biostudies-literatureRichter JAustrian Science Fund FWF12182https://www.ebi.ac.uk/biostudies/studies/S-EPMC6093868biostudies-literatureUnknown8(1)Resolving functions of closely linked genes is challenging or nearly impossible with classical genetic tools. Four members of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) family are clustered on Arabidopsis chromosome five. To resolve the potentially redundant functions of this subclass of CrRLK1Ls named MEDOS1 to 4 (MDS1 to 4), we generated a single CRISPR/Cas9 transformation vector using a Golden Gate based cloning system to target all four genes simultaneously. We introduce single mutations within and deletions between MDS genes as well as knock-outs of the whole 11 kb gene cluster. The large MDS cluster deletion was inherited in up to 25% of plants lacking the CRISPR/Cas9 construct in the T2 generation. In contrast to described phenotypes of already characterized CrRLKScientific reportsMultiplex mutagenesis of four clustered CrRLK1L with CRISPR/Cas9 exposes their growth regulatory roles in response to metal ions.PMC6093868I 1725-B16I 1725Berger MRichter JBorowska MSchoft VNeuhold JHauser MTWatson JMStasnik PStolt-Bergner PfalseMultiplex mutagenesis of four clustered CrRLK1L with CRISPR/Cas9 exposes their growth regulatory roles in response to metal ions.Resolving functions of closely linked genes is challenging or nearly impossible with classical genetic tools. Four members of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) family are clustered on Arabidopsis chromosome five. To resolve the potentially redundant functions of this subclass of CrRLK1Ls named MEDOS1 to 4 (MDS1 to 4), we generated a single CRISPR/Cas9 transformation vector using a Golden Gate based cloning system to target all four genes simultaneously. We introduce single mutations within and deletions between MDS genes as well as knock-outs of the whole 11 kb gene cluster. The large MDS cluster deletion was inherited in up to 25% of plants lacking the CRISPR/Cas9 construct in the T2 generation. In contrast to described phenotypes of already characterized CrRLK2018-01-01T00:00:00Z2018 Aug2025-04-19T10:58:12.78Z2019-03-26T23:51:44ZS-EPMC60938683011186510.1038/s41598-018-30711-3