Novel CRISPRi effector for gene repression research

Learn how our CRISPRi platform uses a novel dCas9 fusion protein and synthetic sgRNA to provide an improved workflow for gene characterization and arrayed screening.

Over the last decade, CRISPR-Cas9 systems have proven to be invaluable for deciphering gene function. CRISPR-Cas9 has been predominantly used in loss-of-function (LOF) studies to disrupt gene function through the creation of small insertions and deletions (INDELS) at the targeted DNA cut site. More recently, CRISPR modulation systems such as CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) have been developed which rely on nuclease deactivated Cas9 (dCas9) to bring either transcriptional activators or transcriptional repressors to a target gene to alter transcription.

The highly programmable and specific targeting of CRISPR systems have led to their widespread adoption for pooled lentiviral screening. This approach has been complementary to arrayed LOF screening, typically performed with siRNAs or small molecules. In an arrayed screen one gene is targeted per well of cells whereas in a pooled screen an assortment (library) of genes are targeted in a single population of cells. The advantages of arrayed screening include the ability to clearly identify genotype-phenotype correlations, the generation of multi-parametric data, and its suitability to short timepoint experiments.1 CRISPR-Cas9 systems have been applied to arrayed screening but there has been limited use of CRISPRi.

CRISPRi single guide RNAs (sgRNAs) are generally vector-based making it expensive and time-consuming to generate the arrayed libraries needed for large screens. Like siRNAs, sgRNAs can be chemically synthesized at high throughput to generate arrayed libraries of synthetic sgRNAs that are easily deliverable to many cell types and can be chemically modified for enhanced stability. However, performing CRISPRi with synthetic sgRNAs requires a potent dCas9 effector to ensure there is sufficient repression throughout the course of the assay to produce robust LOF phenotypes.

Horizon develops a novel fusion protein and sgRNA workflow

To improve CRISPRi technology, Mills et al. (2022) screened a number of repressor domains and determined Sal-like protein 1 (SALL1) and Sin3a corepressor complex component (SDS3) to be the most highly effective repressors. The domains were used to engineer a novel deactivated Cas9 fusion protein (dCas9-SALL1-SDS3) that mediates robust target knockdown with synthetic sgRNA. The novel protein underwent extensive evaluation of its gene repression potency against different targets, in different cell types, and compared to existing CRISPRi effectors. The workflow used synthetic sgRNA to enable arrayed screening rather than simply pooled screening.

CRISPR journal publication image
Recent publication in The CRISPR Journal authored by the Horizon R&D team.

The key findings include:

  • dCas9-SALL1-SDS3 significantly improves CRISPR-mediated target gene repression compared to dCas9-KRAB and dCas9-KRAB- MeCP2, the most widely used first- and second-generation CRISPRi effectors.
  • The use of dCas9-SALL1-SDS3 with synthetic sgRNA increases target gene repression while exhibiting high target specificity.
  • Synthetic sgRNA can be used with in vitro-transcribed dCas9-SALL1-SDS3 mRNA for short-term delivery into primary cells, including human induced pluripotent stem cells (hiPSC) and primary T cells.
  • The use of dCas9-SALL1-SDS3 orthogonally to siRNA demonstrates the ability of the system to be used in arrayed-format screening.
  • dCas9-SALL1-SDS3 was found to interact with histone deacetylase and Swi-independent three complexes, which are the endogenous functional effectors of SALL1 and SDS3.

Benefits for research

This novel effector and workflow forms the new Dharmacon CRISPRi platform. It’s designed to provide researchers with important advantages over existing workflows.

Potency and Specificity: The novel protein induces high levels of repression that exceed existing effectors. It also exhibits specificity that is comparable to dCas9-KRAB, making it a highly precise tool for LOF studies.

Application Diversity: The platform is highly effective for diverse gene targets and cell types, including therapeutically relevant cell types such as hiPSC and primary T cells.

Co-Characterization: dCas9-SALL1-SDS3 can be used orthogonally to siRNA to provide even more robust data for functional gene characterization.

Deep Data: The arrayed screening workflow provides deeper, more targeted data than pooled screening. It is also amenable to high-throughput screening which saves time and provides more data per run.

Workflow Efficiency: sgRNA production requires less time and effort than siRNA expression, saving time and allowing for efficient use of the platform for small scale experiments.


The Dharmacon CRISPRi platform is an improved loss-of-function (LOF) method for deciphering gene function.

Learn more about the Dharmacon CRISPRi platform development in this open access article in The CRISPR Journal.

Related Applications


  1. Annie Zhang Bargsten, et al. 2020. The First Step To A Successful Crispr Screen: Making The Right Choice Between Arrayed Or Pooled Library. On the Horizon.
  2. Clarence Mills, et al. 2022. A Novel CRISPR Interference Effector Enabling Functional Gene Characterization with Synthetic Guide RNAs. The CRISPR Journal. Open Access.