CRISPR-Cas3 induces broad and unidirectional genome editing in human cells

Hiroyuki Morisaka,Huaigeng Xu,Junji Takeda,Tomoji Mashimo,Takashi Yamamoto,Yayoi Kunihiro,Peter Gee,Yuya Okuzaki,Shinichiro Nakada,Yuki Naito,Noriko Sasakawa,Akitsu Hotta,Kazuto Yoshimi,Ekasit Sonpho,Shigetoshi Sano

doi:10.1038/s41467-019-13226-x

Abstract

Although single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5′-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.

Highlights

Single-component Class 2 clustered regularly interspaced short palindromic repeats (CRISPR) systems, such as type II Cas[9] or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multicomponent Class 1 CRISPR has been less developed
The type I-E CRISPR system was reported to induce the degradation of plasmid DNA in vitro[23,24] as well as transcriptional silencing in E. coli[25,26,27], it is unclear whether it mediates DNA cleavage for genome editing in eukaryotic cells, such as human cells
We demonstrate that the type I-E CRISPR, E. coli Cas complex for antiviral defense (Cascade), Cas[3], and precrRNA, but not mature CRISPR RNA (crRNA), possesses robust and efficient cleavage activity against plasmid DNA and endogenous genomic DNA in human cells

Summary

Introduction

Single-component Class 2 CRISPR systems, such as type II Cas[9] or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multicomponent Class 1 CRISPR has been less developed. Cas[3], which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5′-ARG protospacer adjacent motif (PAM), without prominent offtarget activity This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs) These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system. We demonstrate that the type I-E CRISPR, E. coli Cascade, Cas[3], and precrRNA, but not mature crRNA, possesses robust and efficient cleavage activity against plasmid DNA and endogenous genomic DNA in human cells. In contrast to the CRISPR-Cas[9] system, this distinctive feature of CRISPR-Cas3mediated genome editing might broaden the application of genome editing by facilitating efficient gene knockouts and/or knockins, as well as future therapeutic applications

Methods

Results

Conclusion