Genome Engineering & High-Throughout Phenotyping in Plants

The CRISPR/Cas system is the major tool for targeted mutagenesis in eukaryotes to induce either double-strand breaks (DSBs) or single-strand breaks at any genomic sites. We were able to demonstrate heritable targeted mutagenesis in Arabidopsis thaliana as well as the first application of a Cas9 nickase in plants. We also developed an in planta gene targeting (GT) strategy in which both the GT vector and the target locus are activated simultaneously via DSB induction during plant development. Today, our constructs from the Puchta lab are used in more than 200 plant labs worldwide.

Precise genome engineering and synthetic biology are of tremendous interest for modern agriculture. Therefore, we need to understand both the systems that we can leverage to introduce site-specific changes into a genome as well as the systems that we are hoping to engineer. The green alga Chlamydomonas reinhardtii is the leading single-celled eukaryotic model organism to study basic cell processes in plants but also the animal lineage. The Jonikas lab developed yeast-style methods for propagation and maintenance of hundreds of thousands of Chlamydomonas strains. An indexed and mapped library of >85,000 mutants covering 83% of all Chlamydomonas genes was generated to address a wide range of fundamental questions in cell biology. The abundance of individual mutants can be tracked in pools through deep sequencing of unique DNA barcodes. We have developed a pipeline to perform 50 genome-wide mutant screens within 4 weeks and we aim for more than 250 screens in the near future.