Date of Graduation
Bachelor of Science in Biomedical Engineering
CRISPR-Cas9 technology has widely been used as a viable genome engineering platform to make site-specific insertion, deletion, and breaks. The nuclease dead version of Cas9 or dCas9 can be used for the activation and repression of target gene sites using specific activation or repression domains. In this study, CRISPR guide RNAs were designed for a CRISPR inhibition approach to repress the transcriptional activity of the target genes. An expression plasmid vector composed of a U6 promoter sequence, BbsI restriction sites, and a chimeric gRNA sequence was digested, and the phosphorylated forward and reverse gRNAs were ligated with the plasmid vector. The expression plasmids were transformed in E.coli cells and plated on Agar plates for selective extraction of the expression plasmids with the desired gRNA sequence from the bacterial cells. The presence of gRNAs was confirmed in the expression vectors through Sanger sequencing, and the cloned gRNAs in the expression vectors were transfected into HEK293 cells along with dCas9-KRAB through a lipid-mediated delivery method. The gRNAs were designed for NF-κB, NFKBIZ, and Caveolin-1 genes, among which the gRNAs for NF-κB were transfected to the HEK293 cells. The target genes were chosen because of their upregulated expression at impaired wound healing conditions. NF-κB is associated with several physiological pathways, gene expression, and protein functions involved in wound healing. The upregulation of NF-κB is associated with the negative proliferative phase, extended inflammatory response, and altered phagocytic functions of macrophages (Khanna et al., 2010). An increased expression of NFKBIZ is linked with the upregulation of IL-6 gene expression causing extended inflammation (Trinh et al., 2008), (Johnson et al., 2020). Over expression of Cav1 is known to affect the movement of keratinocytes and epithelial cells, affecting wound closure (Jozic et al., 2019).
CRISPRi, dCas9, guide RNA, wound healing, transfection, NF-κB, NFKBIZ, Cav1
Khatiwada, P. (2021). Guide RNAs preparation for in-vitro CRISPR-Cas9 complex delivery targeting genes that affect wound healing.. Biomedical Engineering Undergraduate Honors Theses Retrieved from https://scholarworks.uark.edu/bmeguht/98