CRISPRi dCas9-SALL1-SDS3

CRISPRmod CRISPR interference (CRISPRi) allows researchers to down regulate specific gene function by blocking transcription, without editing the DNA. In the Horizon CRISPRi system, a deactivated Cas9 nuclease (dCas9) is fused at the C-terminal end to two transcriptional repressors (SALL1 and SDS3). Visit our CRISPRi applications page to learn more.

CRISPRi dCas9-SAL1-SDS3 is available in mRNA and lentiviral formats.

  • CRISPRi dCas9-SALL1-SDS3 mRNA
    Co-transfect or electroporate with CRISPRi synthetic sgRNA, then enrich cell populations with fluorescence or antibiotic resistance selection options. This format is best suited for rapid, transient gene repression studies.
  • CRISPRi dCas9-SALL1-SDS3 lentiviral particles
    Generate your own stable populations of dCas9-SALL-SDS3 expressing cells, which can then be delivered either synthetic or lentiviral CRISPRi sgRNA. This format is ideal for screening or for extended time point assays.

CRISPRi for gene repression

CRISPR-Cas9 systems are not only for creating genomic double-strand breaks, it can also be used to target promoter regions to inhibit transcription. Endogenous expression of a gene can be specifically and efficiently down regulated using dCas9-SALL1-SDS3 in combination with a gene specific guide RNA in either a synthetic or lentiviral format.

Optimization and enrichment

Enrichment for gene interference can be accomplished using dCas9-SALL1-SDS1 mRNA reagents that co-express either EGFP or PuroR (puromycin resistance) with dCas9-SALL1-SDS3. mRNA can be co-transfected or electroporated along with CRISPRi synthetic guide RNA, enabling visualization of successful delivery and increased knockdown levels.

Promoter options

Promoter activity varies by cell type. Therefore, choosing an optimal promoter for your cells is important for robust gene knockdown. Purified high-titer dCas9-SALL1-SDS3 lentiviral particles are available with three different promoter options (hCMV, mCMV, or hEF1a).

SMARTchoice promoter options for expressing dCas9-SALL1-SDS1 
Promoter Description
hCMV human cytomegalovirus immediate early promoter
mCMV mouse cytomegalovirus immediate early promoter
hEF1α human elongation factor 1 alpha promoter
CRISPRi dCas9-SALL1-SDS3 mRNA

Purified dCas9-SALL1-SDS3 mRNA for transient expression. Fluorescent and puromycin resistance options available for sorting, enrichment, or visualization of delivery.

CRISPRi dCas9-SALL1-SDS3 lentiviral particles

Purified lentiviral particles for generation of stable dCas9-SALL1-SDS3 expressing cell populations.

 

CRISPRi using dCas9-SALL1-SDS1 shows improved gene repression with greater consistency over time when compared to dCas9-KRAB
CRISPRi gene repression is observed 24 hours post-transfection and is maximal 48-72 hours post transfection
CRISPRi遺伝子転写抑制はトランスフェクションの24時間後に観察され、トランスフェクションの48〜72時間後に最大になる
CRISPRmod CRISPRi化学合成sgRNAは、dCas9-KRABおよびdCas9-SALL1-SDS3発現細胞の双方で、トランスフェクション後48〜72時間で最大の抑制を達成します。dCas9-KRABまたはdCas9-SALL1-SDS3を安定して発現するU2OS細胞を、10,000細胞／ウェルでプレーティングし、DharmaFECT 4トランスフェクション試薬を使用して、CBX1、HBP1、またはSEL1Lを標的とするデザイン済みCRISPRi化学合成sgRNA（25nM）のプールフォーマットでトランスフェクトしました。トランスフェクションの24、48、72、96、120、および144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、ハウスキーピング遺伝子としてGAPDHを用いた∆∆ Cq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。

CRISPRi synthetic sgRNAs achieve maximal repression at 48-72 hours post-transfection in both dCas9-KRAB and Cas9-SALL1-SDS3-expressing cells. U2OS cells stably expressing integrated dCas9-KRAB or dCas9-SALL1-SDS3 were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with pools of pre-designed synthetic sgRNA (25nM) targeting CBX1, HBP1, or SEL1L. Cells were harvested at 24, 48, 72, 96, 120, and 144 hours post-transfection, total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative expression of each target gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control (NTC).