Precise CAG repeat contraction in a Huntington\u2019s Disease mouse model is enabled by gene editing with SpCas9-NG

Seiya Oura,Masahito Ikawa,Yoshitaka Nagai,Taichi Noda,Seiji Hitoshi,Naoko Morimura,Osamu Nureki,Hiroshi Nishimasu

doi:10.1038/s42003-021-02304-w

Abstract

The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a research hotspot in gene therapy. However, the widely used Streptococcus pyogenes Cas9 (WT-SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting targetable disease mutations. To address this issue, we recently reported an engineered SpCas9 nuclease variant (SpCas9-NG) recognizing NGN PAMs. Here, as a feasibility study, we report SpCas9-NG-mediated repair of the abnormally expanded CAG repeat tract in Huntington’s disease (HD). By targeting the boundary of CAG repeats with SpCas9-NG, we precisely contracted the repeat tracts in HD-mouse-derived embryonic stem (ES) cells. Further, we confirmed the recovery of phenotypic abnormalities in differentiated neurons and animals produced from repaired ES cells. Our study shows that SpCas9-NG can be a powerful tool for repairing abnormally expanded CAG repeats as well as other disease mutations that are difficult to access with WT-SpCas9.

Highlights

The clustered regularly interspaced palindromic repeats (CRISPR)/Cas[9] system is a research hotspot in gene therapy
Before targeting expanded CAG repeat tracts, we examined the genome editing efficiency with SpCas9-NG in three different cells, HEK293T cells, embryonic stem (ES) cells, and zygotes
Thirty-six hours after transfection, we observed green fluorescence appearing after homology-directed repair (HDR) (Fig. 1a)

Summary

Introduction

The clustered regularly interspaced palindromic repeats (CRISPR)/Cas[9] system is a research hotspot in gene therapy. The widely used Streptococcus pyogenes Cas[9] (WTSpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting targetable disease mutations. To address this issue, we recently reported an engineered SpCas[9] nuclease variant (SpCas9-NG) recognizing NGN PAMs. Here, as a feasibility study, we report SpCas9-NG-mediated repair of the abnormally expanded CAG repeat tract in Huntington’s disease (HD). The widely used Cas[9] from Streptococcus pyogenes (SpCas9) requires NGG PAM for its target recognition[6], thereby restricting targetable genomic loci To address this issue, we recently reported an engineered SpCas[9] nuclease variant (SpCas9-NG) recognizing NGN-PAMs7, broadening the range of targetable disease mutations. Our method has the potential to offer an alternative option for repairing expanded repeat sequences

Objectives

Methods

Results

Conclusion