An analysis of possible off target effects following CAS9/CRISPR targeted deletions of neuropeptide gene enhancers from the mouse genome.
An analysis of possible off target effects following CAS9/CRISPR targeted deletions of neuropeptide gene enhancers from the mouse genome.
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DOI：
10.1016/j.npep.2016.11.003
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发表时间：
2017-08
期刊：
影响因子：
2.9
通讯作者：
MacKenzie A
中科院分区：
文献类型：
--
作者：
Hay EA;Khalaf AR;Marini P;Brown A;Heath K;Sheppard D;MacKenzie A
我们已经成功地使用比较基因组学来确定人类基因组中可能的调节元件，这些元件有助于神经肽（如丙氨酸）和受体（如CB1）的组织特异性表达。然而，先前无法从小鼠基因组中快速删除这些元素阻碍了对其体内功能的最佳评估。使用CAS9/CRISPR基因组编辑技术解决了这个问题，该技术使用一种称为CAS9的细菌内切酶，与专门设计的引导RNA （gRNA）分子结合，切割小鼠基因组的特定区域。然而，关于“脱靶”效应的报道，即CAS9内切酶能够切割目标位点以外的位点，限制了这项技术的吸引力。我们通过细胞质微注射gRNA和CAS9 mRNA进入1细胞小鼠胚胎，快速生成增强子敲除小鼠细胞系。目前的研究描述了我们对这些增强子敲除系的基因组的分析，以检测可能的脱靶效应。生物信息学分析用于鉴定最可能的假定脱靶位点，并设计PCR引物，从创始人增强子缺失小鼠系的基因组DNA中扩增这些序列。然后对扩增的DNA进行测序，并与小鼠基因组序列进行对比，以检测脱靶效应。使用这种方法，我们无法在使用分析中使用的四种grna中的任何一种的三个方正系基因组中检测到脱靶效应的任何证据。这项研究表明，转基因小鼠的脱靶效应问题被夸大了，CAS9/CRISPR代表了一种从小鼠基因组中删除假定的神经肽基因增强子序列的高效和准确的方法。
We have successfully used comparative genomics to identify putative regulatory elements within the human genome that contribute to the tissue specific expression of neuropeptides such as galanin and receptors such as CB1. However, a previous inability to rapidly delete these elements from the mouse genome has prevented optimal assessment of their function in-vivo. This has been solved using CAS9/CRISPR genome editing technology which uses a bacterial endonuclease called CAS9 that, in combination with specifically designed guide RNA (gRNA) molecules, cuts specific regions of the mouse genome. However, reports of “off target” effects, whereby the CAS9 endonuclease is able to cut sites other than those targeted, limits the appeal of this technology. We used cytoplasmic microinjection of gRNA and CAS9 mRNA into 1-cell mouse embryos to rapidly generate enhancer knockout mouse lines. The current study describes our analysis of the genomes of these enhancer knockout lines to detect possible off-target effects. Bioinformatic analysis was used to identify the most likely putative off-target sites and to design PCR primers that would amplify these sequences from genomic DNA of founder enhancer deletion mouse lines. Amplified DNA was then sequenced and blasted against the mouse genome sequence to detect off-target effects. Using this approach we were unable to detect any evidence of off-target effects in the genomes of three founder lines using any of the four gRNAs used in the analysis. This study suggests that the problem of off-target effects in transgenic mice have been exaggerated and that CAS9/CRISPR represents a highly effective and accurate method of deleting putative neuropeptide gene enhancer sequences from the mouse genome.
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影响因子：
9.3
作者：
Shanley L;Lear M;Davidson S;Ross R;MacKenzie A
通讯作者：
MacKenzie A
影响因子：
4.6
作者：
Horii T;Arai Y;Yamazaki M;Morita S;Kimura M;Itoh M;Abe Y;Hatada I
通讯作者：
Hatada I
DOI：
10.1038/mtna.2015.37
发表时间：
2015-11-17
期刊：
Molecular therapy. Nucleic acids
影响因子：
--
作者：
Zhang XH;Tee LY;Wang XG;Huang QS;Yang SH
通讯作者：
Yang SH
DOI：
10.1126/science.aad5227
发表时间：
2016-01-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Slaymaker IM;Gao L;Zetsche B;Scott DA;Yan WX;Zhang F
通讯作者：
Zhang F
影响因子：
14.8
作者：
Ran, F. Ann;Hsu, Patrick D.;Zhang, Feng
通讯作者：
Zhang, Feng