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Self-processing of ribozyme-flanked rNAs into guide rNAs in vitro and in vivo for CrISPr-mediated genome editing

Journal of Integrative Plant Biology 2014年第4期56卷 343-349页

作者： yangbin Gao yunde zhao Section of Cell and Developmental Biology University of California San Diego9500 Gilman DriveLa Jolla CA 92093-0116 USA

CrISPr/Cas9 uses a guide rNA （grNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the grNAs often render Cas9/grNA inactive. So far, production of grNA in vivo has only been achieved by using the U6 and U3 snrNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing grNAs both in vitro and in vivo. We design an artificial gene named rGr that, once transcribed, generates an rNA molecule with ribozyme sequences at both ends of the designed grNA. We show that the primary transcripts of rGr undergo self-catalyzed cleavage to generate the desired grNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. rGr can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CrISPr is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any grNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the grNAs/CrISPr. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.

关键词： CAS9 CrISPr DNA digestion grNA genome genomeediting in vitro transcription ribozyme

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Two homologous INDOLE-3-ACETAIVHDE (IAM) HyDrOLASE genes are required for the auxin effects of IAM in Arabidopsis

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Journal of Genetics and Genomics 2020年第3期47卷 157-165页

作者： yangbin Gao Xinhua Dai yuki Aoi yumiko Takebayashi Liping yang Xiaorui Guo Qiwei zeng Hanchuanzhi yu Hiroyuki Kasahara yunde zhao Section of Cell and Developmental Biology University of California San DiegoLa JollaCA92093-0116USA rIKEN Center for Sustainable resource Science Kanagawa230-0045Japan School of Life Sciences Jilin Normal UniversitySiping136000China Key Laboratory of Plant Ecology Northeast Forestry UniversityHarbin150040China State Key Laboratory of Silkworm Genome Biology Institute of Sericulture and Systems BiologySouthwest UniversityChongqing400716China Institute of Global Innovation research Tokyo University of Agriculture and TechnologyTokyo183-8509Japan Department of Bioregulation and Biointeraction Graduate School of AgricultureTokyo University of Agriculture and TechnologyFuchu183-8509Japan

Indole-3-acetamide(IAM)is the first confirmed auxin biosynthetic intermediate in some plant pathogenic *** applied IAM or production of IAM by overexpressing the bacterial iaaM gene in Arabidopsis causes auxin overproduction ***,it is still inconclusive whether plants use IAM as a key precursor for auxin ***,we reported the isolation IAM HyDrOLASE 1(IAMH1)gene in Arabidopsis from a forward genetic screen for IAM-insensitive mutants that display normal auxin ***1 has a close homolog named IAMH2 that is located right next to IAMH1on chromosomeⅣin *** generated iamh1 iamh2 double mutants using our CrISPr/Cas9gene editing *** showed that disruption of the IAMH genes rendered Arabidopsis plants resistant to IAM treatments and also suppressed the iaaM overexpression phenotypes,suggesting that IAMH1 and IAMH2 are the main enzymes responsible for converting IAM into indole-3-acetic acid(IAA)in *** iamh double mutants did not display obvious developmental defects,indicating that IAM does not play a major role in auxin biosynthesis under normal growth *** findings provide a solid foundation for clarifying the roles of IAM in auxin biosynthesis and plant development.

关键词： Auxin Auxin biosynthesis Indole-3-acetamide Arabidopsis CrISPr IAMH1 IAMH2

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