Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model.
Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model.
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Keops复合基因的急性多sGrNA敲低重现了稳定的敲除斑马鱼模型的小头畸形表型。
DOI：
10.1371/journal.pone.0191503
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Hildebrandt F
中科院分区：
文献类型：
--
作者：
Jobst-Schwan T;Schmidt JM;Schneider R;Hoogstraten CA;Ullmann JFP;Schapiro D;Majmundar AJ;Kolb A;Eddy K;Shril S;Braun DA;Poduri A;Hildebrandt F
直到最近，吗啉代寡核苷酸已广泛应用于斑马鱼作为一种快速有效的功能丧失检测方法。然而，与稳定的敲除系相比，脱靶效应和重现性问题影响了它们的进一步使用。在这里，我们采用了一种急性 CRISPR/Cas 方法，使用多个单引导 RNA 同时靶向两个示例基因（osgep 或 tprkb）中的不同位置，以增加在注射的 F0 代中两个等位基因上产生突变的可能性，并实现与吗啉代相似的效果，但具有稳定系的可重复性。这种多单向导RNA方法导致每个等位基因上至少一个突变的中位可能性>99%，并且深度测序揭示了sgRNA特异性插入/删除特征。免疫印迹显示 Osgep 和 Tprkb 蛋白显着减少。对于这两个基因，急性多 sgRNA 敲除重现了我们之前在稳定敲除系中观察到的小头畸形表型和存活率降低，尽管急性多 sgRNA 敲除的情况较温和。最后，我们通过深度测序量化诱变程度，并提供数学模型来量化双等位基因功能丧失突变的机会。我们的研究结果可以推广到针对任何感兴趣的斑马鱼基因的急性和稳定的 CRISPR/Cas 靶向。
Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest.
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DOI：
10.1126/science.aaf5573
发表时间：
2016-08-05
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Abudayyeh OO;Gootenberg JS;Konermann S;Joung J;Slaymaker IM;Cox DB;Shmakov S;Makarova KS;Semenova E;Minakhin L;Severinov K;Regev A;Lander ES;Koonin EV;Zhang F
通讯作者：
Zhang F
影响因子：
46.9
作者：
通讯作者：
--
DOI：
10.1073/pnas.1308335110
发表时间：
2013-08-20
影响因子：
11.1
作者：
Jao, Li-En;Wente, Susan R.;Chen, Wenbiao
通讯作者：
Chen, Wenbiao
影响因子：
11.8
作者：
Kok, Fatma O.;Shin, Masahiro;Ni, Chih-Wen;Gupta, Ankit;Grosse, Ann S.;van Impel, Andreas;Kirchmaier, Bettina C.;Peterson-Maduro, Josi;Kourkoulis, George;Male, Ira;DeSantis, Dana F.;Sheppard-Tindell, Sarah;Ebarasi, Lwaki;Betsholtz, Christer;Schulte-Merker, Stefan;Wolfe, Scot A.;Lawson, Nathan D.
通讯作者：
Lawson, Nathan D.
影响因子：
46.9
作者：
Morgens DW;Deans RM;Li A;Bassik MC
通讯作者：
Bassik MC