CRISPR/Cas Efficiency Analysis Protocol

Plant material was harvested for DNA extraction with the CTAB method [73 ]. Either the first true leaf pairs or entire seedlings were harvested, depending if the material was upscaled or not. A region around the CRISPR/Cas target site was PCR amplified using ALLin Red Taq Master Mix, 2X (highQu). The PCR products were analysed via agarose gel electrophoresis and purified by bead purification with HighPrep PCR (MAGBIO). The purified samples were sent for Sanger sequencing (Mix2seq; Eurofins Scientific) and analysed using ICE (https://ice.synthego.com/#/) and/or EDITR [41 (link)]. The ICE KO-score represents the proportion of cells that have either a frameshift or 21+ bp indel. See Supporting Tables for the list of primer and target sequences. The number of individuals analysed is specified for each experiment.

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Blomme J., Develtere W., Köse A., Arraiza Ribera J., Brugmans C., Jaraba-Wallace J., Decaestecker W., Rombaut D., Baekelandt A., Daniel Fernández Fernández Á., Van Breusegem F., Inzé D, & Jacobs T. (2022). The heat is on: a simple method to increase genome editing efficiency in plants. BMC Plant Biology, 22, 142.

Publication 2022

Mix2seq

Agarose gel electrophoresis Crispr Ctab Frameshift Indel Leaf Plant Primer Seedlings

Corresponding Organization :

Other organizations : Ghent University, Ege University, VIB-UGent Center for Plant Systems Biology

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Variable analysis

independent variables

Whether the first true leaf pairs or entire seedlings were harvested

dependent variables

The proportion of cells that have either a frameshift or 21+ bp indel, as measured by the ICE KO-score

control variables

CTAB method for DNA extraction
ALLin Red Taq Master Mix, 2X (highQu) for PCR amplification
Bead purification with HighPrep PCR (MAGBIO) for PCR product purification
Sanger sequencing (Mix2seq; Eurofins Scientific) for sample analysis

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