Abstract
An ideal rAAV gene editing system not only effectively edits genes at specific site, but also prevents the spread of the virus from occurring off-target or carcinogenic risks. This is important for gene editing research at specific site in vivo. We report a single rAAV containing SaCas9 and guide RNAs under the control of subtle EF1a and tRNA promoters. The capacity of rAAV was compressed, and the editing efficiency was similar to that of the classical Cas9 system in vitro and in vivo. And we inserted the sequence of the green fluorescent protein eGFP into rAAV. The number of cells infected with the rAAV and the region in which the rAAV spreads were known by the fluorescent expression of eGFP in cells. In addition, we demonstrated that myostatin gene in the thigh muscles of C57BL/10 mice was knocked out by the rAAV9-SaCas9 system to make muscle mass increased obviously. The protein eGFP into rAAV has significant implications for our indirect analysis of the editing efficiency of SaCas9 in the genome of the target tissue and reduces the harm caused by off-target editing and prevents other tissue mutations. The rAAV system has substantial potential in improving muscle mass and preventing muscle atrophy.
Highlights
The CRISPR/Cas9 system has become a commonly used technology in medicine and life sciences (Li et al 2018)
We focused on the design of a compact CRISPR/Staphylococcus aureus Cas9 (SaCas9) vector, pX601-EF1a:SaCas9-eGFP-tRNA:sgRNA plasmid suitable for associated virus (AAV) delivery
The pX601-EF1a:SaCas9-eGFPtRNA:sgRNA plasmid contains an EF1a promoter controlling expression of the SaCas9 coding region fused to a self-cleaving P2A sequence attached to an eGFP sequence, and it contains a constitutively expressing sgRNA expression cassette controlled by a tRNA promoter
Summary
The CRISPR/Cas system has become a commonly used technology in medicine and life sciences (Li et al 2018). It has been used to genetically modify potential clinical treatments for diseases (Mollanoori and Teimourian 2018; Yang et al 2016). Genome editing in tissues in vivo is affected by many factors, including the choice of vector, the efficiency of editing proteins and the influence of the internal environment. Adeno-associated virus (AAV) as the vector for Cas provides a promising genomic correction approach (Moreno et al 2018). The technique allows genetic editing of specific muscle areas of inherited muscular dystrophy to improve atrophy symptoms (Crudele and Chamberlain 2019; Tabebordbar et al 2016). Many of the exciting tools developed from
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