Manipulating plant RNA-silencing pathways to improve the gene editing efficiency of CRISPR/Cas9 systems.
Manipulating plant RNA-silencing pathways to improve the gene editing efficiency of CRISPR/Cas9 systems.
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DOI：
10.1186/s13059-018-1529-7
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发表时间：
2018-09-28
期刊：
影响因子：
12.3
通讯作者：
Zhu JK
中科院分区：
文献类型：
--
作者：
Mao Y;Yang X;Zhou Y;Zhang Z;Botella JR;Zhu JK
CRISPR/Cas9系统由用于靶标识别的单向导RNA和用于DNA切割的Cas9蛋白组成，具有彻底改变农业和医学的潜力。尽管人们已经做了大量的工作来提高CRISPR/Cas9的基因编辑活性，但人们对这种细菌系统在真核宿主细胞中的调控知之甚少，特别是在转录后水平。在这里，我们评估了两个CRISPR/Cas9组件的表达水平和一组参与RNA沉默的拟南芥突变体的基因编辑效率。我们发现，在转录后基因沉默途径中有缺陷的突变体显示出显著更高的Cas9和sgRNA转录水平，导致比野生型对照更高的诱变频率。因此，通过将AGO 1-RNAi盒引入CRISPR/Cas9载体来沉默AGO 1可以提高基因编辑效率。来自番茄丛矮病毒的病毒抑制子p19的共表达以抑制植物RNA沉默途径显示了由p19引起的表型效应的严重性与CRISPR/Cas9系统对两种不同靶基因AP 1和TT 4的基因编辑效率之间的强相关性。该系统在促进检测T1植物中的CRISPR/Cas9诱导的突变以及通过简单地视觉观察由p19引起的症状严重程度来鉴定无转基因T2植物方面具有有用的实际应用。我们的研究表明，CRISPR/Cas9基因编辑效率可以通过减少植物中的RNA沉默来提高。本文的在线版本（10.1186/s13059-018-1529-7）包含补充材料，可供授权用户使用。
The CRISPR/Cas9 system, composed of a single-guide RNA for target recognition and a Cas9 protein for DNA cleavage, has the potential to revolutionize agriculture as well as medicine. Even though extensive work has been done to improve the gene editing activity of CRISPR/Cas9, little is known about the regulation of this bacterial system in eukaryotic host cells, especially at the post-transcriptional level. Here, we evaluate the expression levels of the two CRISPR/Cas9 components and the gene editing efficiency in a set of Arabidopsis mutants involved in RNA silencing. We find that mutants defective in the post-transcriptional gene-silencing pathway display significantly higher Cas9 and sgRNA transcript levels, resulting in higher mutagenesis frequencies than wild-type controls. Accordingly, silencing of AGO1 by introduction of an AGO1-RNAi cassette into the CRISPR/Cas9 vector provides an increase in gene editing efficiency. Co-expression of the viral suppressor p19 from the tomato bushy stunt virus to suppress the plant RNA-silencing pathway shows a strong correlation between the severity of the phenotypic effects caused by p19 and the gene editing efficiency of the CRISPR/Cas9 system for two different target genes, AP1 and TT4. This system has useful practical applications in facilitating the detection of CRISPR/Cas9-induced mutations in T1 plants as well as the identification of transgene-free T2 plants by simple visual observation of the symptom severity caused by p19. Our study shows that CRISPR/Cas9 gene editing efficiency can be improved by reducing RNA silencing in plants. The online version of this article (10.1186/s13059-018-1529-7) contains supplementary material, which is available to authorized users.
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影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
5.4
作者：
Raja, Priya;Sanville, Bradley C.;Bisaro, David M.
通讯作者：
Bisaro, David M.
通过反式编码的小 RNA 和宿主因子 RNase III 进行 CRISPR RNA 成熟。
影响因子：
64.8
作者：
Deltcheva, Elitza;Chylinski, Krzysztof;Sharma, Cynthia M.;Gonzales, Karine;Chao, Yanjie;Pirzada, Zaid A.;Eckert, Maria R.;Vogel, Joerg;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Mali P;Yang L;Esvelt KM;Aach J;Guell M;DiCarlo JE;Norville JE;Church GM
通讯作者：
Church GM
影响因子：
4.6
作者：
Miki D;Zhu P;Zhang W;Mao Y;Feng Z;Huang H;Zhang H;Li Y;Liu R;Zhang H;Qi Y;Zhu JK
通讯作者：
Zhu JK