Verification of DNA motifs in Arabidopsis using CRISPR/Cas9-mediated mutagenesis
Li, C., Chen, C., Chen, H., Wang, S., Chen, X., Cui, Y. (2018). Verification of DNA motifs in Arabidopsis using CRISPR/Cas9-mediated mutagenesis, 16(8), 1446-1451. http://dx.doi.org/10.1111/pbi.12886
© 2018 The Authors Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. Transcription factors (TFs) and chromatin-modifying factors (CMFs) access chromatin by recognizing specific DNA motifs in their target genes. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) has been widely used to discover the potential DNA-binding motifs for both TFs and CMFs. Yet, an in vivo method for verifying DNA motifs captured by ChIP-seq is lacking in plants. Here, we describe the use of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) to verify DNA motifs in their native genomic context in Arabidopsis. Using a single-guide RNA (sgRNA) targeting the DNA motif bound by REF6, a DNA sequence-specific H3K27 demethylase in plants, we generated stable transgenic plants where the motif was disrupted in a REF6 target gene. We also deleted a cluster of multiple motifs from another REF6 target gene using a pair of sgRNAs, targeting upstream and downstream regions of the cluster, respectively. We demonstrated that endogenous genes with motifs disrupted and/or deleted become inaccessible to REF6. This strategy should be widely applicable for in vivo verification of DNA motifs identified by ChIP-seq in plants.
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