A Rapid and Cheap Methodology for CRISPR/Cas9 Zebrafish Mutant Screening

被引:21
作者
D'Agostino, Ylenia [1 ]
Locascio, Annamaria [1 ]
Ristoratore, Filomena [1 ]
Sordino, Paolo [1 ]
Spagnuolo, Antonietta [1 ]
Borra, Marco [2 ]
D'Aniello, Salvatore [1 ]
机构
[1] Staz Zool Anton Dohrn, Biol & Evolut Marine Organisms BEOM, I-80121 Naples, Italy
[2] Staz Zool Anton Dohrn, Res Infrastruct Marine Biol Resources RIMAR, I-80121 Naples, Italy
关键词
Zebrafish knock-out mutants; CRISPR/Cas9; qPCR; Derivative melting curve; Mutation screening; CAS SYSTEM; GENOME; NUCLEASE; ANIMALS;
D O I
10.1007/s12033-015-9905-y
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The introduction of new genome editing tools such as ZFNs, TALENs and, more recently, the CRISPR/Cas9 system, has greatly expanded the ability to knock-out genes in different animal models, including zebrafish. However, time and costs required for the screening of a huge number of animals, aimed to identify first founder fishes (F0), and then carriers (F1) are still a bottleneck. Currently, high-resolution melting (HRM) analysis is the most efficient technology for large-scale InDels detection, but the very expensive equipment demanded for its application may represent a limitation for research laboratories. Here, we propose a rapid and cheap method for high-throughput genotyping that displays efficiency rate similar to the HRM. In fact, using a common ViiA (TM) 7 real-time PCR system and optimizing the parameters of the melting analysis, we demonstrated that it is possible to discriminate between the mutant and the wild type melting curves. Due to its simplicity, rapidity and cheapness, our method can be used as a preliminary one-step approach for massive screening, in order to restrict the scope at a limited number of embryos and to focus merely on them for the next sequencing step, necessary for the exact sequence identification of the induced mutation. Moreover, thanks to its versatility, this simple approach can be readily adapted to the detection of any kind of genome editing approach directed to genes or regulatory regions and can be applied to many other animal models.
引用
收藏
页码:73 / 78
页数:6
相关论文
共 16 条
[1]   Repositories share key research tools [J].
Baker, Monya .
NATURE, 2014, 505 (7483) :272-272
[2]   A Primer for Morpholino Use in Zebrafish [J].
Bill, Brent R. ;
Petzold, Andrew M. ;
Clark, Karl J. ;
Schimmenti, Lisa A. ;
Ekker, Stephen C. .
ZEBRAFISH, 2009, 6 (01) :69-77
[3]  
Chiang P., 1996, GENOME RES, V6, P1023
[4]   CRISPR/Cas, the Immune System of Bacteria and Archaea [J].
Horvath, Philippe ;
Barrangou, Rodolphe .
SCIENCE, 2010, 327 (5962) :167-170
[5]   A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis [J].
Huang, Mo Chao ;
Cheong, Wai Chye ;
Lim, Li Shi ;
Li, Mo-Huang .
ELECTROPHORESIS, 2012, 33 (05) :788-796
[6]   Efficient genome editing in zebrafish using a CRISPR-Cas system [J].
Hwang, Woong Y. ;
Fu, Yanfang ;
Reyon, Deepak ;
Maeder, Morgan L. ;
Tsai, Shengdar Q. ;
Sander, Jeffry D. ;
Peterson, Randall T. ;
Yeh, J-R Joanna ;
Joung, J. Keith .
NATURE BIOTECHNOLOGY, 2013, 31 (03) :227-229
[7]   Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system [J].
Jao, Li-En ;
Wente, Susan R. ;
Chen, Wenbiao .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2013, 110 (34) :13904-13909
[8]   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity [J].
Ran, F. Ann ;
Hsu, Patrick D. ;
Lin, Chie-Yu ;
Gootenberg, Jonathan S. ;
Konermann, Silvana ;
Trevino, Alexandro E. ;
Scott, David A. ;
Inoue, Azusa ;
Matoba, Shogo ;
Zhang, Yi ;
Zhang, Feng .
CELL, 2013, 154 (06) :1380-1389
[9]   The CRISPR-Cas immune system: Biology, mechanisms and applications [J].
Rath, Devashish ;
Amlinger, Lina ;
Rath, Archana ;
Lundgren, Magnus .
BIOCHIMIE, 2015, 117 :119-128
[10]   The new CRISPR-Cas system: RNA-guided genome engineering to efficiently produce any desired genetic alteration in animals [J].
Seruggia, Davide ;
Montoliu, Lluis .
TRANSGENIC RESEARCH, 2014, 23 (05) :707-716