The TRC shRNA Design Methods and Rules
Overview
We design shRNA molecules with an algorithm. Our algorithm uses several criteria to rank potential 21mer targets within each human and mouse Refseq transcript. The algorithm applies a set of rules, including those derived from the siRNA literature, our cloning scheme, constraints on the synthesis of the oligonucleotides and others. In applying the algorithm, our aim is to achieve a balance of two competing goals: make hairpins that effectively knockdown the target transcript and, as best possible, design hairpins that knockdown only one gene and not other so-called 'off-target' genes. Each goal presents distinct challenges. The criteria for predicting effective knockdown with either siRNA or shRNA are not well understood. Our rules are primarily derived from the siRNA literature; how well these rules apply to shRNA design is unclear. Genome evolution constrains target specificity. Many genes are part of extensive gene families, which may make targeting any one gene difficult. Functionally distinct genes share many motifs. Our knowledge of transcript structure and variants is still very incomplete as well. For all these reasons and more, we construct 5 shRNAs for each transcript with the expectation of getting a range of knockdown efficiencies across the set and at least one or two which knockdown effectively.
Users of this database should be aware that in order to have consistent and reliable annotation, the TRC consortium decided early on to use NCBI's REFSEQ collection of transcripts as the definitive source of information for the primary target sequence for the design of shRNA molecules.
As a general rule in the construction of the library, we construct shRNA molecules targeting just the first Refseq transcript reported from each NCBI gene. In part due to our design process, see below, the majority of the shRNAs target all known transcript variants.
A brief narrative of the candidate selection process
Get the Candidate Sequences
For each human and mouse Refseq transcript, we generate all 21mers starting 25 bp after the beginning of the CDS up to those starting 150 bp from the end of the transcript. Each 21mer is called a 'candidate'.
Score the Candidate Sequences For Knockdown Efficiency
Each candidate is given an "original score" by applying a set of rules that either penalize or reward features predicting successful knockdown and clone-design considerations, and then calculating the product of all the penalties/rewards. The individual rules are listed below. The candidates are then sorted by score and all those above a minimum score are stored.
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