Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing

Summary

The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out clones.

Abstract

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, specifically to study the function of new genes/proteins associated with a disease, identified during the genetic diagnosis. For the development of such cell lines, two major issues have to be untangled: insertion of the CRISPR tools (the Cas9 and the guide RNA) with high efficiency into the chosen cells, and restriction of the Cas9 activity to the specific deletion of the chosen gene. The protocol described here is dedicated to the insertion of the CRISPR tools in difficult to transfect cells, such as muscle cells. This protocol is based on the use of lentiviruses, produced with plasmids publicly available, for which all the cloning steps are described to target a gene of interest. The control of Cas9 activity has been performed using an adaptation of a previously described system called KamiCas9, in which the transduction of the cells with a lentivirus encoding a guide RNA targeting the Cas9 allows the progressive abolition of Cas9 expression. This protocol has been applied to the development of a RYR1-knock out human muscle cell line, which has been further characterized at the protein and functional level, to confirm the knockout of this important calcium channel involved in muscle intracellular calcium release and in excitation-contraction coupling. The procedure described here can easily be applied to other genes in muscle cells or in other difficult to transfect cells and produce valuable tools to study these genes in human cells.

Introduction

With the progress of gene sequencing and the identification of mutations in genes of unknown functions in a specific tissue, the development of relevant cellular models to understand the function of a new target gene and confirm its involvement in the related pathophysiological mechanisms constitutes an essential tool. In addition, these models are of major importance for future therapeutic developments^1,2, and constitute an interesting alternative to the development of knock-out animal models in straight line with the international recommendations for reduction in the use of animals in experimentation. Gene e....

Protocol

Muscle biopsies were obtained from the Bank of Tissues for Research (Myobank, a partner in the EU network EuroBioBank, Paris, France) in accordance with European recommendations and French legislation. Written informed consent was obtained from all individuals. Immortalized myoblasts were kindly produced by Dr. V. Mouly (Myology Institute, Paris, France), and the protocols were approved by Myology Institute ethic committee (MESRI, n AC-2019-3502).

1. CRISPR guide design

1. Identify the region of the gene to be deleted. Search for its genomic sequence using genome browser tools such as ensembl.org or genome.ucsc.edu....

Results

This protocol was applied to immortalized myoblasts from a healthy subject¹⁵ (so-called HM cells, for human myoblasts), in which the RyR1 has been previously characterized¹⁶, in order to knock out the RYR1 gene encoding the RyR1 protein. The design of the guides RNA was made to delete the sequence encompassing part of exon 101 and intron 101 of the gene. Deletion of part of exon 101 is foreseen to result in disruption of the reading frame. In addition, exon 101 enc.......

Discussion

A major step on the way to the characterization of genes of unknown function involved in pathologies is the development of relevant cellular models to study the function of these genes. The use of gene editing using CRISPR/Cas9 is an exponentially growing field of research, and the development of knock-out models as presented here is among its most widely used applications. In this context, we propose here a versatile protocol to develop a human cell line knock-out in any gene of interest, allowing the characterization o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-CACNA1S antibody	Sigma-Aldrich	HPA048892	Primary antibody
Blp I	NE BioLabs	R0585S	Restriction enzyme
CalPhos Mammalian Transfection Kit	Takara	631312	Transfection kit
Easy blot anti Mouse IgG	GeneTex	GTX221667-01	HRP secondary antibody
Easy blot anti Rabbit IgG	GeneTex	GTX221666	HRP secondary antibody
Fluo-4 direct	Molecular Probes	F10472	Calcium imaging
GAPDH(14C10) Rabbit mAb	Cell Signaling Technology	#2118	Primary antibody
HindIII	Fermentas	ER0501	Restriction enzyme
InFusion HD Precision Plus	Takara	638920	Ligation kit
MasterMix Phusion High Fidelity with GC	ThermoFisher Scientific	F532L	Mix for PCR reaction with High fidelity Taq polymerase and dNTPs
Myosin Heavy Chain antibody	DHSB	MF20	Primary antibody
NucleoBond Xtra Maxi EF	Macherey-Nagel	REF 740424	Maxipreparation kit for purification of plasmids
NucleoSpin Gel and PCR Clean-up	Macherey-Nagel	740609	DNA purification
NucleoSpin Tissue	Macherey-Nagel	740952	Kit for DNA extraction from cell
One Shot Stbl3 Chemically Competent E. coli	ThermoFisher Scientific	C737303	Chemically competent cells
Plasmid #87904	Addgene	87904	Lentiviral plasmid encoding the SpCas9 (for LV-Cas9)
Plasmid #87919	Addgene	87919	Lentiviral backbone for insertion of cassette with guides (for LV-guide-target)
Plasmid #12260	Addgene	12260	Lentiviral plasmid encoding lentiviral packaging GAG POL
Plasmid #8454	Addgene	8454	Lentiviral plasmid encoding envelope protein for producing lentiviral and MuLV retroviral particles
V5 Tag Monoclonal Antibody	Invitrogene	R96025	Primary antibody
XL10-Gold Ultracompetent Cells	Agilent	200317	Chemically competent cells
Xma I	NE BioLabs	R0180S	Restriction enzyme