Name: CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery (Video) | JoVE Journal
Uploaded: 2025-05-30T13:40:00.000+00:00
Duration: 7 min 49 s
Description: Learn CRISPR epigenome editing in human cells using plasmid DNA transfection and mRNA nucleofection. This protocol enables targeted gene modulation without toxic DNA breaks for safer gene expression control.

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5 minutes

We thank the members of the Nu&#241;ez lab, especially Rithu Pattali and Izaiah Ornelas, for developing and optimizing the protocols described in this manuscript.

J.K.N. is an inventor of patents related to the CRISPRoff/on technologies, filed by The Regents of the University of California.

This protocol details two different transient delivery methods for CRISPR epigenome editors: plasmid DNA transfection and mRNA nucleofection. Both techniques have unique advantages, disadvantages, and general considerations (Figure 1F).
Plasmid DNA transfection leads to robust epigenome editor expression, and we include a BFP fusion within the epigenome editor constructs that allows for the detection and quantification of transfection efficacy using flow cytometry...

Though the genomic content of every cell in our body is nearly identical, the transcriptional profile of each cell type differs greatly. Epigenetic modifications on DNA and histone proteins are key regulators of transcriptional expression. Transcriptionally active euchromatin is hallmarked by distinct epigenetic marks compared to compact, transcriptionally inactive heterochromatin. For example, heterochromatic regions are defined by repressive histone modifications, including trimethylation on lysine 9 of histone 3 (H3K9me3), trimethylation on lysine 27 of histone H3 (H3K27me3), and DNA methylation on cytosines next to guanines (CpG) at gene promoters1. Genomic regions of active gene expression are defined typically by histone acetylation and trimethylation on lysine 4 of histone 3 (H3K4me3)1.
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) revolution has generated a wealth of tools that enable programmed altering of genomic sequences. CRISPR technology is based on a prokaryotic defense mechanism capable of cleaving nucleic acids at programmable target sequences. CRISPR nucleases2,3,4, base editors5,6, and prime editors7,8 can change the DNA sequence of mammalian genomes through DNA cutting and repair of these breaks. Though effective, these strategies can cause DNA breaks at off-target sites9,10 and large-scale genomic structural mutations11,12,13,14,15. Alternative CRISPR-based tools enable tractable modulation of gene activation and repression without changing the underlying DNA sequence. These tools harness a nuclease deficient Cas9 (dCas9), allowing for DNA binding at target sites dictated by sgRNA sequence, in combination with effector proteins that alter the chromatin landscape16,17,18. Effector proteins, such as epigenetic writers, readers, and erasers, can be directly fused to dCas9 or recruited by a peptide scaffold fused to dCas9, such as SunTag, or an RNA scaffold on the sgRNA, such as the MS2-MCP system16,17,18. Examples of programmable transcription control tools include CRISPR activation (CRISPRa)19,20,21 and CRISPR interference (CRISPRi)22,23. CRISPRa functions by directly recruiting the transcription machinery, increasing target transcriptional gene expression19. In contrast, CRISPRi represses transcription by establishing H3K9me3, a repressive epigenetic mark22.
Advances in epigenome editing have enabled broad use of these tools across scientific fields. Fusions of different effector domains and proteins have expanded the toolkit of available epigenome editors18,24,25,26,27,28,29,30. Additionally, epigenome editors are used to decipher the roles of epigenetic modifications31,32,33,34,35,36, effectors37,38,39, and effector mutations28,40,41 in gene regulation. Specifically, CRISPRi and CRISPRa are used in functional genomics screens for a variety of biological processes, including cell survival42 and cell fate43,44,45,46,47. Furthermore, epigenome editing holds therapeutic potential for ex vivo cell engineering and in vivo therapies18.
Here, we describe methods for applying two dCas9-based epigenome editors for programmable transcriptional repression in human cell lines: CRISPRi22 and CRISPRoff48. CRISPRi is a fusion of dCas9 to the repressive KRAB domain from a zinc finger protein such as ZNF10 (KOX1) and ZIM322,23. When CRISPRi is targeted to a specific gene promoter, the KRAB domain recruits an H3K9me3 writer, SETDB1, to repress the target gene (Figure 1A). When CRISPRi is expressed transiently, the established H3K9me3 at the target locus is not maintained, and gene expression is restored over time32,48. To conduct stable knockdowns using CRISPRi such as in functional genomics applications, constitutive expression of CRISPRi in the cells along with the sgRNA is essential. Recently, CRISPRoff was engineered to program heritable epigenome editing48. CRISPRoff is a single protein fusion of dCas9 to the KRAB domain and de novo DNA methyltransferase complex, DNMT3A and DNMT3L. A transient pulse of CRISPRoff in human cells programs deposition of H3K9me3 and DNA methylation at the targeted genes, which leads to long-term repression of target genes by the maintenance of DNA methylation and H3K9me3 (Figure 1B)48. Furthermore, epigenome edits can be reversed. For example, a gene that is stably silenced by CRISPRoff can be reactivated by TET1-dCas9 which can enzymatically remove the DNA methylation marks at target loci49.
