A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing

Abstract

Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify diseaserelevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of nonhuman proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans.

Genome Editing for CNS Disorders

“…Transient expression strategies have been mainly developed for CRISPR/Cas-based tools either through the delivery of RNPs or ON/OFF expression systems. These include self-inactivation systems (Merienne et al, 2017;Li et al, 2018) and the use of drug inducible promoters, such as the doxycycline (dox)-induced Tet or the Tamoxifendependent Cre promoters (Zhang et al, 2019). However, the optimization of self-inactivation kinetics and the requirement of additional molecules to regulate promoters will delay the translation of these strategies to the clinic.…”

CRISPR-Cas System: An Approach With Potentials for COVID-19 Diagnosis and Therapeutics

“…Cas14 cjCas9, ScCas9 etc. have been discovered which can also be used for the intended use and can also be delivered by rAAV (Kim et al, 2017;Ibraheim et al, 2018;Karvelis et al, 2019;Li et al, 2019). Though there are merits of viral vectors but in vivo delivery using viral vectors suffer from limitations like immunogenicity and duration of Cas expression.…”

Treating Cystic Fibrosis with mRNA and CRISPR

“…To address this, researchers have developed methods to inactivate the expression cassette, or its Cas9 product, allowing for the reduction and modulation of cell exposure to the nuclease over time in vivo. 51,52 The co-delivery of Cas9 mRNA and guide RNA to facilitate editing can bypass some of the drawbacks of delivering protein, using a plasmid, or using a viral vector. For example, mRNA is transient, allowing for editing to take place during a specific timeframe instead of being long lived.…”