CRISPR/Cas9-targeted removal of unwanted sequences from small-RNA sequencing libraries

作者：Andrew A. Hardigan, Brian S. Roberts, Dianna E. Moore, Ryne C. Ramaker, Angela Jones, R Myers · 发表于：Nucleic Acids Research · 年份：2019 · DOI：10.1093/nar/gkz425 · 被引用次数：42 · 研究领域：CRISPR and Genetic Engineering、MicroRNA in disease regulation、Advanced biosensing and bioanalysis techniques

In small RNA (smRNA) sequencing studies, highly abundant molecules such as adapter dimer products and tissue-specific microRNAs (miRNAs) inhibit accurate quantification of lowly expressed species. We previously developed a method to selectively deplete highly abundant miRNAs. However, this method does not deplete adapter dimer ligation products that, unless removed by gel-separation, comprise most of the library. Here, we have adapted and modified recently described methods for CRISPR/Cas9-based Depletion of Abundant Species by Hybridization ('DASH') to smRNA-seq, which we have termed miRNA and Adapter Dimer-DASH (MAD-DASH). In MAD-DASH, Cas9 is complexed with single guide RNAs (sgRNAs) targeting adapter dimer ligation products, alongside highly expressed tissue-specific smRNAs, for cleavage in vitro. This process dramatically reduces adapter dimer and targeted smRNA sequences, can be multiplexed, shows minimal off-target effects, improves the quantification of lowly expressed miRNAs from human plasma and tissue derived RNA, and obviates the need for gel-separation, greatly increasing sample throughput. Additionally, the method is fully customizable to other smRNA-seq preparation methods. Like depletion of ribosomal RNA for mRNA-seq and mitochondrial DNA for ATAC-seq, our method allows for greater proportional read-depth of non-targeted sequences.