CRISPR/Cpf1-mediated DNA-free plant genome editing.
CRISPR/Cpf1-mediated DNA-free plant genome editing.
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DOI：
10.1038/ncomms14406
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发表时间：
2017-02-16
影响因子：
16.6
通讯作者：
Kim SG
中科院分区：
文献类型：
--
作者：
Kim H;Kim ST;Ryu J;Kang BC;Kim JS;Kim SG
Cpf 1是一种V型CRISPR效应子，识别富含胸苷的前间隔区邻近基序，并在单个CRISPR RNA（crRNA）的指导下在靶位点诱导粘性双链断裂。在这里，我们表明，Cpf 1可以作为一种工具，无DNA编辑的植物基因组。我们描述了将具有体外转录或化学合成的靶向特异性crRNA的重组Cpf 1蛋白递送到从大豆和野生烟草中分离的原生质体中。设计的crRNA是独特的，并且在整个大豆参考基因组中不具有相似的序列（≤3个错配）。靶向深度测序分析显示，在大豆中的FAD 2旁系同源物和野生烟草中的AOC中成功诱导了突变。与SpCas 9不同，Cpf 1主要在靶位点诱导各种核苷酸缺失。在大豆基因组中的潜在脱靶位点未检测到显著突变。这些结果表明，Cpf 1-crRNA复合物是一种有效的无DNA基因组编辑工具，用于植物基因组编辑。Cpf 1是一种V型CRISPR效应蛋白，与Cas9相比具有不同的靶向和指导RNA需求，因此为精确基因组编辑提供了额外的工具。在这里，Kim等人表明Cpf 1核糖核蛋白可以被引入原生质体中，并用于植物中的无转基因编辑。
Cpf1, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif and induces cohesive double-stranded breaks at the target site guided by a single CRISPR RNA (crRNA). Here we show that Cpf1 can be used as a tool for DNA-free editing of plant genomes. We describe the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco. Designed crRNAs are unique and do not have similar sequences (≤3 mismatches) in the entire soybean reference genome. Targeted deep sequencing analyses show that mutations are successfully induced in FAD2 paralogues in soybean and AOC in wild tobacco. Unlike SpCas9, Cpf1 mainly induces various nucleotide deletions at target sites. No significant mutations are detected at potential off-target sites in the soybean genome. These results demonstrate that Cpf1–crRNA complex is an effective DNA-free genome-editing tool for plant genome editing. Cpf1 is a type V CRISPR effector protein that has different target and guide RNA requirements compared to Cas9, thus offering an addition tool for precision genome editing. Here Kim et al. show that Cpf1 ribonucleoprotein can be introduced into protoplasts and used for transgene-free gene editing in plants.