The Application Of Two Gene-targeted Knockout Tecnologies, TALEN And CRISPR,in Arabidopsis

In recent years, with the development and application of multiple specific nucleases, the efficiency and accuracy of genome editing technology is significantly improved, and widespread application of this technology should make breakthough in many fields, such as the study of gene function, the therapy of genetic diseases and the development of genetically modified crops and so on.TALEN (transcription activator like effectors nuclease) and CRISPR (clustered regμlatory interspersed short palindromic repeat) are currently two approaches of the most effective gene-targeted knockout technologies, and both of them contain artifically recombinated nucleases, which cut DNA targetedly, resulting in double-strands DNA breaks (DSB) and then DNA repair pathways of non-homologous end joining (NHEJ) or homologous recombination (HR). The NHEJ pathway prone to cause the deletion, insertion and substitution of DNA bases, leading to gene-targeted knockout.Three Arabidopsis genes, IAA2, SIRT2and SAUR41, are under investigation in our laboratory.In order to deepen the study and to obtain null mutants, we have designed and constructed a series of TALEN and CRISPR vectors targeting these genes. On the other hand, in Agrobacterium-mediated transformation of Arabidopsis flowers by the vacuum infiltration method, the application of TALEN and CRISPR technologies have not well-established, and we aim to improve them.We first obtained the T1generation of transgenic Arabidopsis lines for TALEN and CRISPR of target genes. Then, in leaf tissues, the rate of somatic mutation was evaluated by the T7endonuclease digestion. We also induced callus tissues from the T1generation of transgenic lines, and detected somatic mutation rate in callus cells. Finally, we screened germline mutaion in the T2generation transgenic plants and the plants regenerated from callus.We found that, TALEN of the IAA2gene produced the highest somatic mutagenesis frequencies as high as31.6%in callus cells; CRISPR of the SAUR41gene resulted to the highest somatic mutagenesis frequencies of16.1%in leaf cells. Compared with CRISPR, TALEN prone to cause a larger fragment deletion of the targeted genes, and the DSB repair mechanism seemed much more complicated. Compared to leaf cells, the mutation rate of callus cells was approximately2times higher. Thus, plant regeneration from callus can significantly improve the efficiency of mutant screen. T1transgenic plants were chimera contain somatic mutations and germline mutations, and the germline mutation rate was very low. TALEN and CRISPR systems using germ cell specific promoter may improve the rate of germline mutations.