If you need to repress gene expression, our CRISPRi system will provide potent and robust results. Here’s how.
Introducing CRISPRmod CRISPRi for gene silencing
One of the first CRISPR interference (CRISPRi) systems comprises dCas9 fused to the Krüppel associated box (KRAB) repressor1. However, this system does not always silence genes enough for a robust phenotype to be observed. We decided to find a stronger repressor.
A better CRISPRi system: dCas9 fused to SALL1-SDS3
Our CRISPRi system utilizes a machine learning-designed guide RNA to target a deactivated Cas9 (dCas9) fused to domains from two powerful transcriptional repressors, SALL1 and SDS32, to a gene promoter region. The result: a significant decrease in expression levels of the targeted gene.
KRAB vs SALL1-SDS3
Data shows: dCas9-SALL1-SDS3 often outperforms dCas9-KRAB
Looking at 4 genes in 3 different cell types (Figure 1), dCas9-SALL1-SDS3 frequently knocks down gene expression significantly more than dCas9-KRAB, using the same guide RNAs.
Figure 1. dCas9-SALL1-SDS3 often represses gene expression more than dCas9-KRAB.
Pooled synthetic CRISPRi sgRNAs targeting BRCA1, PSMD7, SEL1L, and ST3GAL4 were delivered to K562, A375, and Jurkat cells expressing dCas9-KRAB or dCas9-SALL1-SDS3. After 72 hours, relative gene expression was measured by qPCR. All data were normalized to the corresponding non-targeting controls (NTCs).
Time Matters: Faster Knockdown, Longer Silencing
We compared the silencing of 3 genes over time, using the same sgRNAs with either dCas9-SALL1-SDS3 or dCas9-KRAB (Figure 2).
Figure 2. dCas9-SALL1-SDS3 results in stronger and longer-lasting knockdown than dCas9-KRAB.
U2OS cells stably expressing integrated dCas9-KRAB or dCas9-SALL1-SDS3 were transfected with synthetic sgRNA pools targeting CBX1, HBP1, or SEL1L. Cells were harvested at 24, 48, 72, 96, 120, and 144 hours post-transfection, and relative gene expression was measured using RT-qPCR. The relative expression of each target gene was normalized to a corresponding NTC.
Repression with dCas9-SALL1-SDS3 reaches up to a 70-80% reduction in expression levels, in contrast to dCas9-KRAB which gives around 50-70% reduction for the same 3 genes. Additionally, silencing with dCas9-SALL1-SDS3 is long-lasting: after 144 hours (6 days), expression remains around 40% of the baseline. Compare that to dCas9-KRAB-mediated silencing, where gene expression returns to 90-100% of baseline gene expression at the same timepoint (Table 1).
| Gene Silencing | dCas9-KRAB | dCas9-SALL1-SDS3 |
|---|---|---|
| 72 hour timepoint | 50-70% repression | 70-80% repression |
| 144 hour timepoint | 0-10% repression | 60% repression |
Table 1. Head-to-head comparison of gene repression over time with dCas9-KRAB and dCas9-SALL1-SDS3.
Need Extended Repression?
For those experiments requiring extended knockdown, our lentiviral all-in-one CRISPRi constructs deliver. With dCas9-SALL1-SDS3, gene expression stays repressed at 75-90% of baseline up to 2 weeks after transduction in 4 cell lines (figure 3).
Figure 3. Long term transcriptional repression with CRISPRmod All-in-one dCas9-SALL1-SDS3.
Relative mRNA expression of PPIB 7 days (left) or 14 days (right) post-transduction of HEK293T, Raji, THP1, and U2OS cells with either All-in-one NTC lentiviral particles or All-in-one lentiviral particles expressing an sgRNA targeting PPIB. The All-in-one lentiviral particles expressed PuroR for selection.
Choose the best dCas9-SALL1-SDS3 format for your experiments
Whether you’re after potent short-term or long-term knockdown, explore our game changing dCas9-SALL1-SDS3 and pre-designed CRISPRi sgRNA options.
References
- Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013 Jul 18;154(2):442-51. doi: 10.1016/j.cell.2013.06.044. Epub 2013 Jul 11. PMID: 23849981; PMCID: PMC3770145. https://pubmed.ncbi.nlm.nih.gov/23849981/
- Mills C, Riching A, Keller A, Stombaugh J, Haupt A, Maksimova E, Dickerson SM, Anderson E, Hemphill K, Ebmeier C, Schiel JA, Levenga J, Perkett M, Smith AVB, Strezoska Z. A Novel CRISPR Interference Effector Enabling Functional Gene Characterization with Synthetic Guide RNAs. CRISPR J. 2022 Dec;5(6):769-786. doi: 10.1089/crispr.2022.0056. https://pubmed.ncbi.nlm.nih.gov/36257604/