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

Hayley Goullée,Joshua S Clayton,Gianina Ravenscroft,Alistair R R Forrest,Rhonda L Taylor,Nigel G Laing

doi:10.1186/s13395-021-00278-1

Hayley Goullée, Joshua S Clayton + Show 4 more

Open Access

https://doi.org/10.1186/s13395-021-00278-1

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Abstract

BackgroundCRISPR/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.MethodsSince 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.ResultsCells 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).ConclusionThis 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.

Highlights

CRISPR/Cas9 is an essential tool in the field of genomics
In support of previous reports, these data show that skMDCs seeded on Matrigel have a higher rate of proliferation compared to cells grown on uncoated wells
Our results show that seeding myoblasts on Matrigel at low confluency results in higher levels of transfection compared to Matrigel negative conditions

Summary

Introduction

CRISPR/Cas is an essential tool in the field of genomics. First described in 2012 [1], this system uses a programmable guide RNA (gRNA) to target the Cas endonuclease to a specific DNA sequence for cleavage.Such precision editing of the genome has accelerated the development of disease models, genetic therapies, and our understanding of the genome as a whole [1,2,3,4,5].Cas can be delivered to cells as DNA (via plasmid), mRNA or protein. First described in 2012 [1], this system uses a programmable guide RNA (gRNA) to target the Cas endonuclease to a specific DNA sequence for cleavage. Such precision editing of the genome has accelerated the development of disease models, genetic therapies, and our understanding of the genome as a whole [1,2,3,4,5]. Cas can be delivered to cells as DNA (via plasmid), mRNA or protein The latter method, first described in 2014 by Kim et al [6], involves the delivery of a purified Cas enzyme pre-complexed with the gRNA to form a Cas9/gRNA ribonucleoprotein (RNP) complex. This study aimed to maximize CRISPR/Cas transfection and editing in primary human myoblasts

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