Date Published: March 5, 2019
Publisher: Public Library of Science
Author(s): Omar T. Hammouda, Frank Böttger, Joachim Wittbrodt, Thomas Thumberger, Stephan C.F. Neuhauss.
In the era of CRISPR gene editing and genetic screening, there is an increasing demand for quick and reliable nucleic acid extraction pipelines for rapid genotyping of large and diverse sample sets. Despite continuous improvements of current workflows, the handling-time and material costs per sample remain major limiting factors. Here we present a robust method for low-cost DIY-pipet tips addressing these needs; i.e. using a cellulose filter disc inserted into a regular pipet tip. These filter-in-tips allow for a rapid, stand-alone four-step genotyping workflow by simply binding the DNA contained in the primary lysate to the cellulose filter, washing it in water and eluting it directly into the buffer for the downstream application (e.g. PCR). This drastically cuts down processing time to maximum 30 seconds per sample, with the potential for parallelizing and automation. We show the ease and sensitivity of our procedure by genotyping genetically modified medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos (targeted by CRISPR/Cas9 knock-out and knock-in) in a 96-well plate format. The robust isolation and detection of multiple alleles of various abundancies in a mosaic genetic background allows phenotype-genotype correlation already in the injected generation, demonstrating the reliability and sensitivity of the protocol. Our method is applicable across kingdoms to samples ranging from cells to tissues i. e. plant seedlings, adult flies, mouse cell culture and tissue as well as adult fish fin-clips.
Gene-editing tools such as CRISPR/Cas9 have revolutionized genome editing in most model organisms [1–3]. The easy application of this targeting approach in organisms as well as cell culture is driving extensive high-throughput applications such as genetic screens [4,5]. Common to all genome targeting applications is the need for rapid subsequent genotyping. Following PCR amplification of the targeted loci, among the most commonly used assays for indel detection are mismatch cleavage assays (using T7 Endonuclease I or Cel-based Surveyor assay), high-resolution melting analysis (HRMA) and sequencing [6,7] in combination with Tracking of Indels by Decomposition (TIDE, ). However, the high-throughput approaches face a bottle neck when it comes to genomic DNA (gDNA) extraction, which usually requires a time- and material-consuming protocol or is not immediately applicable to various sample material, e.g. medaka embryos.
Teleost fish have been extensively used for high-throughput approaches [16–19]. The recent advances in CRISPR/Cas9 mediated genome editing [3,20] as well as the establishment of the medaka inbred panel  as a population genomic resource have led to rising demand on high throughput genotyping for phenotype-genotype correlation.