This protocol will detail two delivery methods for the transient expression of epigenome editors: plasmid DNA transfection and mRNA nucleofection. Additionally, we outline how to use flow cytometry to assess epigenome editing efficacy at two endogenous genes, CLTA and CD55. These methods can be adapted and applied to other epigenome editing experiments using additional editors or can be used for targeting different genes.
<img alt="CRISPRi/CRISPRoff gene silencing diagram; transient, heritable editing; transfection, nucleofection method." class="xfigimg" src="/files/ftp_upload/68089/68089fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Schematic of CRISPRi and CRISPRoff epigenome editing mechanism and workflow. (A) Linear schematics of the sgRNA transgene and CRISPRi epigenome editor. The addition of CRISPRi and sgRNA allows for the addition of the repressive H3K9me3 histone mark to silence the target locus. The highest level of silencing is achieved early after CRISPRi addition, and the gene target is generally reactivated after a few passages. (B) Linear schematics of the sgRNA transgene and CRISPRoff epigenome editor. The addition of CRISPRoff to cells targeting a gene of interest leads to the addition of repressive H3K9me3 along with DNA methylation at CpG sites to silence the target gene. Silencing by CRISPRoff is heritable - the high level of silencing is achieved early during transfection and persists over multiple cell divisions. (C) Overview of timeline for epigenome editing via transfection method. On day 0, the cells are plated for transfection. On day 1, the editor and guide plasmid can be introduced into the cells via transfection. On day 3, the cells will be assessed for BFP expression via flow cytometry. The percent of BFP is used as a normalizing factor to determine the final silencing efficacy of the experiment for each condition. On day 6 onwards, the cells are analyzed for silencing the reporter of interest as the highest level of silencing will be achieved on this day. (D) Transfection method overview wherein epigenome editor and sgRNA plasmids are added to cells in a dropwise manner. (E) Overview of the timeline for epigenome editing via the nucleofection methods. In this protocol, mRNA is nucleofection into the cells on day 0. The cells are assessed for silencing on day 3 post-nucleofection using flow cytometry analysis. (F) Overview of the nucleofection protocol. Appropriate amounts of cells and mRNA are mixed and added into nucleofector cuvettes. If the sgRNA is introduced by nucleofection, it can also be added to this mixture. The cuvettes are put into the nucleofector, and appropriate pulse codes are used to introduce the mRNA into cells. Post-nucleofection, the cells are plated and passaged for analysis in later days. (G) Comparison between plasmid transfection and mRNA nucleofection strategies for epigenome editing. <a href="https://www.jove.com/files/ftp_upload/68089/68089fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>4D-Nucleofector</td><td>Lonza</td><td>AAF-1003</td><td></td></tr><tr><td>96-well tissue culture plates</td><td>Corning</td><td>3596</td><td></td></tr><tr><td>96-well U-bottom Plate</td><td>Corning</td><td>351177</td><td></td></tr><tr><td>APC anti-human CD55 Antibody</td><td>BioLegend</td><td>311312</td><td></td></tr><tr><td>BD FACSymphony A1 Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Bleach</td><td>Waxie</td><td>11003428432</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5425</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Thermo Scientific</td><td>Countess 3</td><td></td></tr><tr><td>CRISPRoff transfection plasmid</td><td>Addgene&amp;nbsp;</td><td>167981</td><td></td></tr><tr><td>Diluent 2 Hematology Reagent for Flow Cytometry (Sheath fluid)</td><td>Thermo Scientific</td><td>23-029-361</td><td></td></tr><tr><td>DMEM, High Glucose</td><td>Thermo Scientific</td><td>11965118</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14-190-250</td><td></td></tr><tr><td>Eppendorf tubes</td><td>Thomas scientific</td><td>1159M35</td><td></td></tr><tr><td>FBS</td><td>Avantor Seradigm</td><td>89510-186</td><td></td></tr><tr><td>Lonza Walkersville&amp;nbsp;SF Cell Line 4D-Nucleofector X Kit L</td><td>Fisher Scientific</td><td>NC0281111</td><td></td></tr><tr><td>mMESSAGE mMACHINE&amp;trade; T7 ULTRA Transcription Kit</td><td>Thermo Fisher</td><td>AM1345</td><td></td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31985070</td><td></td></tr><tr><td>PCR strip tubes</td><td>USA Scientific&amp;nbsp;</td><td>1402-4700</td><td></td></tr><tr><td>Penicillin-Streptomycin-Glutamine</td><td>Gibco</td><td>10378016</td><td></td></tr><tr><td>pLG1 sgRNA expression plasmid</td><td>Addgene&amp;nbsp;</td><td>217306</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>22-400-105</td><td></td></tr><tr><td>SF Cell Line 96-well Nucleofector&amp;reg;&amp;nbsp;Kit</td><td>Lonza</td><td>V4SC-2096</td><td></td></tr><tr><td>Tissue culture incubator</td><td>PHCbi</td><td>MCO-170AICUVDL-PA</td><td></td></tr><tr><td>TransIT-LTI transfection reagent</td><td>Mirus</td><td>MIR 2306</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Gibco</td><td>25200114</td><td></td></tr></tbody></table>

