Ribosomal RNA depletion from bacteria for RNA-seq using CRISPR-associated Cas9

Background

Ribosomal RNA (rRNA) accounts for ~90% of total cellular RNA. If your study is not researching rRNA, the abundance of these molecules can make it difficult to sequence RNA of interest. In eukaryotic organisms, the first step of RNA sequencing (RNA-seq) after RNA extraction is ribosomal depletion, which usually selectively captures the non-rRNA sequences by using oligonucleotides to anneal to the polyadenylated tails of messenger RNA (mRNA). While this method is useful for general RNA-seq of eukaryotic species, the mRNA of prokaryotes lacks the required polyadenylation. This type of rRNA depletion can also be insufficient for degraded DNA like that derived from formalin-fixed, paraffin-embedded (FFPE) samples. Ribosomal depletion of organisms like bacteria and viruses requires other methods.

Prezza et al. adapted a method called Depletion of Abundant Sequences by Hybridization (DASH) that involves DNA cleavage using CRISPR-associated Cas9. “Hybridization” in the method name refers to the hybridization of Cas9-bound guide RNA (gRNA) with target DNA. DASH uses in vitro Cas9 nuclease, directed by gRNA, to cleave and destroy high-abundance, unwanted cDNA sequences, while leaving desirable cDNA sequences intact. This study applies the DASH method to deplete bacterial cDNA derived from rRNA sequences in preparation for RNA-seq.

Experiment

Prezza et al. isolated RNA from 2 bacterial species using TRIzol™ reagent. DNase I removed genomic DNA, and RNA quality was confirmed using an Agilent 2100 Bioanalyzer™ (Agilent Technologies). Target sequences of rRNA genes were chosen using a custom Python script (available on Github) and filtered based on GC content in addition to other criteria. The custom target sequences were ordered as oPools™ Oligo Pools from Integrated DNA Technologies, Inc., (IDT) and used to generate double-stranded DNA (dsDNA) templates. Single guide RNA (sgRNA) pools were derived from the dsDNA templates. Total RNA was used to generate cDNA before performing DASH, that cleaved rRNA-derived cDNA fragments. The resulting cDNA libraries were amplified to further enrich for the regions of interest and sequenced on Illumina systems before being processed and analyzed.

“We were very satisfied with our oPools Oligo Pools, especially for their fast turnaround time and high output amount. We also sequenced the gRNAs transcribed from the DNA template pool that we ordered and all of the expected sequences were there.”

—Dr Gianluca Prezza

Results

The researchers were able to deplete rRNA from both bacterial species, and they optimized depletion efficiency by increasing the number of sgRNAs and their concentrations. Commercial kits typically are unable to remove rRNA from very low amounts of total RNA. A key feature of the DASH method is that it is performed after RNA has been converted to cDNA. Therefore, the method does not detract from input RNA that may be required for library prep, allowing removal of more than half of the rRNA with as little as 1 ng of input RNA when paired with library prep kits designed for low input samples. Increased coverage of non-ribosomal RNA was achieved with an even smaller input amount of ~400 pg. Prezza et al. suggested that the protocol could be further improved by testing alternative Cas nucleases. IDT recommends Alt-R™ S.p. HiFi Cas9 Nuclease V3.