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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  CrRLK1L s 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  CrRLK  1  L  mutants, quadruple  mds  knock-outs were fully fertile, developed normal root hairs and trichomes and responded to pharmacological inhibition of cellulose biosynthesis similar to wildtype. Recently, we demonstrated the role of four  CrRLK1L  in growth adaptation to metal ion stress. Here we show the involvement of  MDS  genes in response to Ni 2+  during hypocotyl elongation and to Cd 2+  and Zn 2+  during root growth. Our finding supports the model of an organ specific network of positively and negatively acting  CrRLK1L s.

Multiplex mutagenesis of four clustered CrRLK1L with CRISPR/Cas9 exposes their growth regulatory roles in response to metal ions