2018:

 

Swift Large-scale Examination of Directed Genome Editing (SLEDGE Hammer)
Omar T. Hammouda,  Thomas Thumberger, Joachim Wittbrodt
doi: https://doi.org/10.1101/479261

Abstract

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 the 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 three-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 and zebrafish embryos (targeted 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 filter-in-tips. Furthermore, our method is applicable across kingdoms with samples ranging from cells to tissues (e.g. plant seedlings, adult flies, mouse cell culture and tissue as well as adult fish fin-clips).