Dr Aimee Van Der Reis1, Dr Richard O'Rorke1, Dr Geoffrey Thomson2, Dr Annabel Whibley3, Professor Andrew Jeffs1
1University Of Auckland, Auckland, New Zealand, 2Yale University, New Haven, United States of America, 3Bragato Research Institute, Auckland, New Zealand
Biography:
Aimee van der Reis is a postdoctoral fellow based at the University of Auckland. Aimee's interests lie in using modern molecular techniques to unpack complex questions typically associated with the marine environment. She is particularly interested in using DNA metabarcoding for marine conservation and management purposes. She is currently involved in a Marsden research project that is investigating whether crustose coralline algae act as a seed bank in marine ecosystems.
Abstract:
The sequencing outputs from DNA metabarcoding studies are frequently overwhelmed by unwanted reads. CRISPR-Cas9, often referred to as "genetic scissors", is a powerful tool that allows scientists to cut and modify DNA with high precision. However, despite the wide adoption of this technology across the biological sciences, CRISPR-Cas9 has been underexplored as a step in DNA metabarcoding methodology for preventing the sequencing of unwanted organisms. CRISPR-Cas9 has been shown to successfully cut amplicons of unwanted organisms (e.g., hosts in microbiome and dietary studies) after PCR. Cutting after PCR has one advantage, which is that cutting can be optimized as amplicon size is known and thus molarity of PCR products can easily be determined. Post-PCR amplicon cutting, however, is not typically ideal as amplification of the unwanted organism has already taken place, which creates uncertainties. For example, PCR errors can remove the CRISPR-Cas9 target preventing cutting, and PCR stochasticity can eliminate low copy sequences-of-interest that are overwhelmed by unwanted sequences. Thus, even though wanted amplicons are preferentially sequenced post-cutting, the uncertainty of having actually captured desired organisms during PCR remains. Here we present a CRISPR-Cas9 DNA metabarcoding method that will prevent unwanted organism DNA amplification during PCR. Using regurgitated pellets from New Zealand king shags we showcase the effectiveness of applying a single CRISPR-Cas9 step between DNA extraction and PCR to substantially increase the amplification of wanted (dietary) organisms. We demonstrate its efficacy across a gradient of CRISPR-Cas9 reaction conditions, which will help guide other researchers interested in adopting this technology to get better value out of their DNA metabarcode sequencing.