­In vivo high-efficiency somatic homology directed repair

Background

Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases, but precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements. For example, CRISPR/Cas9-based genome editing technologies provide powerful tools for genetic manipulation and delivery of Cas9 and a homology directed repair (HDR) template using adeno-associated virus (AAV; CASAAV-HDR) was recently shown to enable the creation of precise genomic edits even in postmitotic cells.

Technology Overview

Because there is a need to identify novel loci that allow high efficiency genome editing, Boston Children’s Hospital researchers developed a methodology for in vivo genome editing that allows for somatic insertion of large blocks of genetic code (several hundred base pairs) into cell types transduced by adeno-associated virus. In this approach, an adeno-associated virus is used to deliver a sgRNA, which targets the untranslated region (UTR) of the selected gene, and a template for homology directed repair to cells expressing the Cas9 nuclease. Results showed that in mouse cardiomyocytes some regions of the genome, such as the Myl2 and Pln loci, were particularly amenable to high-efficiency editing (>50% of cardiomyocytes edited for Myl2). Additionally, the technique had an in vivo high efficiency editing in both neonatal and adult mouse cardiomyocytes. This approach can be used to create disease models, and the high-efficiency loci may be used to therapeutically drive permanent and stable expression of a transgene. Cas9 could be delivered via a second AAV vector in a therapeutic context and this approach could be therapeutically deployed in skeletal muscle and hepatocytes in addition to cardiomyocytes.

Applications

• Gene editing
• Creation of disease models

Advantages

• The efficiency of this in vivo editing is much higher than any previously reported.