Research Techniques Made Simple: Delivery of the CRISPR/Cas9 Components into Epidermal Cells

Abstract

CRISPR/Cas9 technology is a powerful tool used to alter the genetic landscape of various hosts. This has been exemplified by its success in the transgenic animal world where it has been utilized to develop novel mouse lines modeling numerous disease states. The technology has helped to develop both in vitro and in vivo systems that simulate diseases within the fields of epithelial biology, skin cancer biology, dermatology, and beyond. Importantly, the delivery of the single-guide RNA/Cas9 editing complex to the host cell is key for its success. In this paper, we discuss the various methods that have been utilized as delivery techniques for CRISPR/Cas9 components, the benefits and pitfalls of each, and how successful they have been at genetically modifying epidermal cells. In addition, we acknowledge recent advances in the field of dermatology that have harnessed these methods to better understand epidermal biology, identify potential therapeutic targets, or serve as novel methods to treat disease states.

Investigations into the FLG Null Phenotype: Showcasing the Methodology for CRISPR/Cas9 Editing of Human Keratinocytes

“…To overcome these limitations, genomic engineering by CRISPR and CRISPR/Cas9 (Cong et al, 2013;Gasiunas et al, 2012;Jinek et al, 2012;Mali et al, 2013) could be a powerful technique. Yet, the introduction of the CRISPR/Cas9 machinery into the notoriously difficult-to-transfect KCs has been proven troublesome, although many options are seemingly available (Shi et al, 2021). Some CRISPR/Cas9related work is performed in primary KCs, although most of the published research utilizes immortalized KCs (Smits et al, 2022).…”

CLDN1 knock out keratinocytes as a model to investigate multiple skin disorders

“…11,12 A CRISPR/Cas9 system containing three single-guide RNAs (sgRNA) targeting the first exon of CLDN1 and recombinant Cas9 nuclease was used to knockout claudin-1 expression (Figure 1A). A ribonucleoprotein complex of Cas9 protein and sgRNAs was chosen because there is low off-target editing due to the short half-life of Cas9 protein in cells, 13 in contrast to other delivery methods such as plasmids or viral vectors, 14 which have prolonged expression of the gene editing complex. Additionally, multiple sgRNAs were chosen to ensure high knockout efficiency from a single editing event.…”

New Therapeutics for Extracellular Vesicles: Delivering CRISPR for Cancer Treatment

“…Lentiviruses infect both dividing and non-dividing cells and are able to insert transgenes into host genomes for stable and long-term expression [ 63 ]. To assemble a lentivirus for CRISPR/Cas9 delivery, a three-plasmid system is widely used, where psPAX2 is deployed for viral packaging, pMD2.G for VSV-G envelope expression, and lentiCRISPRv2 for Cas9 and sgRNA loading [ 64 , 65 , 66 , 67 ]. In contrast to small-sized AAVs, lentiviruses measure 80–100 nm in diameter, which allows a genome size of up to 9.7 k bases.…”