CRISPR modulation

Modified CRISPR-Cas9 systems for activation or repression of endogenous gene transcription


Adding new dimensions to the CRISPR toolbox, the CRISPRmod family of gene modulation tools promote (CRISPRa) or repress (CRISPRi) target gene transcription, without cutting the DNA.

Benefits & applications

  • Endogenous modulation – control expression within a gene’s native context
  • CRISPR level specificity – leverage PAM-anchored targeting
  • Multiplexing – simultaneously activate or inhibit multiple genes
  • Orthogonal validation – use in parallel with CRISPR knockout or RNAi for robust datasets

Explore CRISPRi

Knock down target gene expression with CRISPRi

Explore CRISPRa

Activate endogenous gene expression with CRISPRa


What is CRISPRmod?

CRISPRmod is Horizon's family of CRISPR activation and CRISPR interference reagents, used for targeted modulation of endogenous gene expression.

In the central dogma of molecular biology, DNA is translated to RNA, which is then translated into protein (figure 1). Researchers can modulate this pathway at multiple points through loss- or gain-of-function experiments to better understand gene function.

Existing CRISPR knockout and HDR knock-in techniques allow researchers to edit (make heritable changes to) cellular DNA, while traditional RNAi techniques degrade mRNA, thereby preventing translation.

CRISPRmod offers a fresh gene modulation method, regulating expression at the transcriptional level. The Horizon CRISPRmod portfolio includes both CRISPRa and CRISPRi systems, to respectively up- or down-regulate target gene expression

The central dogma of biology: DNA to RNA to Protein


Why use CRISPRmod? 

CRISPRmod enables a new generation of CRISPR-based experiments for endogenous gene modulation. CRISPRmod also offers an orthogonal method for confirming phenotypes observed from other gene modulation methods. 

  • Activate or inhibit genes within their native context for more biologically relevant models
  • Simultaneously modulate multiple genes to better understand a pathway or network 
  • Orthogonally validate CRISPR knockout or siRNA results for more robust datasets


How does CRISPRmod work?

Both CRISPRmod systems (CRISPRa and CRISPRi) use a deactivated Cas9 construct fused to either activators or repressors, to leverage the power of PAM-anchored targeting for transcriptional modulation (figure 2). CRISPRmod simply brings the precision of CRISPR to gene modulation.

The native Cas9 DNA-cutting functionality has been obliterated by point mutations in the RuvCI and HNH nuclease domains (W. Cheng et al.), changing the protein to a deactivated, or dead, Cas9 (dCas9). This dCas protein is further engineered for targeted transcriptional modulation through the fusing of activator or repressor domains to the C-terminus.

    CRISPRa system
    CRISPRi system
Diagram of CRISPRa based transcriptional activation
Diagram of CRISPRi-based transcriptional repression
Diagram of CRISPRi-based transcriptional repression

Figure 2.  Schematic diagrams showing a) CRISPRa machinery binding upstream of TSS to activate transcription, and b) CRISPRi machinery binding downstream of target gene TSS to block transcription.


Multiplexed gene modulation 

CRISPRmod offers a unique method for the simultaneous modulation of multiple genes. Each guide RNA binds to its specific DNA target independently and relies on an exogenously supplied dCas9 fusion protein, thereby minimizing any competition for endogenous pathways. Here we show  the effectiveness of multiplexing CRISPRi within iPSC cells (figure 3).

Synthetic sgRNAs enable easy multiplexing for the simultaneous repression of multiple genes

Synthetic sgRNAs enable easy multiplexing for the simultaneous repression of multiple genes
Synthetic sgRNAs enable easy multiplexing for the simultaneous repression of multiple genes
Figure 6. 個々のCRISPRmod CRISPRi sgRNAを混合して単一の試薬にしたプールを用いて、標的遺伝子の転写抑制を強化したり、多重化して複数の遺伝子の同時抑制を可能にしたりすることができます。dCas9-SALL1-SDS3を安定して発現するWTC-11ヒトiPS細胞に、Lonza 96ウェルシャトルシステムを用いて、PPIB、SEL1L、およびRAB11Aを標的とする化学合成sgRNAを導入しました。各遺伝子ターゲットに対して最も有効なデザイン済みsgRNAを選択し、個別に、またはガイドあたり3 µMの濃度でマルチターゲットプール(3つの異なる遺伝子をターゲットとするsgRNAの混合物)の一部として使用しました。ヌクレオフェクションの72時間後に細胞を回収し、全RNAを単離し、RT-qPCRを使用して相対的な遺伝子発現を測定しました。各標的遺伝子の相対的発現は、ACTBをハウスキーピング遺伝子として用いたΔΔCq法で計算し、non-targeting コントロール(NTC)に対して正規化しました。

