Efficient Cloning and Sequence Validation of Repetitive and High GC-Content Short Hairpin RNAs.

Ramadevi Chilamkurthy, Adam A White, Adrian A Pater, Philip J Jensik, Keith T Gagnon

Human gene therapy 2022 Aug

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Short hairpin RNAs, or short hairpin RNAs (shRNAs), are a proven tool for gene knockdown and a promising therapeutic approach for suppression of disease-associated genes. The efficient preparation of shRNA-expressing vectors can sometimes become a bottleneck due to the complexity of shRNA hairpin sequence and structure, especially for repetitive or high GC-content targets. Here, we present improved shRNA cloning and validation methods that enabled efficient and rapid cloning of several shRNAs targeting disease-associated repeat expansions, including GGGGCC, CAG, CTG, CCTG, and CGG into modified pLKO.1 vectors. Improvements included shRNA insert design and preparation, recombination-based cloning, and sequencing-based validation that included Sanger and nanopore long-read sequencing. This improved method should enable practical, efficient cloning of nearly any shRNA sequence.

Citation

Ramadevi Chilamkurthy, Adam A White, Adrian A Pater, Philip J Jensik, Keith T Gagnon. Efficient Cloning and Sequence Validation of Repetitive and High GC-Content Short Hairpin RNAs. Human gene therapy. 2022 Aug;33(15-16):829-839