端、tracrRNA-crRNA互补区、一个五（尿苷酸）序列、crRNA的3′末端和tracrRNA中一个无序的茎2区。这些优化措施协同地使平均的插入缺失频率增加了867倍。优化后的Un1Cas12f1系统在通过质粒载体、PCR扩增子和腺相关病毒（AAV）传递时，能够在人类细胞中进行高效、特异的基因组编辑。由于Un1Cas12f1在原生质粒之外进行切割，它可以被用来有效地创建大的缺失。工程化的Un1Cas12f1系统显示出与SpCas9相当的效率和与AsCas12a相似的特异性。

据了解，基因治疗将受益于一个微型的CRISPR系统，它适合于小型AAV的基因组，并且在真核细胞中具有高切割活性和特异性。目前发现的最紧凑的CRISPR相关核酸酶之一是古细菌Un1Cas12f1。 然而，Un1Cas12f1及其变体在真核细胞中的活性非常低。

附：英文原文

Title: Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus

Author: Kim, Do Yon, Lee, Jeong Mi, Moon, Su Bin, Chin, Hyun Jung, Park, Seyeon, Lim, Youjung, Kim, Daesik, Koo, Taeyoung, Ko, Jeong-Heon, Kim, Yong-Sam

Issue&Volume: 2021-09-02

Abstract: Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5′ terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA–crRNA complementary region, a penta(uridinylate) sequence, the 3′ terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.

DOI: 10.1038/s41587-021-01009-z

Source: https://www.nature.com/articles/s41587-021-01009-z

Nature Biotechnology：《自然—生物技术》，创刊于1996年。隶属于施普林格·自然出版集团，最新IF：68.164
官方网址：https://www.nature.com/nbt/
投稿链接：https://mts-nbt.nature.com/cgi-bin/main.plex