Site-directed mutagenesis in <i>Arabidopsis thaliana</i> using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles

다국어 초록 (Multilingual Abstract)

<P><B><B><I>Main conclusion</I></B></B></P><P><B>Dividing tissue-targeted site-directed mutagenesis using RGEN of CRISPR/Cas system produces heritable mutations in</B><B><I>Arabidopsis thaliana.</I></B></P><P><B>Abstract</B></P><P>Site-directed genome engineering in higher plants has great potential for basic research and molecular breeding. Here, we describe a method for site-directed mutagenesis of the <I>Arabidopsis</I> nuclear genome that efficiently generates heritable mutations using the RNA-guided endonuclease (RGEN) derived from bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated) protein system. To induce mutagenesis in proliferating tissues during embryogenesis and throughout the plant life cycle, the <I>single guide RNA</I> (<I>sgRNA</I>) and Cas9 DNA endonuclease were expressed from the <I>U6 snRNA</I> and <I>INCURVATA2</I> promoters, respectively. After <I>Agrobacterium</I>-mediated introduction of T-DNAs encoding RGENs that targets <I>FLOWERING LOCUS T</I> (<I>FT</I>) and <I>SQUAMOSA PROMOTER BINDING PROTEIN</I>-<I>LIKE 4</I> genes, somatic mutagenesis at the targeted loci was observed in T1 transformants. In the results of FT-RGEN, T1 plants often showed late flowering indicative of the presence of large somatic sectors in which the <I>FT</I> gene is mutated on both chromosomes. DNA sequencing analysis estimated that about 90 % of independent chromosomal DNA fragments carried mutations in the analyzed tissue of a T1 plant showing late flowering. The most frequently detected somatic polymorphism showed a high rate of inheritance in T2 plants, and inheritance of less frequent polymorphisms was also observed. As a result, late-flowering plants homozygous for novel, heritable null alleles of <I>FT</I> including a 1 bp insertion or short deletions were recovered in the following T2 and T3 generations. Our results demonstrate that dividing tissue-targeted mutagenesis using RGEN provides an efficient heritable genome engineering method in <I>A. thaliana</I>.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1007/s00425-014-2180-5) contains supplementary material, which is available to authorized users.</P>