Optimization of DNA, RNA and RNP Delivery for Efficient Mammalian Cell Engineering Optimization of DNA, RNA and RNP Delivery for Efficient Mammalian Cell Engineering using CRISPR/Cas9

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. The cell then repairs the resulting DSBs using either homology-directed repair (HDR) or the errorprone non-homologous end joining (NHEJ) pathway. Both DNA repair pathways can be leveraged in different ways to introduce desired modifications at the target 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 Cas9-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.