Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida.
Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida.
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DOI：
10.1038/srep38169
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发表时间：
2016-12-01
影响因子：
4.6
通讯作者：
Toki S
中科院分区：
文献类型：
--
作者：
Endo A;Masafumi M;Kaya H;Toki S
CRISPR/Cas9系统如今被广泛应用于在包括植物在内的各种生物体中进行基因组编辑。来自普雷沃氏菌和弗朗西斯氏菌1（Cpf 1）的CRISPR是一种新表征的RNA引导的核酸内切酶，与Cas9相比具有两个不同的特征。首先，Cpf 1利用富含胸苷的前间隔区邻近基序（PAM），而Cas9优选富含胍的PAM。Cpf 1可以用作序列特异性核酸酶，靶向Cas9难以进入的基因组中富含AT的区域。其次，Cpf 1产生具有5′突出端的DNA末端，而Cas9在切割后产生平端DNA末端。“粘性”DNA末端应增加使用互补DNA末端将所需DNA片段插入Cpf 1切割位点的效率。因此，Cpf 1可能是精确基因组工程的有力工具。为了评估Cpf 1是否可以应用于植物基因组编辑，我们选择了来自Francisella novicida（FnCpf 1）的Cpf 1，它识别已知Cpf 1蛋白中较短的PAM（TTN），并将其应用于烟草和水稻的靶向诱变。我们的研究结果表明，在转基因植物中发生了靶向突变表达FnCpf 1与crRNA。靶区域的缺失是最常见的突变。我们的研究结果表明，FnCpf 1可以成功地应用于植物基因组工程。
CRISPR/Cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. CRISPR from Prevotella and Francisella 1 (Cpf1) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. First, Cpf1 utilizes a thymidine-rich protospacer adjacent motif (PAM) while Cas9 prefers a guanidine-rich PAM. Cpf1 could be used as a sequence-specific nuclease to target AT-rich regions of a genome that Cas9 had difficulty accessing. Second, Cpf1 generates DNA ends with a 5′ overhang, whereas Cas9 creates blunt DNA ends after cleavage. “Sticky” DNA ends should increase the efficiency of insertion of a desired DNA fragment into the Cpf1-cleaved site using complementary DNA ends. Therefore, Cpf1 could be a potent tool for precise genome engineering. To evaluate whether Cpf1 can be applied to plant genome editing, we selected Cpf1 from Francisella novicida (FnCpf1), which recognizes a shorter PAM (TTN) within known Cpf1 proteins, and applied it to targeted mutagenesis in tobacco and rice. Our results show that targeted mutagenesis had occurred in transgenic plants expressing FnCpf1 with crRNA. Deletions of the targeted region were the most frequently observed mutations. Our results demonstrate that FnCpf1 can be applied successfully to genome engineering in plants.
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影响因子：
46.9
作者：
Kleinstiver BP;Tsai SQ;Prew MS;Nguyen NT;Welch MM;Lopez JM;McCaw ZR;Aryee MJ;Joung JK
通讯作者：
Joung JK
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46.9
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Hendel A;Bak RO;Clark JT;Kennedy AB;Ryan DE;Roy S;Steinfeld I;Lunstad BD;Kaiser RJ;Wilkens AB;Bacchetta R;Tsalenko A;Dellinger D;Bruhn L;Porteus MH
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56.9
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Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
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CRISPR-CAS9介导的基因失活的高度活性SGRNA的合理设计。
影响因子：
46.9
作者：
Doench, John G.;Hartenian, Ella;Graham, Daniel B.;Tothova, Zuzana;Hegde, Mudra;Smith, Ian;Sullender, Meagan;Ebert, Benjamin L.;Xavier, Ramnik J.;Root, David E.
通讯作者：
Root, David E.
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4.9
作者：
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通讯作者：
Matsuoka M