Molecular Therapy. 2021 Jun 24
Zhiquan Liu, Siyu Chen, Wanhua Xie, Yuning Song, Jinze Li, Liangxue Lai,and Zhanjun Li
Products used in the paper Details Operation
AAV vector packaging Production of AAV8-SpaCas9 vectors was performed by Packgene Biotech (China). Request Quote

Research Field: genome editing system

Keywords: CRISPR, SpaCas9, base editor, Acr, AAV, Pcsk9

AAV Serotype: AAV8

Dose: 2 x 10^11 GCs

Routes of Administration: via the tail vein

Animal or cell line strain: ICR mice


Compact CRISPR-Cas9 systems that can be packaged into an adeno-associated virus (AAV) show promise for gene therapy. However, the requirement of protospacer adjacent motifs (PAMs) restricts the target scope. To expand this repertoire, we revisited and optimized a small Cas9 ortholog derived from Streptococcus pasteurianus (SpaCas9) for efficient genome editing in vivo. We found that SpaCas9 enables potent targeting of 5′-NNGYRA-3′ PAMs, which are distinct from those recognized by currently used small Cas9s; the Spa-cytosine base editor (CBE) and Spa-adenine base editor (ABE) systems efficiently generated robust C-to-T and A-to-G conversions both in vitro and in vivo. In addition, by exploiting natural variation in the PAM-interacting domain, we engineered three SpaCas9 variants to further expand the targeting scope of compact Cas9 systems. Moreover, mutant mice with efficient disruption of the Tyr gene were successfully generated by microinjection of SpaCas9 mRNA and the corresponding single guide RNA (sgRNA) into zygotes. Notably, all-in-one AAV delivery of SpaCas9 targeting the Pcsk9 gene in adult mouse liver produced efficient genome-editing events and reduced its serum cholesterol. Thus, with distinct PAMs and a small size, SpaCas9 will broaden the CRISPR-Cas9 toolsets for efficient gene modifications and therapeutic applications.

Popular Services

AAV Packaging Service

AAV Analytical Service

Vector Design