Disruption of splicing-regulatory elements using CRISPR/Cas9 to rescue spinal muscular atrophy in human iPSCs and mice.

Jinjing Li ,Yidi Sun,Cheng Tang,He Li,Lu-Lu Lai,Erwei Zuo,Xiaowen Shen,Ning Wang,Hui Yang,Shuang Wu,Qifang Wang,Hai-Zhu Chen,Qijie Zhang ,Lixiang Ma ,Xinxin Guo ,Adrian R Krainer ,Min Lin ,Ying‐Qian Lu ,Linqi Shi ,Xinhua Lin ,Xin Hu ,Weiwen Ying ,Chunmei Zhou ,Wan‐Jin Chen

doi:10.1093/nsr/nwz131

Abstract

We here report a genome-editing strategy to correct spinal muscular atrophy (SMA). Rather than directly targeting the pathogenic exonic mutations, our strategy employed Cas9 and guide-sgRNA for the targeted disruption of intronic splicing-regulatory elements. We disrupted intronic splicing silencers (ISSs, including ISS-N1 and ISS + 100) of survival motor neuron (SMN) 2, a key modifier gene of SMA, to enhance exon 7 inclusion and full-length SMN expression in SMA iPSCs. Survival of splicing-corrected iPSC-derived motor neurons was rescued with SMN restoration. Furthermore, co-injection of Cas9 mRNA from Streptococcus pyogenes (SpCas9) or Cas9 from Staphylococcus aureus (SaCas9) alongside their corresponding sgRNAs targeting ISS-N1 into zygotes rescued 56% and 100% of severe SMA transgenic mice (Smn−/−, SMN2tg/−). The median survival of the resulting mice was extended to >400 days. Collectively, our study provides proof-of-principle for a new strategy to therapeutically intervene in SMA and other RNA-splicing-related diseases.

Highlights

The majority of human protein-coding genes are able to undergo alternative pre-mRNA splicing and pathogenic mutations that affect splicing are prevalent [1]
No double-strand breaks (DSBs) were detected with sgRNA2 or sgRNA3 (Supplementary Fig. 3A), so we used sgRNA1 targeting ISS-N1 and SpCas9 vectors for transfection of spinal muscular atrophy (SMA)-2 induced pluripotent stem cells (iPSCs) followed by an enhanced green fluorescence selection protocol
survival motor neuron 2 (SMN2) splicing correction and survival motor neuron (SMN)-FL restoration were seen once one copy of the CAG or AAAG motif had been disrupted in SMN2-ISSs of SMA iPSCs

Summary

Introduction

The majority of human protein-coding genes are able to undergo alternative pre-mRNA splicing and pathogenic mutations that affect splicing are prevalent [1]. Our results showed that CRISPR/Cas9-based disruption of two SMN2 SREs (ISS-N1 and ISS + 100) rescued the SMA phenotypes in human induced pluripotent stem cells (iPSCs) and in germline-corrected SMA mice.

Results

Conclusion