Genome editing by RNA-guided nucleases, such as SpCas9, 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 sgRNAs. We used this toolkit to explore the limits of multiplexing in two major model species, and report on isolation of transgene-free octuple Nicotiana benthamiana and duodecuple (12x) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on production of toxic 5-fluorouracyl in 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 identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (~112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A.thaliana, and our results indicate Cas9 availability is the limiting factor in such higher order multiplexing applications. We identify 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 investigate groups of candidate genes.

中文翻译：

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高效的多重编辑：一次生成8个Nicotiana benthamiana和12个拟南芥突变体

通过RNA引导的核酸酶（例如SpCas9）进行的基因组编辑已用于许多不同的植物物种中。但是，尚没有充分文献证明可以通过多路技术同时靶向多个独立基因座的程度。在这里，我们基于高度内含子优化的zCas9i基因开发了一个工具包，该工具包可组装表达多达32个sgRNA的核酸酶构建体。我们使用该工具包探索了两个主要模型物种中的多重化限制，并报告了单代中无转基因八倍体烟草本氏烟和十二指肠（12x）拟南芥突变株的分离。我们为本氏猪笼草开发了新的反选择标记，最重要的是Sl-FAST2，与成熟的拟南芥种子荧光标记和FCY-UPP相当，基于在前体存在下产生有毒的5-氟尿嘧啶。我们针对9种不同sgRNA阵列中的8个基因，并依靠FCY-UPP选择非转基因T1，我们鉴定出了效率极高的本氏猪笼草突变株：所有分析过的植物的所有基因均带有突变（〜112/116目标网站已修改）。此外，我们通过拟南芥中的24个sgRNA阵列靶向了12个基因。拟南芥的效率明显较低，我们的结果表明，Cas9的可用性是此类高阶多路复用应用程序的限制因素。我们通过表型筛选和扩增子测序的组合来确定一个十二指肠突变株。