Twelve genes at one blow: multiplex genome editing with CRISPR/Cas.
Twelve genes at one blow: multiplex genome editing with CRISPR/Cas.
复制标题
一击完成 12 个基因：使用 CRISPR/Cas 进行多重基因组编辑
DOI：
10.1111/tpj.15228
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Marillonnet
中科院分区：
文献类型：
--
作者：
Stuttmann;Barthel;Martin;Erickson;Kretschmer;Berner;Keilwagen;Marillonnet
通过RNA引导的核酸酶（如SpCas 9）进行的基因组编辑已用于许多不同的植物物种。然而，在多大程度上可以通过多重化同时靶向多个独立的基因座还没有很好的文献记载。在这里，我们开发了一个基于高度内含子优化的zCas 9 i基因的工具包，该基因允许组装表达多达32个单向导RNA（sgRNA）的核酸酶构建体。我们使用该工具包探索了两个主要模式物种中多重化的限制，并报告了在单代（分别为T1和T2）中分离无转基因的八倍体（8×）本氏烟草和十二倍体（12×）拟南芥突变株系。我们为N开发了新的反选择标记。最重要的是S1-FAST 2，与成熟的拟南芥种子荧光标记相当，以及FCY-UPP，基于在前体存在下产生毒性5-氟尿嘧啶。用9种不同sgRNA的阵列靶向8个基因，并依靠FCY-UPP选择非转基因T1，我们鉴定了N。具有极高效率的本氏诱变株系：所有分析的植物在所有基因中携带突变（约112/116个靶位点被编辑）。此外，我们通过A中的24个sgRNA的阵列靶向12个基因。thaliana.在拟南芥中效率显著较低，我们的结果表明Cas9的可用性是这种高阶多路复用应用的限制因素。我们通过表型筛选和扩增子测序相结合的方法鉴定了一个十二倍突变株系。所提供的资源和结果为如何使用多重技术来产生复杂的基因型或功能性地询问候选基因组提供了新的视角。
Genome editing by RNA‐guided nucleases, such asSpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron‐optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene‐free octuple (8×)Nicotiana benthamianaand duodecuple (12×)Arabidopsis thalianamutant lines in a single generation (T1and T2, respectively). We developed novel counter‐selection markers forN. benthamiana, most importantlySl‐FAST2, comparable to the well‐established Arabidopsis seed fluorescence marker, and FCY‐UPP, based on the production of toxic 5‐fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY‐UPP for selection of non‐transgenic T1, we identifiedN. benthamianamutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs inA. thaliana. Efficiency was significantly lower inA.thaliana, and our results indicate Cas9 availability is the limiting factor in such higher‐order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle