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Codon Optimization of Gene Editing CRISPR‐SaCas9 Plasmid Increases Protein Expression in Human Liver Cells to Boost in vivo Therapeutic Knockout Application Efficiency
Author(s) -
Groshong Tyler L,
Madejski Irene R,
Sundaresan Avirath,
Gupta Ishir G,
Holmes Grace D,
De Luke
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.07105
Subject(s) - plasmid , crispr , cas9 , green fluorescent protein , transfection , gene , biology , microbiology and biotechnology , gene delivery , western blot , in vivo , genetics
The CRISPR‐Cas9 gene editing technology shows great promise in revolutionizing the treatment of genetic disease; however, the current focus on treatment by ex vivo delivery is limited to blood diseases. In vivo treatment can substantially extend the clinical reach of the technology, but is limited by a challenging delivery process: the large size of the Cas9 gene (~4500bp) inhibits delivery by the small viral vectors and lipid nanoparticles used. We propose that a codon‐optimized saCas9 plasmid will lead to significantly higher protein expression than the presently used genes; this can compensate for the low delivery efficiency and has great potential to improve the viability of in vivo CRISPR‐Cas9 therapies. Here we present Western Blot fluorescence data showing that optimization leads to increased Cas9 production and subsequently increased CRISPR‐Cas9 editing efficiency. A commonly used parent saCas9 sequence was selected from Addgene’s public plasmid database. A novel, codon‐optimized gene sequence was engineered from the parent using GenScript’s OptimumGene TM algorithm. Both the optimized gene and parent gene were transfected into human HepG2 liver cells, and Western Blot/ELISA protein quantification of the optimized plasmid compared with three sample plasmids demonstrated that the codon‐optimized gene expressed the saCas9 protein at ~3.5× the rate of the commercial sequence. The same set of optimized and commercial plasmids were then transfected into GFP‐stable HepG2 cells in parallel with GFP‐specific sgRNA; the cells transfected with optimized saCas9 demonstrated a significantly higher GFP knockout rate than cells transfected with commercially available control plasmids, as measured through GFP detection stay and Western Blot on GFP proteins. The augmented Cas9 protein expression supported by the novel saCas9 sequence can better enable in vivo CRISPR‐Cas9 treatments. Support or Funding Information This work was supported by The Nueva School.Comparison of Commercial and Optimized SaCas9 ExpressionSaCas9 Protein Standard Curve