CRISPR-TSKO facilitates efficient cell type-, tissue-, or organ-specific mutagenesis in Arabidopsis

First author: Ward Decaestecker; Affiliations: Ghent University (根特大学): Ghent, Belgium

Corresponding author: Thomas B. Jacobs

Detailed functional analyses of many fundamentally-important plant genes via conventional loss-of-function approaches are impeded by severe pleiotropic phenotypes. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a CRISPR-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis, CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The underlying modular cloning system allows for efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens new avenues to discover and analyze gene functions in spatial and temporal contexts of plant life while avoiding pleiotropic effects of system-wide loss of gene function.

由于许多基础重要的植物基因功能缺失会导致严重多效性表型，因此严重阻碍了研究人员对这些基因进行深入的功能鉴定。尤其是一些对于基础细胞功能或者生殖必需的基因，这些基因的功能突变会导致纯合子代的败育，使得进一步的功能研究停滞不前。为了克服这种限制，本文作者开发了一套基于CRISPR的组织特异性基因敲除系统，命名为CRISPR-TSKO，能够在特定的细胞类型、组织以及器官中产生体细胞突变。在拟南芥根冠、气孔细胞系或整个侧根中，对于必需基因的CRISPR-TSKO突变产生了预期的缺陷表型。这套方法底层的克隆系统使得研究者在第一代转基因植株中有效选择、鉴定和功能分析成为可能。CRISPR-TSKO系统为在时空上发现、鉴定和分析植物基因的功能开辟了新的途径，同时避免了全系统基因功能突变带来的多效性影响。

通讯：Thomas B. Jacobs (http://www.vib.be/en/research/scientists/Pages/Thomas-Jacobs-Lab.aspx)

个人简介：2014年，美国佐治亚大学，博士；2014-2016年，美国Boyce Thompson研究所，博士后。

研究方向：植物基因组编辑。

doi: http://dx.doi.org/10.1101/474981

Journal: bioRxiv

First Posted: November 20, 2018

An inducible genome editing system for plants

First author: Xin Wang; Affiliations: University of Helsinki (赫尔辛基大学): Helsinki, Finland

Corresponding author: Ari Pekka Mähönen

Conditional manipulation of gene expression is a key approach to investigating the primary function of a gene in a biological process. While conditional and cell-type specific overexpression systems exist for plants, there are currently no systems available to disable a gene completely and conditionally. Here, we present a novel tool with which target genes can be efficiently conditionally knocked out at any developmental stage. The target gene is manipulated using the CRISPR-Cas9 genome editing technology, and conditionality is achieved with the well-established estrogen-inducible XVE system. Target genes can also be knocked-out in a cell-type specific manner. Our tool is easy to construct and will be particularly useful for studying genes which have null-alleles that are non-viable or show strong developmental defects.

有条件的改变基因的表达是研究基因的生物学功能的主要方法。植物中目前已有的是有条件的、细胞类型特异性过表达系统，但还没有系统能够有条件的使得基因完全丧失其功能。本文中，作者开发了一套新的工具能够在任何发育阶段高效有条件的敲除靶基因。在该系统中，作者通过CRISPR-Cas9基因组编辑技术来敲除基因，而条件性则是通过一个建立完备的雌激素诱导XVE系统来实现。靶基因的敲除同样能够在特定的细胞类型中实现。本文所开发的系统十分容易构建，并且对于具有无效等位基因或者突变后发育有严重缺陷的基因的功能研究尤其有用。

通讯：Ari Pekka Mähönen (https://researchportal.helsinki.fi/en/persons/ari-pekka-mähönen)

个人简介：2006年，芬兰赫尔辛基大学，博士；荷兰乌得勒支大学，博士后。

研究方向：以拟南芥的根为模型研究植物维管形成层的发育。

doi: http://dx.doi.org/10.1101/779140

Journal: bioRxiv

First Posted: September 23, 2019

Live imaging-assisted domain-specific CRISPR genome editing at single cell resolution in plants

First author: Ting Li; Affiliations: Gregor Mendel Institute of Molecular Plant Biology (孟德尔分子植物生物学研究所): Vienna, Austria

Corresponding author: Elliot M. Meyerowitz

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has been widely used for genome engineering in a wide range of organisms, but much of the development of CRISPR-based genome editing has been aimed toward improving its efficiency and accuracy, so as to obtain genetic materials carrying known and stably heritable genome modifications. Precise spatiotemporal control over genome editing technology at cell type resolution is a key challenge for gene function studies. Some tissue-specific CRISPR genome editing methods relying on phenotypic characterization and fluorescent immune-staining techniques have been developed for biomedical research and gene therapy, they function by spatially controlling expression of Cas9. Recent work establishes the presence and location of mutational events at a single cell level in Arabidopsis roots and stomata. Here we present an efficient domain-specific CRISPR-Cas9 system combined with a high resolution live-imaging based screening strategy, applied in the shoot apical meristem of Arabidopsis thaliana. Using the system we investigate PIN-FORMED1 (PIN1) protein functions in tissue morphogenesis and PIN1 mechanical stress response in a cell layer-specific fashion. We find that reported failure to generate new primordia in epidermal PIN1 knockout SAMs is due to a reduction in mechanical stress differences in the sub-epidermal layer. The methods described are applicable to spatial-temporal gene manipulation in plants.

成簇规律间隔短回文重复序列CRISPR技术已经被广泛用于各种生物的基因编辑，但是许多基于CRISPR的基因组编辑工具的开发致力于提升其工作效率和精度，以便获得携带已知和稳定的可遗传基因组修饰的遗传材料。在细胞类型层面，从时空上精确控制基因编辑技术是基因功能研究的巨大挑战。一些依赖于表型特征和荧光免疫染色技术的组织特异性CRISPR基因组编辑方法已经被开发出来，用于医药或基因理论研究，这些技术主要通过时空控制Cas9的表达发挥作用。近期有一些工作在拟南芥根和气孔单个细胞层面建立了基因组编辑系统。本文中，作者报道了一个有效结构域特异性CRISPR-Cas9系统，结合了基于高分辨率实时成像的筛选策略，并应用于拟南芥的茎尖分生组织。利用该系统，作者研究了PIN1蛋白在组织形态建成中的作用以及特定细胞层的PIN1机械胁迫响应。作者发现报告株系不能在表皮PIN1敲除SAM中形成新的原基是由于表皮下层的机械应力差异降低所导致的。该方法能够应用于植物基因时空表达的修改。

通讯：Elliot M. Meyerowitz (https://www.bbe.caltech.edu/people/elliot-meyerowitz)

个人简介：1973年，哥伦比亚大学，学士；1977年，耶鲁大学，博士；斯坦福大学，博士后。

研究方向：植物茎尖分生组织。

doi: http://dx.doi.org/10.1101/793240

Journal: bioRxiv

First Published: October 04, 2019