Extended Data Fig. 9: CRISPR editing of maize cis-regulatory regions influences phenotype.
From: Transcription factor binding divergence drives transcriptional and phenotypic variation in maize
a, Genome browser screenshot of TSH1 locus showing binding in upstream promoter region by many TFs. Grey shaded areas upstream of and within CRM169681 indicate regions that were deleted in alleles shown in b. b, Schematic showing two independent CRISPR alleles with deletions and inversions that eliminate at least five TF binding sites (colored bars; colors match TFs shown in genome browser screenshot in a). Lightly shaded grey areas indicate regions deleted in both alleles. c, Images of mature tassels for WT, two tsh1 promoter CRISPR alleles, and tsh1-ref mutant (coding region mutation51). Both CRISPR promoter alleles show outgrowth of the tassel sheath leaf (white arrows) that is not present in the WT (black arrow). Tassel branching is reduced in the tsh1-ref, and the CRISPR promoter alleles (white brackets) relative to WT (black bracket). d, SEMs of immature tassels of the CRISPR promoter alleles showing bract outgrowth (white arrowheads). e, qRT-PCR analysis of CRISPR promoter alleles showing reduced expression in immature tassels relative to WT immature tassels. Data are presented as mean values. Error bars represent standard deviation of three biological replicates for each edited allele (two biological replicates for wildtype). Data points correspond to individual biological replicates. Statistical significance determined by two-sided t-test. f, CRISPR editing of BIF2 3’UTR ARF binding sites. A cis-regulatory module (CRM18765) is situated downstream of the BIF2 gene. Within this CRM region, there is a strong ARF peak containing five ARF binding motifs (three TGTCs and two GACAs)38. Three single guide RNAs (gRNAs) were designed for CRISPR-Cas9 editing that specifically targeted the ARF motifs. Three deletion alleles were obtained. Homozygous plants of these alleles exhibited a weak bif2 phenotype with various degrees of severity (BIF2-crm1cr1 and BIF2-crm1cr3 are more severe than BIF2-crm1cr2) during the early ear development stage, characterized by partial barren patches (white arrows) on the ear primordia as seen by SEM.
