When studying gene function, a common approach is to reduce or block gene expression and then analyze the phenotype. For over a decade, RNA interference (RNAi) has been the king in this field. However, the emergence of new technologies, especially CRISPR technology, is gradually undermining the dominance of RNAi. The rapid development of technology provides increasing assistance to biological research, but also brings a somewhat perplexing question: "Which technology should be chosen?"
RNAi
RNAi (RNA interference) technology, a method that can apply to almost all eukaryotes, uses small double-stranded RNA (dsRNA) to efficiently and specifically degrade homologous mRNA in cells to block gene expression. RNAi only knocks down gene expression (KD).
The basic process of RNAi is as follows: The Dicer enzyme cuts the dsRNA/shRNA into siRNA fragments of 21-23 nucleotides in size. The siRNA double strand then combines with a ribozyme complex in the cytoplasm to form an RNA-induced silencing complex (RISC). The helicase unwinds the siRNA double strand into a single strand, with the guide strand in the single strand binding to the target mRNA through base complementary pairing. With ATP supply, the endonuclease slicer in RISC cuts the target mRNA, degrades it, and prevents its translation, thereby causing post-transcriptional silencing of the target gene. Two standard methods of introducing RNAi into cells are shRNA (formed by intracellular transcription) and siRNA (chemically synthesized).
Characteristics of RNAi technology
High efficiency: RNAi pathway in cells exhibits a cascade amplification effect. A minimal amount of dsRNA can produce a strong RNAi effect through the cycle of initiation stage - effect stage - amplification stage to maintain efficient interference.
Specificity: RNAi specifically degrades homologous mRNA only, leaving the expression of other mRNAs unaffected. This precise targeting ensures accurate silencing of the target gene. However, it's worth noting that the off-target effect of RNAi, particularly its ability to induce the silencing of non-target mRNAs with limited sequence complementarity, has been a subject of increasing attention since its discovery.
Position effect: The influence of double-stranded RNA (dsRNA) at various positions of the Tissue Factor (TF) on gene silencing efficiency was studied by Holen et al. The results indicate that dsRNA prefers the binding site of mRNA based on its sequence. Specifically, mRNA with lower GC content exhibits a more effective silencing effect.
Transmissibility: It can cross cell boundaries and be transmitted and maintained over long distances between different cells and even organisms, allowing RNAi to spread throughout the organism and to be transmitted to offspring.
Simplicity: A significant advantage of RNAi is that the silencing mechanism exists in almost all eukaryotes. Therefore, there is no need to manipulate the target cell line in advance genetically. Simple siRNA transfection can lead to a loss-of-function phenotype.
It seems that the RNAi mechanism is mainly active in the cytoplasm. Nuclear transcripts (such as long non-coding RNA or lncRNA) may be more difficult to target effectively. CRISPR technology can target nuclear transcripts.
CRISPRko and CRISPRi
The CRISPR system has two technologies to down-regulate gene expression: CRISPRko and CRISPRi.
CRISPRko uses Cas9 protein combined with sgRNA to generate double-stranded breaks in target DNA. When cells repair double-stranded breaks through the non-homologous end joining (NHEJ) pathway, a frameshift in the coding region will occur, making the protein produced by translation lose its function. The achieved effect is knockout. The target site for sgRNA binding should be designed in the exon region and not too close to the amino or carboxyl terminus of the protein. Whether the target site for sgRNA binding is on the sense or the antisense strand does not affect the final effect.
CRISPRi uses dCas9 protein combined with sgRNA to bind near the transcription start site and inhibit gene transcription. The achieved effect is knockdown. The KRAB protein is generally fused at the C-terminus of dCas9 to enhance the inhibitory effect. The target site for sgRNA binding should be designed near the transcription start site (TSS).
Compared with RNAi, using the same amount of effector RNA, CRISPR seems to produce more consistent and robust gene knockout/knockdown effects, and the off-target impact is much lower than that of RNAi.
The following table summarizes the similarities and differences between RNAi and CRISPR technologies.
| RNAi | CRISPRi | CRISPRko | |
| Type of Loss-of-function-phenotype | Reversible | Reversible | Irreversible |
| Experimental simplicity | Simple | Medium | Medium |
| Cost | Low | Low | Low |
| Off-target | High | Low | Low |
| Location of function | Cytoplasm | Nucleus | Nucleus |
| Functioning molecule | siRNA | dCas9+sgRNA | Cas9+sgRNA |
References
1. Boettcher M, McManus MT. Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR. Mol Cell. 2015 May 21;58(4):575-85. doi: 10.1016/j.molcel.2015.04.028. PMID: 26000843; PMCID: PMC4441801.
2. Wang F, Guo T, Jiang H, Li R, Wang T, Zeng N, Dong G, Zeng X, Li D, Xiao Y, Hu Q, Chen W, Xing X, Wang Q. A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines. Mol Genet Genomics. 2018 Jun;293(3):769-783. doi: 10.1007/s00438-018-1420-y. Epub 2018 Jan 30. PMID: 29383448.
Souce: NovoPro 2024-08-28