4.3 Article

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

Journal

SKELETAL MUSCLE
Volume 11, Issue 1, Pages -

Publisher

BMC
DOI: 10.1186/s13395-021-00278-1

Keywords

CRISPR; Gene editing efficiency; Primary human myoblasts; Matrigel; Confluency

Categories

Funding

  1. Australian Government
  2. Australian National Health and Medical Research Council (NHMRC) [APP1117510, APP1122952]
  3. NHMRC Fellowship [APP1154524]
  4. NHMRC [APP1146321]

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This study demonstrates a simple and effective method to maximize CRISPR/Cas9-mediated gene editing in primary human myoblasts by delivering Cas9 ribonucleoprotein complexes on Matrigel-coated wells at low confluency. This approach achieved significantly higher editing efficiency compared to commercial recommendations, showing potential for improving genetic manipulation of skeletal muscle cells and other cell types.
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 (similar to 40%) or high (similar to 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.

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