shRNA vs gRNA:
RNAi technology used to be popular in biological research, especially in high-throughput genetic screening, gene function study, clinical treatments for antivirus and cancers. However, the RNAi technology has been questioned because of its off-target effect. In recent years, the emerging CRISPR gene editing technology is widely used in almost every biological field. Because of its high specificity, low off-target effect and wide application prospects, CRISPR is replacing RNAi as the best choice for most research.
Comparing RNAi & CRISPR:
|Result||Reversible knockdown||Complete and permanent knockout|
|Ease of experiment||Easy||Moderate|
|Off target effect||High||Low|
|Efficiency of silencing||Low||High|
|Range of targeting||mRNA, cytoplasmic IncRNA, and circRNA (ineffective)||Coding genes, non-coding genes, cytoplasmic and intranuclear IncRNA and circRNA.|
What RNAi might cost you?
One of the biggest limitations of the RNAi silencing method is that it suffers from High off-target effects. RNAi might silence unintended RNA targets resulting in modified phenotypes and therefore influence gene function screening experiments. There are three reasons for this high off-target effect:
· siRNA may compete with the intrinsic microRNA in RISC complex, resulting in affecting the function of microRNA and the expression regulation of other genes;
· siRNA also targeted sequences with limited complementarity;
· siRNA may interact with the 3'UTR of unintended RNA, leading to its degradation.
Other than high-off target effect, issues that researchers are facing with are as followings:
· shRNA fails to show any reduction in protein expression level;
· No suppression but overexpression of target genes;
· Transcripts with high turnover are difficult to silence;
· Non-coding sequences are difficult to silence.
Researchers conducted parallel experiments with shRNA libraries and CRISPR gRNA libraries, which proved that the efficiency and reliability of using gRNA libraries to screen genes were much higher than that of shRNA libraries.
In addition to high efficiency and high true positive rate, the off-target effect of CRISPR is very low. Because its specificity depends on two aspects, one is the base pairing between gRNA and target DNA, the other is that the Cas9-gRNA complex only binds to the PAM sequence in the genome. Only when these two conditions are met at the same time can Cas9 cause DSB.
Although RNAi was adopted as a gene silencing technique first, CRISPR has surpassed RNAi in popularity due to several advantages made possible by advancements that have refined CRISPR technology. The main reason for the popularity of CRISPR is it's specific yet versatile nature. At the same time, the choice remains with the user to perform knockouts, knock-ins, or knockdown experiments, making CRISPR extremely versatile.
Ubigene has an advanced platform for molecular and cellular experiments. Our experienced technical team can design high-score shRNA and high-efficiency gRNA for you to meet the needs of your RNAi and CRISPR knockout experiments.
· gRNA and shRNA clones of different species;
· Virus packaging for gRNA or shRNA, including lentiviruses, adenoviruses and AAVs;
· Custom stable cell lines with gene knockout or knockdown;
· Gene knockout and knockdown zebrafish, and related downstream analytical services.
· Combined shRNA over CRISPR/cas9 as a methodology to detect of-target efects and a potential compensatory mechanism
· CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes
· Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes
· Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR