shRNA靶点设计（shRNA target design）

Gene knockdown

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.

shRNA

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.

Designing shRNAs

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.