Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

Raed Ibraheim,Scot A Wolfe,Shakira M Nelson ,Wen Xue,Stacy Maitland,Tomás Rodríguez ,Yusong Cao ,Phillip W L Tai,Dan Wang,Suk Namkung,Guangping Gao,Athma A Pai,Eraj Shafiq Khokhar,Aamir Mir,Zexiang Chen,Nida Javeed,Emmanouela Tsagkaraki,Jiaming Wang,Esther Mintzer,Erik J Sontheimer

doi:10.1038/s41467-021-26518-y

Abstract

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

Highlights

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits
The cell relies on the DNA repair machinery to resolve these double-strand breaks (DSBs) through non-homologous end joining (NHEJ), microhomologymediated end joining (MMEJ), or homology-directed repair (HDR), the latter of which can enable a wide range of precisely determined repair outcomes[16]
We systematically compared editing efficiencies using full-length and 100-nt single-guide RNA (sgRNA) that were made by T7 in vitro transcription (IVT), with and without calf intestinal alkaline phosphatase (CIP) treatment[57], and commercial synthetic guides carrying three 2′-O-methyl phosphorothioate modifications at each end to protect against exonucleases[58]

Summary

Introduction

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes. The establishment of a self-inactivating single-rAAV system capable of precise, versatile, HDR-based genome sequence changes in vivo, combined with the accuracy and wide targeting range of Nme2Cas[9], expand our capabilities to develop more potent therapeutic genome editing platforms

Methods

Results

Conclusion