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

In small RNA (smRNAs) 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 gelseparation, 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 sgRNAs targeting adapter dimer ligation products, alongside highly expressed tissue-specific smRNAs, for cleavage in vitro. This process dramatically reduces (>90%) adapter dimer and targeted smRNA sequences, is multiplexable, 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 nontargeted sequences.