The Development of an AAV-Based CRISPR SaCas9 Genome Editing System That Can Be Delivered to Neurons in vivo and Regulated via Doxycycline and Cre-Recombinase.

Namrata Kumar,Cuauhtemoc Ulises Gonzalez,Anusha Sairavi,Christopher De Solis,Aradhana Aradhana,Jonathan E Ploski,Trieu-Mi J Dao,William Stanford,Sadiqa Thaseen,Nigel D Abraham

doi:10.3389/fnmol.2018.00413

Namrata Kumar, Cuauhtemoc Ulises Gonzalez + Show 8 more

Open Access

https://doi.org/10.3389/fnmol.2018.00413

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Abstract

The RNA-guided Cas9 nuclease, from the type II prokaryotic clustered regularly interspersed short palindromic repeats (CRISPR) adaptive immune system, has been adapted by scientists to enable site specific genome editing of eukaryotic cells both in vitro and in vivo. Previously, we reported the development of an adeno-associated virus (AAV)-mediated CRISPR Streptococcus pyogenes (Sp) Cas9 system, in which the genome editing function can be regulated by controlling the expression of the guide RNA (sgRNA) in a doxycycline (Dox)-dependent manner. Here, we report the development of an AAV vector tool kit utilizing the Cas9 from Staphylococcus aureus (SaCas9). We demonstrate in vitro genome editing in human derived 293FT cells and mouse derived Neuro2A (N2A) cells and in vivo in neurons of the mouse brain. We also demonstrate the ability to regulate the induction of genome editing temporally with Dox and spatially with Cre-recombinase. The combination of these systems enables AAV-mediated CRISPR/Cas9 genome editing to be regulated both spatially and temporally.

Highlights

The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system has become an extremely useful tool in probing biology in both clinical and scientific settings
In our first set of experiments, we focused on designing a CRISPR/Staphylococcus aureus Cas9 (SaCas9) system suitable for associated virus (AAV) delivery
The first AAV plasmid contains an EF-1α short (EFS) promoter controlling expression of the SaCas9 coding region fused to a self-cleaving P2A sequence attached to an HA-Flag-HA epitope tagged Klarsicht, ANC-1, Syne homology (KASH) domain

Summary

Introduction

The CRISPR/Cas system has become an extremely useful tool in probing biology in both clinical and scientific settings. The PAM sequence is a short sequence adjacent to the Cas nuclease cut site that the Cas molecule requires for appropriate binding When these two components are expressed, the sgRNA will bind to Cas and direct it to the sequence complementary to the gRNA, where it will initiate a double-stranded break (DSB). To repair these breaks, cells use an error prone mechanism of nonhomologous end joining (NHEJ) which can lead to disruption of function in the target gene through insertions or deletion of codons, shifts in the reading frame, or result in a premature stop codon triggering nonsense-mediated decay. Templates of donor DNA can be provided to enable homologydirected repair (HDR) and homology-independent targeted integration (HITI) allowing for targeted insertion of genetic sequences via recombination (Cong et al, 2013; Mali et al, 2013; Wang et al, 2013; Suzuki et al, 2016; Zhang et al, 2017)

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