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Codon Optimization of Gene Editing CRISPR‐SaCas9 Augments Protein Expression in Human Liver Cells to Boost in vivo Therapeutic Application
Author(s) -
Cheng Benjamin,
Groshong Tyler,
Madejski Irene,
Sweeney Robert,
De Luke
Publication year - 2019
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.2019.33.1_supplement.495.3
Subject(s) - crispr , cas9 , gene , biology , genome editing , in vivo , genetic enhancement , computational biology , gene delivery , microbiology and biotechnology , genetics
CRISPR‐Cas9 gene editing technology shows great promise in revolutionizing the treatment of genetic disorders; 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 Cas9 gene will support 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. 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™ algorithm. Both the optimized gene and parent gene were transfected into human HepG2 liver cells with high efficiency as visualized by GFP‐tagged saCas9. ELISA protein quantification of two sample groups demonstrated that the codon‐optimized gene expressed the saCas9 protein at 3.5× the rate of the commercial sequence. 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. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .