CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions

Summary

The presented method describes the generation of a CRISPR-mediated gene knockout in the human embryonic stem cell (hESC) line H9, which stably expresses sgRNAs targeting the L2HGDH gene using a highly efficient lentiviral-mediated gene delivery system.

Abstract

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical application of this technique, particularly in pluripotent stem cells, presents certain challenges, such as being time- and labor-intensive and having low editing efficiency. Here, we describe the generation of a CRISPR-mediated gene knockout in a human embryonic stem cell (hESC) line stably expressing sgRNAs for the L2HGDH gene, using a highly efficient and stable lentiviral-mediated gene delivery system. The sgRNAs targeting exon 1 of the L2HGDH gene were chemically synthesized and cloned into the lentiCRISPR v2-puro vector, which combines the constitutive expression of sgRNAs with Cas9 in a highly efficient single-vector system to achieve higher lentiviral titers for hESC infection and stable selection using puromycin. Puromycin-selected cells were further expanded, and single-cell clones were obtained using the limited dilution method. The single clones were expanded, and several homozygous knockout clones for the L2HGDH gene were obtained, as confirmed by a 100% reduction in L2HGDH expression using Western blot analysis. Furthermore, using MSBSP-PCR, the CRISPR mutation site was mapped upstream of the PAM recognition sequence of Cas9 in the selected homozygous clones. Sanger sequencing was performed to analyze the exact insertions/deletions, and functional characterization of the clones was conducted. This method produced a significantly higher percentage of homozygous deletions compared to previously reported non-viral gene delivery methods. Although this report focuses on the L2HGDH gene, this robust and cost-effective approach can be used to create homozygous knockouts for other genes in pluripotent stem cells for gene function studies.

Introduction

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are stem cells with the potential to differentiate into all cell types in the body. These cells serve as valuable tools for studying human development, as well as for understanding the underlying mechanisms of various diseases, thus offering tremendous promise for regenerative medicine, disease modeling, and drug discovery. Such studies involve investigating how specific genes contribute to the development, functioning, and regulation of organisms^1,2.

Various techniques and approaches are employed to decip....

Protocol

The details of the gene sequences, reagents, and equipment used in this study are listed in the Table of Materials.

1. Single guide RNA (sgRNA) design, cloning, and lentiviral vector production

NOTE: Two different sgRNA sequences targeting the exon 1 of the L2HGDH gene, both adapted from Qiu et al.⁶with PAM sites of AGG and TGG for sequences 1 and 2, respectively, are used. Both sgRNAs were 20 bp in le.......

Discussion

This study has standardized a method that enables highly efficient and cost-effective gene deletions in hESCs through CRISPR-Cas9 technology. This method successfully achieved homozygous deletion of the L2HGDH gene in hESCs within 3-4 weeks, starting from hESC infection to single-cell clonal selection and propagation (Table 1). Although CRISPR-Cas9-mediated gene manipulations can be achieved by transient transfections in most cells, this becomes challenging in stem cells due to poor transfection efficien.......

Materials

Name	Company	Catalog Number	Comments
2-MERCAPTOETHANOL	Invitrogen	31350010
38.5 mL, Sterile + Certified Free Open-Top Thinwall Ultra-Clear Tubes	Beckman Coulter	C14292
Accutase	Stem cell technologies	7920
bFGF Recombinant human	Invitrogen	PHG0261
Brachyury Rabbit mAb	Abclonal	A5078
BsmBI-v2	NEB	R0739S
chir99021	Tocris	4423/10
Corning Matrigel Basement Membrane Matrix, LDEV-free	Corning	354234
Cyclopamine	Stem cell technologies	72074
DMEM media	Invitrogen	11995073
DMEM NUTRIENT MIX F12	Invitrogen	11320033
DPBS w/o: Ca and Mg	PAN Biotech	P04-36500
Fetal bovie serum	Invitrogen	10270106
FoxA2/HNF3β	CST	8186
GAPDH (14C10) Rabbit mAb Antibody	CST	2118S
Gentle Cell Dissociation Reagent	Stem cell technologies	7174
HyClone Non Essential Amino Acids (NEAA) 100x Solution	GE healthcare	SH30238.01
Ki-67 (D3B5) Rabbit mAb	CST	9129
KnockOut Serum Replacement	Invitrogen	10828028
L GLUTAMINE, 100x	Invitrogen	2924190090
L2H-BMSBSP-F1	Macrogen		CGTGCGGGTTCGCGTCTGGG
L2HGDH Polyclonal antibody	Proteintech	15707-1-AP
L2HGDH-SgRNA1-F	Macrogen		CACCGCGTGCGG GTTCGCGTCTGGG
L2HGDH-SgRNA1-R	Macrogen		AAACCCCAGACGC GAACCCGCACGC
L2HGDH-SgRNA2-F	Macrogen		CACCGCCCGCGG GCTTTTCGCCGG
L2HGDH-SgRNA2-R	Macrogen		AAACCCGGCGAA AAGCCCGCGGGC
L2H-SeqF1	Macrogen		GCTAAAGAGCGC GGGTCCTCGG
L2H-SeqR1	Macrogen		GTGGACGGGTTG TTCAAAGCCAGAG
L2H-UMSBSP-R1	Macrogen		GTGGACGGGTTG TTCAAAGCCAGAG
LentiCRISPRv2	Addgene	52961
mTesR1 complete media	Stem cell technologies	85850
Nanog Antibody	CST	3580
NEUROBASAL MEDIUM 1x CTS	Invitrogen	A1371201
Neuropan 2 Supplement 100x	PAN Biotech	P07-11050
Neuropan 27 Supplement 50x	PAN Biotech	P07-07200
Oct-4 Antibody	CST	2750
Pax6 (D3A9V) XP Rabbit mAb	CST	60433
PENICILLIN STREPTOMYCIN SOL	Invitrogen	15140122
pMD2.G	Addgene	12259
Polybrene infection reagent	Sigma	TR1003- G
Polyethylenimine, branched	Sigma	408727
psPAX2.0	Addgene	12260
Puromycin	Invitrogen	A1113802
qPCR Lentivirus Titer Kit	Abm	LV900
Rock inhibitor Y-27632 dihydrochloride	Tocris	1254
SB 431542	Tocris	1614/10
Sox2 Antibody	CST	2748
Sucrose	Sigma	57-50-1
TRYPSIN .05% EDTA	Invitrogen	25300062
U6-459F	Macrogen		GAGGGCCTATT TCCCATGATTC
Wizard Genomic DNA Purification Kit	Promega	A1120
XAV 939	Tocris	3748/10