What is the Difference Between CRISPR and RNAi

The main difference between CRISPR and RNAi is that CRISPR takes part in gene knockout whereas RNAi takes part in gene knockdown. Furthermore, CRISPR interferes with the DNA sequence while RNAi interferes with mRNA.

CRISPR and RNAi are two types of approaches used in gene silencing in various types of biotechnological experiments.

Key Areas Covered

1. What is CRISPR
     – Definition, Mechanism, Importance
2. What is RNAi
     – Definition, Mechanism, Importance
3. What are the Similarities Between CRISPR and RNAi
     – Outline of Common Features
4. What is the Difference Between CRISPR and RNAi
     – Comparison of Key Differences

Key Terms

CRISPR, Gene Knockdown, Gene Knockdown, Gene Silencing, RNAi

What is CRISPR

CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences that naturally occur in the genome of prokaryotes including bacteria. These repeats are derived from the viruses that infect prokaryotes. Therefore, they can be used to recognize similar DNA sequences, destroying similar DNA sequences from viruses in the subsequent infections. Thus, CRISPR becomes an antiviral defense system in prokaryotes. Here, an enzyme known as Cas9 (CRISPR-associated protein 9) uses CRISPR as a guide sequence to recognize complementary strands and then, it cleaves the complementary sequence.

Figure 1: CRISPR-Cas9 as a Molecular Tool Introduces Targeted Double-Strand DNA Breaks

However, the CRISPR-Cas9 system is used as a genome-editing tool to develop biotechnological products and to treat genetic disorders. Here, the process changes the genetic code, resulting in knocking out genes. This permanently silences the gene, completely eliminating its function. For that, site-specific, 20 nucleotides, single guide RNA (sgRNA) is used to recognize and bring Cas9 to the target locus. Then, Cas9 cleaves both ends of the DNA, resulting in a double-stranded break.

Figure 2: Genome Editing by CRISPER-Cas9

After that, the two strands can be rejoined either through non-homologous end joining (NHEJ) or homologous recombination (HR) to insert donor DNA in between the two ends. Both NHEJ and HR result in knocking out genes.

What is RNAi

RNAi (RNA interference) is a biological process which regulates gene expression at the post-transcriptional level through degrading target mRNA. It is one of the most widely used approaches to study gene function in reverse genetics. Here, the two main types of small RNA molecules involved in the process are micro RNA (miRNA) and small interfering RNA (siRNA). Another form of small RNA involved in RNAi, mimicking the function of miRNA is short hairpin RNA (shRNA). However, shRNA has to be artificially introduced to the system through delivery systems. Both miRNA and shRNA form double-stranded RNA by hybridization with the target mRNA, which is complementary to the small RNA sequence.

Figure 3: RNAi

Then, an enzyme known as Dicer binds with the RNA duplex, cleaving it into small, double-stranded RNA complexes of 20-25 nucleotides in length. These small complexes are known as siRNA, which binds to another complex named RISC (RNA-induced silencing complex). Finally, the catalytic component of the RISC known as Ago2 (Argonaute 2) cleaves the mRNA strand in the siRNA duplex. Therefore, this process is responsible for inhibiting gene expression. Therefore, it is possible to use RNAi to silence genes temporarily at the RNA level. Hence, it becomes a tool for knocking down genes. More importantly, the loss of function here is reversible.

Similarities Between CRISPR and RNAi

• CRISPR and RNAi are two approaches used in gene silencing experiments in biotechnology.
• The main function of them is to stop gene expression.
• Besides, they are important in the study of the function of genes and for treating genetic disorders.

Difference Between CRISPR and RNAi

Definition

CRISPR refers to the hallmark of a bacterial defense system that forms the basis for CRISPR-Cas9 genome editing technology while RNAi refers to a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Thus, this is the fundamental difference between CRISPR and RNAi.

Found in

Another difference between CRISPR and RNAi is that the CRISPR system naturally occurs in prokaryotes while RNAi naturally occurs in many eukaryotes.

Significance

Above all, the main difference between CRISPR and RNAi is that the CRISPR is a genome editing technology involved in the knocking out of genes while RNAi is a form of post-transcriptional regulation of gene expression involved in the knocking down of gene expression.

Applicability

Moreover, CRISPR is applicable at the DNA level while RNAi is applicable at the RNA level. Hence, this is also a difference between CRISPR and RNAi.

Duration

Furthermore, another difference between CRISPR and RNAi is that the CRISPR silences genes permanently while RNAi silences genes temporarily.

Cost

Also, while CRISPER associates with high cost, RNAi associates with low cost.

Sensitivity

The off-target effects in CRISPR are low while RNAi associates with high rates of off-target effects. This is also a difference between CRISPR and RNAi.

Conclusion

CRISPR is a genome-editing tool responsible for the knockout of genes. It is applicable at the DNA level and brings a permanent gene silencing effect. In comparison, RNAi is a cellular mechanism used for the regulation of gene expression at the post-transcriptional level. Therefore, it is applicable at the RAN level and it temporarily knocks down gene expression by degrading mRNA. Therefore, the main difference between CRISPR and RNAi is the type of effect of gene silencing brought by each approach.

References:

1. Davis, E D. “Knockout by TALEN or CRISPR vs. Knockdown by ShRNA or SiRNA.” Genecopoeia, GeneCopoeia, Inc., 2014, Available Here.

Image Courtesy:

1. “15 Hegasy Cas9 DNA Tool Wiki E CCBYSA” By Guido4 – Own work (CC BY-SA 4.0) via Commons Wikimedia  
2. “16 Hegasy DNA Rep Wiki E CCBYSA” By Guido4 – Own work (CC BY-SA 4.0) via Commons Wikimedia  
3. “RNAi-simplified” By This figure is adapted from one by Matzke MA, Matzke AJM – This figure is adapted from one by Matzke MA, Matzke AJM (2004) Planting the Seeds of a New Paradigm. PLoS Biol 2(5): e133 doi:10.1371/journal.pbio.0020133. (CC BY 2.5) via Commons Wikimedia