shRNA靶点设计（shRNA target design）
VectorBuilder’s shRNA Target Design tool allows you to design short hairpin RNAs (shRNAs) with high knockdown scores to help you achieve highly efficient knockdown of your genes of interest (GOIs).
VectorBuilder applies rules similar to those used by the RNAi consortium (TRC) to design and score shRNAs. For each given RefSeq transcript, we search for all possible 21mers that are considered as candidate target sites. Candidates are excluded if they contain features thought to reduce knockdown efficiency/specificity or clonability, including a run of ≥4 of the same base, a run of ≥7 G or C, GC content <25% or >60%, and AA at the 5’ end. Knockdown scores are penalized for candidates that contain internal stem-loop, high GC content toward the 3’ end, known miRNA seed sequences, or off-target matches to other genes. For genes with alternative transcripts, target sites that exist in all transcripts are given higher scores.
All scores are ≥0, with mean at ~5, standard deviation at ~5, and 95% of scores ≤15. An shRNA with a knockdown score about 15 is considered to have the best knockdown performance and clonability, while an shRNA with a knockdown score of 0 has the worst knockdown performance or is hard to be cloned.
RNA interference (RNAi) has been a widely utilized method of gene modulation for many decades. Short RNA sequences (approximately 21-23 nucleotides) complementary to the RNA of a gene of interest are introduced into target cells. The exogenous RNA strand binds to the complementary endogenous mRNA strand. The cell degrades the double-stranded RNA and translation does not occur, knocking down the performance of the gene. It is important to note that this approach does not completely knock out the gene, as some mRNAs will not be bound and will produce functional protein.
One common RNAi approach utilizes short hairpin RNAs (shRNAs). Here the sequence is designed such that a single transcript folds back on itself and hybridizes, forming a hairpin. This double-stranded RNA complex with its internal loop is transported into the cytoplasm, where it is processed by Dicer and is loaded into the RISC complex. The RISC complex facilitates binding between the silencing RNA and the target mRNA, at which point the mRNA will be cleaved or degraded (Figure 1).
Figure 1. Production and function of forms of RNAi.
Why choose shRNA?
The formation of a hairpin from a single transcript allows for customization in introduction, duration, and regulation of silencing. The plasmid coding for the shRNA can be transfected (e.g. through electroporation or microinjection) or transduced (using viruses such as lentivirus or adenovirus). Because of the option to use viral transduction, shRNA vectors can be tagged and/or integrated into the genome using virus machinery and can be introduced to a wide range of cell types.
VectorBuilder’s shRNA Design tool allows you to input your sequence and receive a list of all possible shRNA sequences in order of knockdown score. Our algorithm takes each 21mer (every sequence of 21 base pairs) and determines (1) its clonability and (2) its specificity. Clonability is influenced by the order and distribution of nucleotides. Repeats, GC content that is too high or too low, and formation of stem loops within the shRNA sequence decrease stability and therefore knockdown score. Specificity ensures that only the target gene is affected, so each candidate sequence is compared to the host genome. If there are regions outside of the gene of interest where the shRNA may bind, the knockdown score is decreased. High knockdown scores theoretically reflect high performance of the shRNA, but some unpredicted interactions can occur, reducing the experimental efficiency. Because there is not a guarantee that the theoretical outcomes completely match the experimental outcomes, journals typically require confirmation of shRNA knockdown using two different shRNA sequences. To maximize the changes of having two shRNA sequences with high targeting efficiency, we offer a 3+1 service, where three shRNA sequences and one control (scramble) sequence is provided.
- Sequences in both GenBank and FASTA formats can be recognized.
- Some returns may target the 3’ UTR, but this region can be as effective in knockdown as the coding region.
- Knockdown scores should be validated experimentally, as actual efficiency could depart significantly from what the scores predict. For this reason, it is recommended to test at least 3+1 service to increase the likelihood of success for effective knockdown using two sequences.