CRISPR gene-editing | Best Choice for Research
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
· 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.