Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Summary

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus¹²⁹ x Mus castaneus).

Abstract

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus¹²⁹ x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Introduction

Transcriptional regulatory elements are critical for spatio-temporal fine tuning of gene expression during development¹ and modification of these elements can result in disease due to aberrant gene expression². Many disease-associated regions identified by genome wide association studies are in non-coding regions and have features of transcriptional enhancers^3-4. Identifying enhancers and matching them with the genes they regulate is complicated as they are often located several kilobases away from the genes they regulate and may be activated in a tissue-specific manner^5-6. Enhancer predictions are commonly based on histone ....

Protocol

1. Designing and Constructing the gRNA

1. To delete transcriptional enhancer regions use two gRNAs, one 5' and one 3' of the region of interest. Use the mouse UCSC genome browser track generated by the Zhang laboratory to identify unique gRNA sequences (http://www.genome-engineering.org¹⁵). Next check these gRNAs and their adjacent PAM for SNPs and indels using online tools provided by the Sanger Institute (www.sanger.ac.uk/sanger/Mouse_SnpViewer/rel-1211)^27-28. To target both alleles with equal efficiency, avoid gRNA/PAM sequences that contain a SNP or indel.
   1. While choosing the gRNA, check for the feasibility of desi....

Results

The protocol described here uses F1 ES cells to study cis-regulation of gene expression in monoallelic enhancer deleted cells generated using CRISPR/Cas9 genome editing (Figure 1). The gRNA and allele-specific primer design for genotyping and gene expression are the key factors in this approach. Each allele-specific primer set must be validated by qPCR to confirm allele specificity. Allele-specific primers that amplify only their respective genomic DNA target are.......

Discussion

CRISPR/Cas9 mediated genome editing technology provides a straightforward, fast and inexpensive method for genome modification. The method detailed here to generate and analyze monoallelic enhancer deletion for functional enhancer characterization takes advantage of SNPs in F1 mouse cells. The advantages of this type of approach are: 1) monoallelic enhancer deletions do not produce confounding effects that occur when a critical enhancer is deleted from both alleles, i.e., a great reduction in the protein levels .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phusion High-Fidelity DNA Polymerase	NEB	M0530S	high fidelity DNA polymerase used in gRNA assembly
Gibson Assembly Master Mix	NEB	E2611L
gRNA_Cloning Vector	Addgene	41824	A target sequence is cloned into this vector to create the gRNA plasmid
pCas9_GFP	Addgene	44719	Codon-optimized SpCas9 and EGFP co-expression plasmid
AflII	NEB	R0520S
EcoRI	NEB	R3101S
Neon Transfection System 100 µL Kit	Life Technologies	MPK10096	Microporator transfection technology
prepGEM	ZyGEM	PT10500	genomic DNA extraction reagent
Nucleo Spin Gel & PCR Clean-up	Macherey-Nagel	740609.5
High-Speed Plasmid Mini Kit	Geneaid	PD300
Maxi Plasmid Kit Endotoxin Free	Geneaid	PME25
SYBR select mix for CFX	Life Technologies	4472942	qPCR reagent
iScript cDNA synthesis kit	Bio-rad	170-8891	Reverse transcription reagent
0.25% Trypsin with EDTA	Life Technologies	25200072
PBS without Ca/Mg2+	Sigma	D8537
0.5 M EDTA	Bioshop	EDT111.500
HBSS	Life Technologies	14175095
1 M HEPES	Life Technologies	13630080
BSA fraction V (7.5%)	Life Technologies	15260037
Max Efficiency DH5α competent cells	Invitrogen	18258012
FBS	ES cell qualified		FBS is subjected to a prior testing in mouse ES cells for pluripotency
DMSO	Sigma	D2650
Glutamax	Invitrogen	35050
DMEM	Life Technologies	11960069
Pencillin/Streptomycin	Invitrogen	15140
Sodium pyruvate	Invitrogen	11360
Non-essential aminoacid	Invitrogen	11140
β-mercaptoethanol	Sigma	M7522
96-well plate	Sarstedt	83.3924
Sealing tape	Sarstedt	95.1994
CoolCell LX	Biocision	BCS-405	alcohol-free cell freezing container
CHIR99021	Biovision	1748-5	Inhibitor for F1 ES cell culture
PD0325901	Invivogen	inh-pd32	Inhibitor for F1 ES cell culture
LIF	Chemicon	ESG1107	Inhibitor for F1 ES cell culture