ShRNA target prediction informed by comprehensive enquiry (SPICE): a supporting system for high-throughput screening of shRNA library.

Kenta Kamatuka,Masahiro Hattori,Tomoyasu Sugiyama

doi:10.1186/s13637-016-0039-8

Kenta Kamatuka, Masahiro Hattori + Show 1 more

Open Access

https://doi.org/10.1186/s13637-016-0039-8

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Abstract

RNA interference (RNAi) screening is extensively used in the field of reverse genetics. RNAi libraries constructed using random oligonucleotides have made this technology affordable. However, the new methodology requires exploration of the RNAi target gene information after screening because the RNAi library includes non-natural sequences that are not found in genes. Here, we developed a web-based tool to support RNAi screening. The system performs short hairpin RNA (shRNA) target prediction that is informed by comprehensive enquiry (SPICE). SPICE automates several tasks that are laborious but indispensable to evaluate the shRNAs obtained by RNAi screening. SPICE has four main functions: (i) sequence identification of shRNA in the input sequence (the sequence might be obtained by sequencing clones in the RNAi library), (ii) searching the target genes in the database, (iii) demonstrating biological information obtained from the database, and (iv) preparation of search result files that can be utilized in a local personal computer (PC). Using this system, we demonstrated that genes targeted by random oligonucleotide-derived shRNAs were not different from those targeted by organism-specific shRNA. The system facilitates RNAi screening, which requires sequence analysis after screening. The SPICE web application is available at http://www.spice.sugysun.org/.Electronic supplementary materialThe online version of this article (doi:10.1186/s13637-016-0039-8) contains supplementary material, which is available to authorized users.

Highlights

Reverse genetics approaches, which enable the determination of gene function by analyzing loss-of-function in a phenotype, have been useful for investigating the role of genes in cells and organisms [1, 2]
Evaluation of a web application The number of small interfering RNA (siRNA) target candidates was compared between shRNA target prediction informed by comprehensive enquiry (SPICE) and other sequence search engines
Evaluation of random short hairpin RNA (shRNA) library using a web application SPICE was developed for searching targets of shRNA obtained using random oligonucleotides

Summary

Introduction

Reverse genetics approaches, which enable the determination of gene function by analyzing loss-of-function in a phenotype, have been useful for investigating the role of genes in cells and organisms [1, 2]. Recent progress in whole genome sequencing and comprehensive expressed complementary DNA (cDNA) sequencing has enabled the use of systematic approaches to uncover the roles of genes that have been categorized as unknown function genes. Reverse genetics approaches, such as gene knockout with homologous recombination and gene knockdown with antisense RNA, have been highly effective; they do not yield rapid results as gene silencing using double-stranded RNA (dsRNA) under RNA interference (RNAi). The use of cDNAderived dsRNA has been limited to invertebrates because long dsRNAs (>30 nucleotides (nt)) evoke interferon responses in vertebrates This problem was solved by using small interfering RNA (siRNA) comprising ~21 nt, i.e., 19 bp with 2-nt 3′ overhangs [5]. The findings led to the development of siRNA-directed reverse genetics methods, which included

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