# Development and Laboratory Validation of a Field‐Deployable CRISPR‐Cas12a eDNA Assay for Phylogeographic Lineage Detection in Arctic Char (Salvelinus alpinus)

## TL;DR

This paper introduces a new CRISPR-based eDNA test that can detect specific genetic lineages of Arctic char, enabling detailed ecological monitoring at a level below the species level.

## Contribution

The study presents the first CRISPR-based eDNA assay for detecting phylogeographic lineages in Arctic char, enabling intraspecific genetic resolution in field settings.

## Key findings

- A CRISPR-Cas12a eDNA assay successfully detected a specific lineage of Arctic char with high sensitivity and specificity.
- LbCas12a outperformed AsCas12a in assay performance under optimized buffer conditions.
- A lateral flow platform enabled portable and rapid detection of the target lineage in laboratory conditions.

## Abstract

Environmental DNA (eDNA) tools are increasingly used for biodiversity monitoring, with most existing assays targeting species‐level identification. However, the use of eDNA to resolve intraspecific genetic variation remains rare and methodologically underdeveloped. This study presents the development and laboratory validation of a novel molecular assay capable of detecting specific phylogeographic lineages, advancing eDNA applications by enabling resolution below the species level. The assay combines Recombinase Polymerase Amplification (RPA) and CRISPR‐Cas12a technologies with a lateral flow platform for field‐ready, on‐site detection. Irish Arctic char (
Salvelinus alpinus
) was selected as the model due to its conservation relevance and post‐glacial lineage diversity in Ireland. Mitochondrial genome sequencing of known Irish lineages identified a Protospacer Adjacent Motif (PAM) site unique to the Atlantic Subclade 1 lineage, allowing clear discrimination from co‐occurring lineages. Two assays were optimised: a species‐specific assay detecting all Arctic char lineages and a lineage‐specific assay targeting Lineage 1. Both showed high sensitivity and specificity under laboratory conditions, with LbCas12a outperforming AsCas12a at optimised buffer concentrations. The lateral flow adaptation, utilising a dual‐labelled FAM‐Biotin probe, enabled portable and rapid detection with minimal equipment. Field validation using eDNA from Irish lakes highlighted the need for improved sampling protocols, as lake‐edge surface samples failed to yield detections. This assay represents the first reported example of a CRISPR‐based eDNA tool for phylogeographic lineage detection in the field. It offers a novel, non‐invasive, and scalable approach to fine‐scale ecological monitoring and establishes a foundation for future conservation tools targeting intraspecific diversity.