Figure 3. Individual CRISPRi sgRNAs can be pooled together in a single reagent to achieve enhanced target gene repression or multiplexed to enable the simultaneous repression of multiple genes.  WTC-11 human iPS cells stably expressing integrated dCas9-SALL1-SDS3 were nucleofected with synthetic sgRNA targeting PPIB, SEL1L, and RAB11A via a Lonza 96-well Shuttle system. The most active pre-designed sgRNA for each gene target was selected and used either individually or as part of a multi-target pool at a concentration of 3 µM per guide. Cells were harvested 72 hours post-nucleofection, total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative expression for each target gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to a non-targeting control (NTC). Three genes were simultaneously repressed without a substantial decrease in target gene repression or marked changes in cell viability and morphology. 


Orthogonal validation

Validated science is good science. Horizon Discovery offers a wide range of reagents that interrogate cellular pathways, allowing inactivation of a gene at the DNA level, modulating RNA transcription, or by degrading mature mRNA transcripts. We are here to help you choose the best method of gene activation or inactivation for your application. Better yet, choose two methods for robust, elegant, validated science.

CRISPRmod presents an ideal opportunity for follow-on studies after RNAi or CRISPRko screening (figure 4). Here we used CRISPRi to follow up on an arrayed siRNA screen we had previously conducted.

Synthetic CRISPRi reagents can be used to orthogonally validate hits from siRNA screens

Synthetic CRISPRi reagents can be used to orthogonally validate hits from siRNA screens
Synthetic CRISPRi reagents can be used to orthogonally validate hits from siRNA screens
Figure 8. 損傷応答アッセイ。DNA二本鎖切断に伴ってh4AX(コアヒストン複合体の構成要素)は急速にリン酸化され、DNA損傷シグナル伝達ネットワークを介して一連の応答を引き起こします。DNA損傷経路に重要なタンパク質をノックダウンすると、修復されていない二本鎖DNA切断が蓄積し、リン酸化h4AX(γ-h4AX)が増加します。以前のRNAiスクリーニングからのヒットを、CRISPRmod CRISPRi試薬を使用して直交的に検証しました。hEF1αプロモーター下でdCas9-SALL1-SDS3を構成的に発現するU2OS細胞に、DharmaFECT 4トランスフェクション試薬を使用してRPA2、RPA1、またはRRM2を標的とするプールフォーマットのsgRNA(50nM)またはON-TARGETplus SMART pools(50nM)をトランスフェクトしました。トランスフェクションの72時間後、細胞を固定し、抗リン酸化h4AX抗体で染色し、ヘキスト染色を使用して核を同定しました。複製プレートを回収し、全RNAを単離し、RT-qPCRを使用して相対的な遺伝子発現を測定しました。各標的遺伝子の相対的発現は、ハウスキーピング遺伝子としてGAPDHを用いたΔΔCq法で計算し、non-targeting コントロール(NTC)に対して正規化しました

Figure 4. Damage Response Assay. h4AX (a constituent of core histone complexes) surrounding double-strand breaks is rapidly phosphorylated, producing a set of responses through the DNA damage signaling network. Knockdown of proteins critical to the DNA damage pathway results in an accumulation of unrepaired double-stranded DNA breaks, and an increase in phosphorylated h4AX (γ-h4AX). Hits from a prior RNAi screen were orthogonally validated using CRISPRi reagents. U2OS cells constitutively expressing dCas9-SALL1-SDS3 under the hEF1α promoter were transfected with pooled sgRNAs (50nM) or ON-TARGETplus SMARTpools (50nM) targeting RPA2, RPA1, or RRM2 using DharmaFECT 4 Transfection Reagent. 72 hours post-transfection, the cells were fixed and stained with an anti-phospho-h4AX antibody, and Hoechst stain was used to identify nuclei. A duplicate plate was harvested, total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative expression for each target gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to non-targeting control (NTC).



Horizon's CRISPRmod product platform

We offer CRISPRmod reagent systems for both transcriptional repression (CRISPRi) and activation (CRISPRa). This incudes a full suite of synthetic guides and mRNA options for easy electroporation/transfection and rapid results, as well as lentiviral options for difficult-to-transfect cells or extended time course experiments. See a product page below or contact our expert scientific support team to find the best product format for your needs.

Transcriptional repression of endogenous gene expression Transcriptional activation of endogenous gene expression
dCas9 protein
dCas9-SALL1-SDS3 dCas9-VPR
  CRISPRi products CRISPRa products


Featured Resources

CRISPRmod reading list

Publications using Horizon CRISPRa or CRISPRi

Article – What is CRISPRa and CRISPRi?

All resources
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