Increasing experimental efficiency in plant genome editing
Improving CRISPR-induced multiplex mutagenesis
May 23, 2024
Ghent 07/05/2024 – CRISPR/Cas9 remains the most powerful tool to generate mutations in plant genomes. Studying the various combinations of mutations has significantly increased the scale of experimental setups, necessitating more space to grow numerous plants. Researchers from VIB-UGent Center for Plant Systems Biology have improved multiplex mutagenesis, which reduces the complexity and cost of large-scale genome editing projects. Their results have been published in The Plant Journal.
CRISPR/Cas experiments are continually increasing in scale, not only in terms of the number of mutants created through precise genome editing but also in terms of the number of genes that can be mutated simultaneously. The lab of Thomas Jacobs from VIB-UGent Center for Plant Systems Biology has developed screens to systematically mutate tens, hundreds, or even thousands of genes at a time. The goal is to enhance the efficiency of inheritable germline mutations, and ultimately reduce the complexity and cost of large-scale genomic editing projects.
To achieve this, the team focused on two key aspects of CRISPR/Cas9 vector design: the promotor to drive Cas9 expression, and the nuclear localization signals (NLS) that direct the protein to the nucleus. By genotyping thousands of Arabidopsis plants, they found that using the RPS5A promotor to express Cas9 led to the highest mutation rate, and that flanking the Cas9 protein with bipartite NLS was the most efficient configuration to create germline mutations. Combining these two elements results in the highest observed multiplex editing efficiency, with 99% of plants harboring at least one knockout mutation and over 80% with 4 to 7 mutations.
This represents a significant advancement in the field of plant genetics and provides a reliable and efficient tool for researchers who focus on complex genetic engineering. What I find particularly interesting is the effect of the NLS. I daresay it had a stronger effect than the promoter. – Dr. Thomas Jacobs, Group leader at VIB-UGent Center for Plant Systems Biology
The optimizations achieved in the study significantly reduce the complexity and cost of large-scale genome editing projects in plant science. To put it in numbers: with their previous vector, a CRISPR screen looking for all double knockouts of just 20 genes was estimated to require a population of about 18,000 plants. With the new vectors, it should take about 3,000 plants.
These optimizations will be useful to generate higher-order knockouts in the germline of Arabidopsis and likely apply to other CRISPR systems as well. - Ward Develtere, PhD student and lead author of the report.
PUBLICATION: https://doi.org/10.1111/tpj.16785
Jonas Blomme for the Thomas Jacobs lab is part of the organizing committee for the upcoming Next-Generation Synthetic Biology conference (5th edition) in December. Registrations are now open.
About the VIB-UGent Center for Plant Systems Biology
The VIB-UGent Center for Plant Systems Biology wants to gain insight into how plants grow and respond to the environment. Scientists study how leaves and roots are formed, which micro-organisms live on and around the plant and which substances the plant makes. They map out the genetic diversity of the plant kingdom. This knowledge can lead to sustainable innovations in agriculture and food.
About VIB
VIB’s core mission is to generate disruptive insights in the molecular underpinning of life and to translate these actively into impactful innovations for patients and society. VIB is an independent research institute where some 1,800 top scientists from Belgium and abroad conduct pioneering basic research. As such, they are pushing the boundaries of what we know about molecular mechanisms and how they rule living organisms such as human beings, animals, plants, and microorganisms. Based on a close partnership with five Flemish universities – Ghent University, KU Leuven, University of Antwerp, Vrije Universiteit Brussel, and Hasselt University – and supported by a solid funding program, VIB unites the expertise of all its collaborators and research groups in a single institute. VIB’s technology transfer activities translate research results into concrete benefits for society such as new diagnostics and therapies and agricultural innovations. These applications are often developed by young start-ups from VIB or through collaborations with other companies. This also leads to additional employment and bridges the gap between scientific research and entrepreneurship. VIB also engages actively in the public debate on biotechnology by developing and disseminating a wide range of science-based information.
More info can be found on www.vib.be.