Improved CRISPR/Cas9 gene editing in primary human myoblasts using low confluency cultures on Matrigel

Abstract

Background CRISPR/Cas9 is an invaluable tool for studying cell biology and the development of molecular therapies. However, delivery of CRISPR/Cas9 components into some cell types remains a major hurdle. Primary human myoblasts are a valuable cell model for muscle studies, but are notoriously difficult to transfect. There are currently no commercial lipofection protocols tailored for primary myoblasts, and most generic guidelines simply recommend transfecting healthy cells at high confluency. This study aimed to maximize CRISPR/Cas9 transfection and editing in primary human myoblasts. Methods Since increased cell proliferation is associated with increased transfection efficiency, we investigated two factors known to influence myoblast proliferation: cell confluency, and a basement membrane matrix, Matrigel. CRISPR/Cas9 editing was performed by delivering Cas9 ribonucleoprotein complexes via lipofection into primary human myoblasts, cultured in wells with or without a Matrigel coating, at low (~ 40%) or high (~ 80%) confluency. Results Cells transfected at low confluency on Matrigel-coated wells had the highest levels of transfection, and were most effectively edited across three different target loci, achieving a maximum editing efficiency of 93.8%. On average, editing under these conditions was >4-fold higher compared to commercial recommendations (high confluency, uncoated wells). Conclusion This study presents a simple, effective and economical method of maximizing CRISPR/Cas9-mediated gene editing in primary human myoblasts. This protocol could be a valuable tool for improving the genetic manipulation of cultured human skeletal muscle cells, and potentially be adapted for use in other cell types.

Production of Genetically Modified Porcine Embryos via Lipofection of Zona-Pellucida-Intact Oocytes Using the CRISPR/Cas9 System

“…For this reason, the use of lipofectamine in oocytes and embryos is starting to be applied and optimized [28]. Lipofection is an efficient method that has been widely used in many kinds of somatic cells to introduce foreign molecules, including the CRISPR/Cas9 system [39,40].…”

Fully functional monocytic MDSC generation from the murine HoxB8 cell line

“…A drawback of using murine BM cells is their heterogeneity, containing many and very different developmental cellular stages. Genome editing of primary cells like BM haematopoietic stem cells and progenitor cells using different targeted genome editing tools such as zinc-finger nucleases, meganucleases or the more recent clustered regularly interspaced short palindromic repeats (CRISPR) has a lot of limitations because of their intrinsic refractory for transfection, their limited life span and inability to undergo serial passaging [41,42]. HoxB8 cells have proven to be a particularly valuable resource in exploring the functional genomics of immune cells.…”

Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci

“…Despite our efforts to incorporate a reporter gene marker in Cas9 for cell sorting, we encountered difficulties in maintaining the sorted cultured cells. This obstacle has also been noted in previously published literature, which indicates that myoblasts tend to lose their differentiation potential after single-cell cloning, and those that are successfully edited often fail to survive the stress of sorting (41). Since generating clonal populations was not feasible, we opted to gather informative data from bulk populations by routinely passaging the cells when they reached 60-70% confluency, before spontaneously differentiating to myotubes.…”