美国波士顿儿童医院和哈佛医学院Jeffrey R. Holt课题组的最新研究，提出了在显性进行性听力损失模型中，等位基因编辑可预防耳聋。 这一研究成果发表在2019年7月出版的国际学术期刊《自然—医学》上。

由于大多数人类显性突变都是单核苷酸取代，研究人员探索了基因编辑策略，在不影响野生型等位基因的情况下有效和选择性地破坏显性突变。然而，单核苷酸识别可能很难实现，因为常用的核酸内切酶，如化脓性链球菌Cas9 (SpCas9)，可以容纳多达7个指导RNA (gRNA)和目标DNA之间的不匹配。此外，一些Cas9酶的原受体邻近基序(PAM)可以耐受与目标DNA的不匹配。为了克服这些限制，研究筛选了14种Cas9/gRNA组合，以特异性和有效地破坏导致显性进行性听力损失的核苷酸替代物，即DFNA36。作为DFNA36的模型，研究组使用了贝多芬小鼠，它在Tmc1中携带一个点突变，Tmc1是听觉所必需的基因，编码内耳毛细胞中机械感觉传导通道的一个形成孔的亚基。

课题组在贝多芬小鼠和人类DFNA36细胞系中发现了一种金黄色葡萄球菌Cas9 (SaCas9-KKH)的PAM变体，它选择性地、有效地破坏了突变等位基因，但没有破坏野生型Tmc1/ TMC1等位基因。腺相关病毒(AAV)介导的SaCas9-KKH传递可在注射一年后预防贝多芬小鼠耳聋。对现有ClinVar基因的分析表明，大约21%的显性人类突变可以用类似的方法进行靶向治疗。

附：英文原文

Title: Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss

Author: Bence Gyrgy, Carl Nist-Lund, Bifeng Pan, Yukako Asai, K. Domenica Karavitaki, Benjamin P. Kleinstiver, Sara P. Garcia, Mikoaj P. Zaborowski, Paola Solanes, Sofia Spataro, Bernard L. Schneider, J. Keith Joung, Gwenalle S. G. Gloc, Jeffrey R. Holt, David P. Corey

Issue&Volume:Volume 25 Issue 7, July 2019

Abstract: Since most dominant human mutations are single nucleotide substitutions, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can be difficult to achieve3 because commonly used endonucleases, such as Streptococcus pyogenes Cas9 (SpCas9), can tolerate up to seven mismatches between guide RNA (gRNA) and target DNA. Furthermore, the protospacer-adjacent motif (PAM) in some Cas9 enzymes can tolerate mismatches with the target DNA. To circumvent these limitations, we screened 14 Cas9/gRNA combinations for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, we used Beethoven mice, which harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner-ear hair cells. We identified a PAM variant of Staphylococcus aureus Cas9 (SaCas9-KKH) that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1 allele, in Beethoven mice and in a DFNA36 human cell line. Adeno-associated virus (AAV)-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to one year post injection. Analysis of current ClinVar entries revealed that ~21% of dominant human mutations could be targeted using a similar approach.

DOI: 10.1038/s41591-019-0500-9

Source:https://www.nature.com/articles/s41591-019-0500-9