crispr epigenome editing in human cells using plasmid dna transfection and mrna nucleofection delivery

CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery

Epigenetics refers to chemical modifications of histone proteins and DNA that can regulate the expression of genes. The human epigenome is altered dynamically during cell differentiation and aging, and many diseases are associated with aberrant epigenome patterning. Recent advances in CRISPR have led to the development of programmable tools to edit epigenetic modifications at targeted genomic loci, enabling precise rewriting of epigenetic modifications in human cells. CRISPR-based epigenome editors rely on catalytically dead Cas9 coupled with epigenetic modifiers that ultimately result in programmed repression or activation of targeted genes in mammalian genomes. Unlike traditional genome editing methods, epigenome editing does not require DNA breaks or changes in the human genome sequence and thus serves as a safer alternative to control&#160;gene expression. In this protocol, we highlight two different methods to perform dCas9-mediated epigenome editing in human cell lines using plasmid DNA transfections and nucleofection of mRNAs encoding CRISPR epigenome editors. We demonstrate programmable epigenome editing to transiently repress genes using CRISPR interference (CRISPRi) and for silencing genes durably for many weeks using CRISPRoff, a fusion of dCas9 to the KRAB domain and de novo DNA methyltransferase complex. We also provide guidance on quantitative methods to measure successful epigenome editing of target genes and key considerations on which epigenome editing tool to use, depending on experimental criteria.

For all epigenome editing experiments, proper controls are critical to assess epigenome editing efficiencies. We recommend using a control sgRNA, which does not target any sequence in the human genome. Using a non-targeting guide control will give confidence that changes at the target loci are driven by the epigenome editor being directed to that site rather than just from epigenome editor overexpression or non-specific binding. Additionally, for reporter gene-based experiments, we suggest using a dCas9-only control to e...

The protocol describes methods for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based epigenome editing in human cell lines using plasmid DNA transfection and mRNA nucleofection.

Epigenetics refers to chemical modifications of histone proteins and DNA that can regulate the expression of genes. The human epigenome is altered ...

crispr-epigenome-editing-human-cells-using-plasmid-dna-transfection

Research

JoVE Journal

Genetics

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Learn CRISPR epigenome editing in human cells using plasmid DNA transfection and mRNA nucleofection. This protocol enables targeted gene modulation without toxic DNA breaks for safer gene expression control.

CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery

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