Division of Plant Sciences, School of Life Sciences, University of Dundee at the JHI, Invergowrie, Dundee, DD2 5DA, Scotland.
Current address: Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
BMC Plant Biol. 2020 Jul 16;20(1):336. doi: 10.1186/s12870-020-02545-7.
Current excitement about the opportunities for gene editing in plants have been prompted by advances in CRISPR/Cas and TALEN technologies. CRISPR/Cas is widely used to knock-out or modify genes by inducing targeted double-strand breaks (DSBs) which are repaired predominantly by error-prone non-homologous end-joining or microhomology-mediated end joining resulting in mutations that may alter or abolish gene function. Although such mutations are random, they occur at sufficient frequency to allow useful mutations to be routinely identified by screening. By contrast, gene knock-ins to replace entire genes with alternative alleles or copies with specific characterised modifications, is not yet routinely possible. Gene replacement (or gene targeting) by homology directed repair occurs at extremely low frequency in higher plants making screening for useful events unfeasible. Homology directed repair might be increased by inhibiting non-homologous end-joining and/or stimulating homologous recombination (HR). Here we pave the way to increasing gene replacement efficiency by evaluating the effect of expression of multiple heterologous recombinases on intrachromosomal homologous recombination (ICR) in Nicotiana tabacum plants.
We expressed several bacterial and human recombinases in different combinations in a tobacco transgenic line containing a highly sensitive β-glucuronidase (GUS)-based ICR substrate. Coordinated simultaneous expression of multiple recombinases was achieved using the viral 2A translational recoding system. We found that most recombinases increased ICR dramatically in pollen, where HR will be facilitated by the programmed DSBs that occur during meiosis. DMC1 expression produced the greatest stimulation of ICR in primary transformants, with one plant showing a 1000-fold increase in ICR frequency. Evaluation of ICR in homozygous T2 plant lines revealed increases in ICR of between 2-fold and 380-fold depending on recombinase(s) expressed. By comparison, ICR was only moderately increased in vegetative tissues and constitutive expression of heterologous recombinases also reduced plant fertility.
Expression of heterologous recombinases can greatly increase the frequency of HR in plant reproductive tissues. Combining such recombinase expression with the use of CRISPR/Cas9 to induce DSBs could be a route to radically improving gene replacement efficiency in plants.
CRISPR/Cas 和 TALEN 技术的进步促使人们对植物基因编辑的机会产生了浓厚的兴趣。CRISPR/Cas 广泛用于通过诱导靶向双链断裂（DSB）来敲除或修饰基因，这些 DSB 主要通过易错的非同源末端连接或微同源介导的末端连接进行修复，导致可能改变或消除基因功能的突变。尽管这些突变是随机的，但它们发生的频率足以允许通过筛选常规识别有用的突变。相比之下，用具有特定特征修饰的替代等位基因或拷贝替换整个基因的基因敲入目前还不太可能实现。同源定向修复（HDR）在高等植物中发生的频率极低，使得筛选有用事件变得不可行。通过抑制非同源末端连接和/或刺激同源重组（HR），可以增加 HDR。在这里，我们通过评估多种异源重组酶在烟草植物中的染色体内同源重组（ICR）中的表达对提高基因替换效率的影响，为提高基因替换效率铺平了道路。
我们在含有高度敏感的β-葡聚糖酶（GUS）基 ICR 底物的烟草转基因系中，以不同组合表达了几种细菌和人类重组酶。使用病毒 2A 翻译重编码系统实现了多种重组酶的协调同时表达。我们发现，大多数重组酶在花粉中显著增加了 ICR，在减数分裂过程中发生的程序性 DSB 将促进 HR。DMC1 表达在初级转化体中产生了对 ICR 的最大刺激，其中一株植物的 ICR 频率增加了 1000 倍。在纯合 T2 植物系中对 ICR 的评估显示，根据表达的重组酶的不同，ICR 增加了 2 到 380 倍。相比之下，在营养组织中 ICR 仅适度增加，并且异源重组酶的组成型表达也降低了植物的育性。
表达异源重组酶可以大大增加植物生殖组织中 HR 的频率。将这种重组酶表达与使用 CRISPR/Cas9 诱导 DSB 相结合，可能是大幅提高植物基因替换效率的途径。
