Multiplied CRISPR/Cas9 targets


- Team Meiosis mechanisms and apomixis (Meiosis), IJPB
- Team Flower and Carpel Development (FCD) / Platform of Translational Research (TransRes), IPS2
- Team Biology of the Cell and Regeneration (BCR), IJPB

Abstract of the project

Genetic redundancy is a strong impediment to basic research and plant breeding. Recent genome editing technologies offer solutions to overcome this major hurdle. We developed tools and methods for multiplexed CRISPR/Cas9 mutagenesis. We implemented the tools for the analysis of the cyclin multigene family in Arabidopsis, for the creation of valuable fruit traits in tomato and for the study of recombination frequency in wheat. In the Cyclin B task, a semi-forward screen targeting simultaneously 9 genes was successfully performed. By a combination of multiple targeting and NGS-based genotyping, any combination of mutations can be identified and analyzed. A series of mutant with fertility defect were identified and several conclusions can be made. One of the two clades of cyclins B (composed of five Cyclins B2.2/B2.3) is dispensable for development and fertility, as shown by the identification of quintuple K.O. However, the degradation of two of these Cyclins B by the APC/C is required for the termination of meiosis. In tomato, we targeted genes associated with flower inflorescence, fruit set and abiotic stresses and key alleles of agronomic importance were generated and characterized. Loss-of-function in plant genes controlling meiotic recombination may increase the frequency of crossovers and alter their distribution, as demonstrated in Arabidopsis, tomato, pea and rice (Mieulet et al., Nature Plant, 2018). We are constructing edited lines in bread wheat to study the role of RECQ4 in this hexaploid species.
All the technical deliverables of the project are made available to the SPS laboratories for targeted mutagenesis in dicotyledonous and monocotyledonous plant species. MultiCrisp has an impact beyond the SPS community as key international collaborations with public and private partners have been implemented based on its results.

Key publications:

Clepet C, Devani RS, Boumlik R, Hao Y, Morin H, Marcel F, Verdenaud M, Mania B, Brisou G, Citerne S, Mouille G, Lepeltier JC, Koussevitzky S, Boualem A, Bendahmane A. (2021) The miR166-SlHB15A regulatory module controls ovule development and parthenocarpic fruit set under adverse temperatures in tomato. Mol Plant. 14(7):1185-1198. doi:10.1016/j.molp.2021.05.005.

Jacob P, Avni A, Bendahmane A. (2018) Translational research: exploring and creating genetic diversity. Trends Plant Sci. 23(1):42-52. doi: 10.1016/j.tplants.2017.10.002.

Roldan MVG, Périlleux C, Morin H, Huerga-Fernandez S, Latrasse D, Benhamed M, Bendahmane A. (2017) Natural and induced loss of function mutations in SlMBP21 MADS-box gene led to jointless-2 phenotype in tomato. Sci Rep. 7(1):4402. doi: 10.1038/s41598-017-04556-1

Communications in conferences:

CREATE: CRISPR-APOBEC1/AID Extensive Allele Mutagenesis and TILLING-mediated variability Enrichment  - GIS Biotechnologies Vertes 2019


Modification date : 22 September 2023 | Publication date : 22 August 2016 | Redactor : MJS