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Optimization of DNA, RNA and RNP Delivery Methods for Efficient CRISPR/Cas9 Mediated Mammalian Cell Engineering
Author(s) -
Khodthong Chuenchanok,
Snow Josh,
Juckem Laura
Publication year - 2016
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.30.1_supplement.571.5
Subject(s) - crispr , cas9 , genome editing , guide rna , biology , ribonucleoprotein , genome engineering , rna , hek 293 cells , recombineering , computational biology , dna , plasmid , gene , microbiology and biotechnology , genetics
The CRISPR/Cas9 genome‐editing platform is a versatile and powerful technology to efficiently create genetically engineered living cells and organisms. This system requires a complex of Cas9 endonuclease protein with a gene‐targeting guide RNA (gRNA) to introduce double‐strand DNA breaks (DSBs) at specific locations in the genome. DSBs are then repaired by the error‐prone Non‐Homologous End Joining (NHEJ) pathway, resulting in insertions and/or deletions (indels) which disrupt the targeted locus. The success of CRISPR genome editing experiments is limited by the intracellular delivery and expression of Cas9 protein and gRNA. Many methods for achieving CRISPR mediated cleavage have been identified and the choice of DNA, RNA or ribonucleoprotein (RNP) format is dictated by experimental goal and cell type. Transfection of each type of molecule requires specific considerations for efficient functional delivery. We performed transfections using different combinations of molecules including: plasmid DNA, messenger RNA, Cas9 protein and gRNA to maximize targeting of the Cyclophilin B (PPIB) gene in HEK 293T/17, U2OS, and other mammalian cell types. Our results extend the utility of the CRISPR/Cas9 system by identifying optimal transfection conditions for intracellular delivery of Cas9 and gRNA in different formats.