Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins

Summary

This protocol describes a non-integrative, episomal CRISPR/dCas9-based system for targeted epigenetic editing in K562 cells, combining dCas9-DNMT3A and dCas9-HDAC1 effectors with specific sgRNAs to induce locus-specific DNA methylation with precision and reduced off-target effects.

Abstract

Investigating the precise role of DNA methylation in gene transcriptional regulation and developing therapies targeting specific gene methylation patterns presents significant challenges due to the scarcity of versatile tools capable of inducing site-specific and long-term epigenetic modifications for modulating gene expression. The study aimed to develop and validate an innovative episomal-based system to facilitate stable DNA methylation at a targeted gene locus, potentially useful for both basic epigenetic research and therapeutic applications. To achieve this, the K562 cell line was co-transfected with two distinct episomal vectors. Both vector types were engineered to express guide RNAs (gRNAs) targeting a 367 bp unmethylated region within the CpG island 326, located upstream of the ZBTB7A gene. Each vector encoded a deactivated form of endonuclease Cas9 (dead or dCas9) fused to either the catalytic domain of DNA methyltransferase DNMT3A (dCas-DNMT3A-CD) or the full-length histone deacetylase HDAC1 (dCas-HDAC1). The dCas sequence included two Nuclear Localization Signals (NLS) to ensure the protein's nuclear import. This dual system expression cassette promotes a chromatin state potentially conducive to long-term epigenetic silencing, promising robust and durable epigenetic results. This intervening approach to the host epigenome via utilization of self-replicating episomal vectors offers several advantages: maintenance and expression of vectors at low copy numbers throughout multiple cell divisions without integration into the host genome, hence minimizing off-target effects and preserving genome integrity. We report the precise and significant increase of DNA methylation at the targeted ZBTB7A CpG island 326. The findings validate that the engineered episomal CRISPR/dCas systems can elicit durable, site-specific DNA methylation. Therefore, this system is a valuable research tool for evaluating the functional effects of targeted methylation changes and a promising platform for developing future epigenetic treatments.

Introduction

Epigenetic regulation, including DNA methylation and histone modifications, plays a pivotal role in controlling gene expression, cell differentiation, and genome stability. Dysregulation of these mechanisms is often involved - either as a cause or a consequence - in various pathological states, particularly in cancer, neurological disorders, and imprinting syndromes^1,2,3. Notably, by precisely intervening to the epigenetic landscape at specific genomic loci using epigenome editing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, gene repression l....

Protocol

1. Design of sgRNAs

1. Identify the desired CpG island or regulatory CpG sites within the promoter region of the gene of interest using a genome browser (e.g., UCSC genome browser). Here, the design was applied on the CpG island 326 (GRCh38)of the ZBTB7A gene.
2. Design single guide RNAs (sgRNAs) using an online CRISPR sgRNA design platform [e.g., CHOPCHOP web tool (version 3)]. When designing sgRNAs, follow the recommendations below to ensure high on-target activity and minimal off-target effects:
   1. Select SpCas9 protein as the endonuclease, using 5'-NGG-3' as the protospacer adjacent motif at the 3....

Results

To investigate whether our targeted epigenetic editing tools could induce site-specific DNA methylation changes at the ZBTB7A CpG island 326, we first generated episomal vectors encoding dCas9 fused to either DNMT3A(CD) or HDAC1, along with various sgRNAs designed with target sites within CpG island 326. We initially co-transfected K562 cells with combinations of these plasmids to evaluate whether simultaneous delivery of sgRNA and effector modules could lead to reproducible epigenetic modifications.

Discussion

One of the most critical steps in this protocol is the design of highly specific sgRNAs. Off-target effects are a known limitation of CRISPR-based epigenetic editing systems, which can often arise from dCas9 binding infidelity, as well as the non-specific activity of the effector domains. To minimize this, sgRNAs must be designed using bioinformatic tools that optimize for high on-target activity and minimal off-target binding³⁰. Employing design tools and validating predicted off-target sites is .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Absolute Q DNA Digital PCR Master Mix (5x)	Applied Biosystems	A52490
AgeI-HF	NEB	R3552
AscI	NEB	R0558
AseI	NEB	R0526
BamHI-HF	NEB	R3136
BD FACS Melody Cell Sorter	BD
Bglll	NEB	R0144
BspEI	NEB	R0540
Deoxynucleotide (dNTP) Solution Mix (10 mM each)	NEB	N0447
DMEM High Glucose w/stable Glutamine, w/Sodium Pyruvate medium	Biowest	L0103
DNA Polymerase I, Large (Klenow) Fragment	NEB	M0210
DNMT3A (CD), HDAC1 sequences	IDT
DreamTaq DNA Polymerase	Thermo Scientific	EP0712
EpiTect	Qiagen	59104	Bisulfite conversion kit
Epitect Bisulfite kit	Qiagen	59104
Esp3I	NEB	R0734
Fetal Bovine Serum	Gibco	A5256701
Geneticin selective antibiotic (G418 sulfate)	Gibco	11811031
Lipofectamine 3000 transfection kit	Invitrogen	L3000015
NEB® 5-alpha Competent E. coli (High Efficiency)	NEB	C2987I
NEB® 5-alpha F'Iq Competent E. coli (High Efficiency)	NEB	C2992H
Nucleospin Tissue	Macherey-Nagel	740952.50
PBS, 10x solution pH 7.4	Lonza	BE17-517Q
Penicillin-Streptomycin solution 100x	Biowest	L0022
pGuide vector	OriGene Technologies	GE100042
Phenol:Chloroform:Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA	Sigma-Aldrich	P2069
Primers, sgRNAs	Eurofins
PyroMark Assay Design	Qiagen	Version 2.0	Bisulfite primer design tool
PyroMark Gold Q24 Reagents	Qiagen	970802
PyroMark PCR kit	Qiagen	978703
PyroMark Q24 MDx	Qiagen		Pyrosequencing workstation
PyroMark Q24 Software	Qiagen	v2.0.8	Pyrosequencing software
Q5® High-Fidelity DNA Polymerase	NEB	M0491
QuantiTect Reverse Transcription Kit	Qiagen	205311
QuantStudio Absolute Q MAP16 Plate Kit	Applied Biosystems	A52865	Digital PCR system
SYBR Green I Nucleic Acid Gel Stain, 10,000x concentrate in DMSO	Invitrogen	S7563
T4 DNA Ligase	NEB	M0202
T4 Polynucleotide Kinase	NEB	M0201
TRIzol Reagent	Invitrogen	15596026