ABSTRACT
Viral-mediated delivery of the CRISPR-Cas9 system is one the most commonly used techniques to modify the genome of a cell, with the aim of analyzing the function of the targeted gene product. While these approaches are rather straightforward for membrane-bound proteins, they can be laborious for intracellular proteins, given that selection of full knockout (KO) cells often requires the amplification of single-cell clones. Moreover, viral-mediated delivery systems, besides the Cas9 and gRNA, lead to the integration of unwanted genetic material, such as antibiotic resistance genes, introducing experimental biases. Here, an alternative non-viral delivery approach is presented for CRISPR/Cas9, allowing efficient and flexible selection of KO polyclonal cells. This all-in-one mammalian CRISPR-Cas9 expression vector, ptARgenOM, encodes the gRNA and the Cas9 linked to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein and the puromycin N-acetyltransferase, allowing for transient, expression-dependent selection and enrichment of isogenic KO cells. After evaluation using more than 12 distinct targets in 6 cell lines, ptARgenOM is found to be efficient in producing KO cells, reducing the time required to obtain a polyclonal isogenic cell line by 4-6 folds. Altogether ptARgenOM provides a simple, fast, and cost-effective delivery tool for genome editing.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Animals , CRISPR-Cas Systems/genetics , Gene Editing/methods , Cell Line , Mammals/geneticsABSTRACT
Fingertip injuries with loss of the nail bed can lead to permanent deformities or absent nail formation. This is a case report of a 17-year-old female who sustained a blunt injury to the left second index finger with nail avulsion. The patient underwent nail reconstruction with a split-thickness graft from the sterile matrix of the left great toe. Postoperatively both the donor and the recipient sites appear to be healing appropriately. Since nail reconstruction with toe graft is rarely performed, this case is of particular interest due to its excellent postoperative outcomes.