RNAi and CRISPRi: On-target knockdown leads to high-confidence datasets

The presence of off-target effects can greatly complicate the interpretation of experimental data. Dharmacon™ ON-TARGETplus™ siRNAs and CRISPRmod CRISPRi system are designed to knock down the target gene with minimal off-target effects and are powerful tools for orthogonal validation strategies resulting in highly specific readouts. 

Researchers are aware of off-target effects caused by an introduction of unnatural components into the cell. However, those can be minimized and there are various strategies to distinguish them from a true phenotype. 

RNAi has a broad spectrum of applications in single assay as well as in screening formats. It has been extensively characterized and optimized by us for decades. Recently, the Dharmacon product family has expanded to include novel CRISPRmod CRISPRi technology reagents, targeting specific locations and blocking transcription without cutting the DNA. This opens another door for target specific knockdown at the transcriptional DNA level, in addition to RNAi reagents controlling the mRNA expression level and thereby preventing translation. 

As we recommend orthogonal validation strategies regarding multiple cellular expression machineries, the combined usage of specific gene silencing methods provides a profound hit validation. 

Addressing the specificity of both techniques, we have performed a comparative off-target signature gene expression analysis of our novel CRISPRmod CRISPRi and well known ON-TARGETplus reagents by targeting SEL1 in USOS cells. The gene expression levels (log2TPM, Transcripts Per Million) of SEL1 knockdown was measured and compared to respective non-targeting controls. 

Despite the differences in the mode of action in both techniques, our CRISPRi and RNAi reagents measurements turned out being highly specific as shown by remarkably Pearson´s correlation coefficients in both gene expression signatures (Figure 1).

Gene expression levels data
Figure 1: Gene expression data for CRISPRi and siRNA experiments
Gene expression levels (log2TPM, Transcripts Per Million) in U2OS cells stably expressing dCas9-SALL1-SDS3 (left) or parental U2OS cells (right) 48 hours post-transfection of a pool of three guide RNAs or a pool of four siRNAs targeting SEL1L (y axis) compared to expression in cells transfected with non-targeting guide RNAs or siRNAs only (x axis). R is Pearson's correlation coefficient, calculated for log transformed values (TPM+1) on all genes except SEL1L. Up-regulated or down-regulated genes with an adjusted p-value < 0.05 and absolute log2 fold change >2 are marked as red and blue, respectively. Differential expression analysis (DESeq) shows minimal off-target gene knockdown with both the dCas9-SALL1-SDS3 CRISPRi system and ON-TARGETplus siRNA. Examination of genomic sequences within 10,000 kb of the target gene and within 1 kb of an off-target guide RNA alignment of up to 4 mismatches indicated the observed differential expression could not be explained by neighborhood effects or nonspecific binding of the guide RNA.

Based on this, we have performed a companion DNA Damage Response Assay suggesting that both methods can also be used for orthogonal validation steps. Replication protein A (RPA) consists of RPA1, RPA2, and RPA3 subunits, described to have a high binding affinity to single-stranded DNA and fulfilling key regulatory functions in the DNA damage response1,2 (Figure 2). 

 
DNA damage response pathway
Figure 2: DNA damage response pathway
Double strand breaks arise as a result of DNA damaging agents or spontaneously as a result of a variety of cellular processes including metabolism, recombination and transposition. Double strand breaks are also created when DNA replication forks stall or approach a nick in template DNA. H2AX (a constituent of core histone complexes) surrounding double strand breaks are rapidly phosphorylated, inducing a set of responses through the DNA damage signaling network, including DNA repair, cell cycle arrest and apoptosis. The RPA complex binds single stranded DNA in both DNA replication and homologous recombination repair structures. RPA knockdown destabilizes replication forks and inhibits homologous recombination repair, resulting in an accumulation of double strand breaks and hence phosphorylated H2AX1.

As expected, the knockdown of RPA1 and RPA2 resulted in an accumulation of unrepaired double strand DNA breaks, which can be monitored via an increased phosphorylated H2AX level (γ-h2AX). Both gene modulation tools showed confirming results in comparison of respective wild-type controls (Figure 3).

DNA Damage Response Assay
Figure 3: DNA Damage Response Assay
H2AX (a constituent of core histone complexes) surrounding double strand breaks are rapidly phosphorylated, inducing a set of responses through the DNA damage signaling network. The knockdown of proteins critical to the DNA damage pathway results in an accumulation of unrepaired double stranded DNA breaks and an increase in phosphorylated H2AX (γ-h2AX). Hits from a prior RNAi screen were orthogonally validated using CRISPRmod 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 antiphospho-H2AX antibody and Hoechst stain was used to identify nuclei. DNA Damage Response Assay was performed with a cellular kit. 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 a non-targeting control (NTC).

These results corroborate the specificity of both gene modulation techniques by targeted repression of DNA transcription (CRISPRi) and mRNA translation (RNAi). Conclusively, the expression of functional proteins can be down-regulated from multiple angles, leading to high- confidence datasets. 
We offer a broad spectrum of our novel CRISPRi reagents besides our widely used RNAi reagents. Horizon´s Scientific Support is a team made up of trained scientists supporting gene editing and gene modulation technologies. We’re happy to discuss your project in English, French, German, Italian, or Spanish; and have teams in both the US and Europe to provide flexible service hours. 

CRISPRi resources

Welcome to the toolbox, CRISPRi - Blog article
CRISPR-mediated transcriptional repression with a novel dCAS9 fusion protein and synthetic guide RNAs - Poster

RNAi resources

Reducing off-target effects in RNA interference experiments – Blog article
Ensure success with appropriate controls in your RNAi experiments – Blog article
siRNA screening: development of hit stratification strategies - Poster
Off-target effects: disturbing the silence of RNA interference (RNAi) – Application note
Dharmacon publications (see RNAi specificity & functionality) – Recommended Reading

 

Jan Korte photo
Written by Jan Korte, Ph.D. , Scientific Support Specialist 2 
Jan has been part of the Scientific Support team since 2017. He is driven by good scientific practice, and energized to see strong data sets supported by a broad spectrum of orthogonal validation strategies. Fascinated by Horizon´s Dharmacon™ RNAi and CRISPR gene editing reagents, as well as the recently developed CRISPRmod family of gene modulation tools (CRISPRa and CRISPRi), he enjoys guiding customers in the combined usage of these different techniques.  Outside of his scientific pursuits, Jan loves exploring new gravel bike tracks and having quality time with friends and family.
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