- CRISPR interference reagents
- dCas9-SALL1-SDS3 mRNA
CRISPRmod CRISPRi dCas9-SALL1-SDS3 mRNA
A lentiviral-free option for dCas9-SALL1-SDS3 expression
Purified dCas9-SALL1-SDS3 mRNA for co-transfection or electroporation with synthetic CRISPRi sgRNA for repression of targeted gene transcription

CRISPRi dCas9-SALL1-SDS3 mRNA expresses a human codon-optimized version of the nuclease-deactivated S. pyogenes Cas9 gene, fused to our proprietary transcriptional repressors (SALL1 and SDS3). When paired with a well-designed guide RNA that targets a gene near the native transcription start site, expression is repressed.
Review our CRISPRi applications page to get an overview of the technology!
Highlights of using CRISPRi dCas9-SALL1-SDS3 mRNA
- No need to generate stable dCas9-SALL1-SDS3-expressing cell lines
- Simply co-transfect CRISPRi synthetic sgRNA and dCas9-SALL1-SDS3 mRNA using DharmaFECT Duo transfection reagent or co-electroporate into cells.
- Co-expression of either EGFP or puromycin resistance allows for easy transfection optimization or enrichment using FACS or antibiotic selection.
- No need for optimal promotor selection, as mRNA is ready for translation
Requirements for a CRISPRi experiment using dCas9-SALL1-SDS3 mRNA
- CRISPRi dCas9-SALL1-SDS3 mRNA
- CRISPRi synthetic sgRNA for your target gene (see Workflow tab)
CRISPRi workflow using synthetic sgRNA and dCas9-SALL1-SDS3 mRNA
Co-transfect or electroporate cells with CRISPRi synthetic sgRNA and CRISPRi dCas9-SALL1-SDS3 mRNA. Then enrich cell populations using fluorescence or puromycin resistance options. This system is best suited for rapid, transient gene repression studies.
Robust, transient gene repression with CRISPRi dCas9-SALL1-SDS3 mRNA and CRISPRi synthetic sgRNA

K562 and Jurkat cells were nucleofected with dCas9-KRAB or dCas9-SALL1-SDS3 mRNA (2 µg), and pooled CRISPRi synthetic sgRNAs (5 µM) targeting PPIB and SEL1L via a Lonza 96-well Shuttle system. WTC-11 hiPS cells were nucleofected with dCas9-KRAB or dCas9-SALL1-SDS3 mRNA (1 µg) and pooled CRISPRi synthetic sgRNA (3 µM) targeting PPPIB or SEL1L via a Lonza 96-well Shuttle system. Cells were harvested 72 hours post-nucleofection. Total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative gene expression for each target gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control (NTC).
Co-transfection of dCas9-SALL1-SDS3 mRNA and CRISPRi synthetic sgRNA provides greater gene repression than transfection into a stable dCas9-SALL1-SDS3 expressing cell line
Data was compiled from multiple experiments and matched for the time point of analysis and delivery method. U2OS cells stably expressing integrated dCas9-SALL1-SDS3 were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with synthetic sgRNA (25 nM) targeting SEL1L, parental U2OS cells were plated at 10,000 cells/well and co-transfected using DharmaFECT Duo Transfection Reagent with dCas9-SALL1-SDS3 mRNA (200 ng) and CRISPRi synthetic sgRNA (25 nM) targeting SEL1L. Jurkat cells stably expressing integrated dCas9-SALL1-SDS3 were nucleofected with CRISPRi synthetic sgRNA (5 µM) targeting SEL1L, parental Jurkats cells were nucleofected with dCas9-SALL1-SDS3 mRNA (2 µg), and CRISPRi synthetic sgRNA (5 µM) targeting SEL1L via a Lonza 96-well Shuttle system. WTC-11 human iPS cells stably expressing integrated dCas9-SALL1-SDS3 were nucleofected with CRISPRi synthetic sgRNA (3 µM) targeting SEL1L, parental WTC-11 hiPS cells were nucleofected with dCas9-SALL1-SDS3 mRNA (1 µg) and CRISPRi synthetic sgRNA (3 µM) targeting SEL1L via a Lonza 96-well Shuttle system. U2OS cells were harvested 48 hours post-transfection, Jurkat and hiPS cells were harvested 72 hours post-nucleofection. Total RNA was isolated and relative gene expression was measured using RT-qPCR. The relative gene expression for SEL1L was calculated with the Cq method using GAPDH or ACTB as the housekeeping gene and normalized to a non-targeting control (NTC).
CRISPRi dCas9-SALL1-SDS3 mRNA co-expressing PuroR allows for enrichment using puromycin selection
K562 cells were nucleofected with Puro dCas9-SALL1-SDS3 mRNA (2 µg), and pooled CRISPRi sgRNA targeting BRCA1, PPIB, CD46, PSMD7, SEL1L, or ST3GAL4 via a Lonza 96-well Shuttle system. At 24 hours post-nucleofection 2 µg/ml of puromycin growth media was added to the cells and a duplicate plate received normal growth media. At 48 hours post-nucleofection 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).WTC-11 hiPS cells were plated at 5,000 cells per well in clear 96-well plates. After 24 hours, cells were co-transfected with CRISPRi PuroR dCas9-SALL1-SDS3 mRNA (200 ng) and pooled CRISPRi synthetic sgRNA (25nM) targeting either PPIB or ST3GAL4 using DharmaFECT Duo Transfection Reagent. At 16 hours post-transfection, 2 µg/ml of puromycin growth media was added to the cells and a duplicate plate received normal growth media. At 24 hours post-transfection the puromycin containing growth media was removed from the cells and replaced with antibiotic-free growth media. At 72 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 ACTB as the housekeeping gene and normalized to a non-targeting control (NTC).
CRISPRi dCas9-SALL1-SDS3 mRNA co-expressing EGFP allows for FACS enrichment
HCT-116 cells were plated at 400,000 cells per well in clear 6-well plates. After 24 hours, cells were co-transfected with EGFP dCas9-KRAB or EGFP dCas9-SALL1-SDS3 mRNA (2.5 µg), and pooled CRISPRi synthetic sgRNA targeting PPIB, PSMD7, or SEL1L (25 nM) using DharmaFECT Duo Transfection Reagent. At 24 hours post-transfection cells were trypsinized and FACS was performed to sort the cells into two categories: GFP negative (GFP Neg), and top 10% GFP expressing (Top 10%). Following sorting, cells were replated in 6-well dishes and allowed to recover. Cells were harvested after 24 hours of recovery, 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).
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