Methods to Modulate the Inflammatory Response through Molecular Targeting‐CRISPR versus Adenovirus.
Authors: Reynoso Marinaliz , Kolb Alexander Louis 
Published: April 2020

Transfection and transduction are the primary methods of molecular manipulation both in vitro and in vivo. Two commonly used methods are clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) transfection and adenoviral transduction. While both methods are used to knockdown or knock‐in a genes of interest, each method differs in the method of cellular incorporation and genomic manipulation. CRISPR/CAS9 transfection utilizes the Cas9 protein to aid and enhance transfection efficiency. Alternatively, adenoviral transduction utilizes a modified adenovirus vector (and other viral infection properties) to incorporate or knockdown the gene of interest. The effectiveness of each method in genomic manipulation is not clearly defined. In our studies, we selectively knocked down myeloid differentiation primary response 88 (MyD88) in Raw 264.7 macrophages using either CRISPR/Cas9 transfection or adenoviral transduction. These cells were then treated with an acute dose (10 ng/mL) of lipopolysaccharide (LPS), a bacterial outer membrane vesicle, for 6 and 24 hours to induce an inflammatory response to which MyD88 serves as an intermediary cytokine. We verified the knockdown of MyD88 using Western Blotting and Image J quantification. Macrophages transfected with a CRISPR MyD88 (CrMKO) produced a 52% knockdown when compared to cells transfected with CRISPR RosA (CrRosA), a control vector, at 30 hours post transfection. Comparatively, macrophages transduced with ad‐GFP‐m‐MyD88 (ad‐MKO), an adenoviral MyD88 knockout vector, produced only a 20% knockdown when compared with control cells transduced with ad‐GFP. At 48 hours post transfection/transduction, both CrMKO cells and ad‐MKO cells demonstrated a 78% reduction in MyD88 relative to control. To determine the downstream effects of MyD88 knockdown by these methods, we measured the secretion of Tumor Necrosis Factor alpha (TNFα) and Interleukin 10 (IL‐10), in cells treated with LPS for 24 hours. The following values are presented as percent reduction when compared to non‐transfected macrophages treated with LPS. In CrRosA cells, there was a 24% reduction in TNFα secretion compared to the 62% reduction in CrMKO cells. In a similar fashion, IL‐10 demonstrated a 24% and 77% reduction in secretion in CrRosA and CrMKO cells, respectively. However, in ad‐MKO cells there was no significant difference in percent reduction in either TNFα or IL‐10 secretion relative to control cells. These data suggest that unlike adenoviral transduction, MyD88 knockdown in macrophages mediated by CRISPR/Cas9 produced a significant decrease in the downstream secretion of both TNFα and IL‐10 (p<0.03 and p<0.05, respectively). Overall, these data suggest CRISPR/Cas9 transfection serves as a better tool for modulating the inflammatory response through molecular targeting.

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