Rapid Construction of Multiplexed CRISPR-Cas9 Systems for Plant Genome Editing
Multiplex CRISPR-Cas9 nuclease mediated genome editing is an efficient method for disrupting gene function in plants. Use of CRISPR-Cas9 has escalated rapidly in recent years and is expected to become routine practice in molecular biology and related fields of research. Due to the relatively novel and widespread adoption of this technology, first-time users may not have regular access to experienced guidance or technical support from peers or mentors. Here, we offer guidance and technical support in the form of a detailed and tested protocol for simultaneous targeting of three separate loci on the TRANSPARENT TESTA 4 (TT4) gene in Arabidopsis thaliana using multiplex CRISPR-Cas9. Although we target multiple loci on a single gene in Arabidopsis, the same approach can be used to target multiple genes or alleles in other plant species as well. We recommend the use of a molecular toolkit to streamline the process and make recommendations for this type of approach. The protocol starts with an overview of the reagents and covers designing of gRNAs and assembly of components into a final T-DNA delivery molecule through Golden Gate cloning and Multisite Gateway LR recombination.
Key wordsCRISPR/Cas9 Plant genome editing Multiplex Golden gate assembly Gateway cloning
This work is supported by startup funds from East Carolina University and a Collaborative Funding Grant from North Carolina Biotechnology Center and Syngenta (2016-CFG-8003) to Y.Q.
- 18.Zhang F, Maeder M, Unger-Wallace E, Hoshaw J, Reyon D, Christian M, Li X, Pierick C, Dobbs D, Peterson T, Joung K, Voytas D (2010) High frequency targeted mutagenesis in Arabidopsis thaliana using zing finger nucleases. Proc Natl Acad Sci U S A 107(26):12028–12033CrossRefPubMedPubMedCentralGoogle Scholar
- 20.Feng Z, Mao Y, Zhang B, Wei P, Yang D-L, Wang Z, Zhang Z, Zheng R, Yang L, Zeng L, Liu X, Zhu J-K (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci U S A 111(12):4632–4637CrossRefPubMedPubMedCentralGoogle Scholar
- 23.Singh R, Kuscu C, Quinlan A, Qi Y, Adli M (2015) Cas9-chromatin binding information enables more accurate CRISPR off-target prediction. Nucleic Acids Res 43(18). doi: 10.1093/nar/gkv575
- 26.De Wilde C, Van Houdt H, De Buck S, Angenon G, De Jaeger G (2000) Depicker A. Plants as bioreactors for protein production: avoiding the problem of transgene silencing 43:347–359Google Scholar