The process for drug discovery and therapeutic development heavily relies on correlating genotype to phenotype. The best way to achieve this is to disrupt gene function and analyze its phenotypic. Researchers can experimentally regulate gene expression and interrogate gene function either at the translational level or at the genetic level using two biological tools, RNAi and CRISPR, respectively.
How do these methods differ from one another? Is one more suitable than the other for certain experiments? In this article, we will answer these questions by comparing the mechanisms of action, advantages, and limitations of RNAi and CRISPR to guide you to an informed decision regarding the best technique to use in your experiments.
Overview of CRISPR and RNAi Technologies
Comparison Between CRISPR and RNAi
Choosing the Right Method for Gene Silencing
The primary difference between RNAi and CRISPR is that RNAi reduces gene expression at the mRNA level (knockdown), while CRISPR completely and permanently silences the gene at the DNA level (knockout).
As gene silencing methods, both knockouts and knockdowns have their own pros and cons. Knockouts of essential genes are lethal, providing only partial information regarding gene function in studies where the gene of interest plays a crucial role in survival of the organism. In such cases, incomplete gene knockdown can provide a better understanding of gene effect on phenotype because the effects of reducing protein levels to different extents can be studied.
Moreover, the reversible nature of knockdowns makes it possible to verify the phenotypic effect by restoring protein expression to normal in the same cells. Importantly, since a knockdown is transient, it can be a safer option than permanent genome editing. These features made RNAi an instant hit with researchers for transiently blocking gene expression.
On the other hand, knockouts are effective in completely blocking protein expression, eliminating any confounding effects from low levels protein expression remaining after knockdown. As CRISPR became popular for its ease of genetic editing, variations in the method and new versions of CRISPR-associated nucleases enabled researchers to use CRISPR for applications beyond gene knockouts.
For instance, CRISPRi allows silencing of genes without permanently knocking out the gene. This is achieved using a dead Cas9 nuclease that physically blocks RNA polymerase and inhibits gene transcription or by editing gene regulators to modulate gene expression. Although the mechanism is different from knocking out genes, the inhibition still occurs at the DNA level.
Recently, researchers have developed nuclease variants that can target RNA instead of DNA, yielding an outcome similar to RNAi.
Comparing Specificity of CRISPR and RNAi
One of the biggest limitations of the RNAi silencing method is that it suffers from high off-target effects. Silencing unintended RNA targets results in modified phenotypes and is therefore detrimental for gene function screening experiments.
The off-target effects in RNAi may be of two types: sequence-independent and sequence-dependent. For instance, multiple studies have shown that siRNAs trigger an interferon activated pathway in certain cell types in a sequence-independent manner, resulting in increased expression of interferon-regulated genes. In 2003, research showed for the first time that siRNA also targeted sequences with limited complementarity. Even today, sequence-based off-target effects remain the most challenging issue in RNAi experiments.
The CRISPR system initially also had some sequence-specific off-target effects. However, in a short timespan, the technology has advanced rapidly in multiple areas. Efficient design tools enabled finding guide RNAs that exhibit minimal off-target effects. The introduction of sgRNA and further chemically modified sgRNAs have also greatly contributed to reducing the off-target effects relative to plasmid and IVT-derived guide RNAs.
Although optimizing siRNA design, concentrations, and chemical modifications have decreased some of the off-target effects of RNAi, a recent comparative study showed that CRISPR has far fewer off-target effects than RNAi.
Thus, CRISPR is likely to continue its rapid growth and replace RNAi for most research applications, including clinical trials.
High-Throughput Genetic Screening: CRISPR vs. RNAi
Although CRISPR and RNAi differ in their mechanisms of action, both methods can independently (or in combination) be used to accomplish certain goals. A perfect example is their application in high-throughput genetic screening. The approach involves disrupting multiple genes in cells and analyzing the resulting phenotypic effects. Results are analyzed by often investigating detectable phenotypic markers such as resistance to antibiotics, activation of a fluorescent marker, or faster cell growth allowed correlating gene and function.
Before the discovery of CRISPR, RNAi was largely used to form libraries for screening gene function. But now CRISPR has emerged as an important tool in target identification and validation studies, particularly with the availability of arrayed synthetic sgRNA libraries. The arrayed format enables easy data deconvolution as compared to pool format and synthetic sgRNA achieves consistently high editing efficiencies.
Synthego’s Arrayed CRISPR Libraries enable confident screening with minimum off-targets and false negatives. Read how these libraries benefitted the lab Kaylene Simpson, Head of VCFG, Peter MacCallum Cancer Center, in this case study.
In summary, although RNAi was adopted as a gene-silencing technique first, CRISPR has several advantages over RNAi, as listed in Table.1. If you are interested in learning more about Synthego Arrayed CRISPR Libraries, feel free to reach out to us. If you are looking for further resources to better understand libraries, check out our libraries resources section.
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