A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish.
Contemporary improvements in the type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system offer a convenient way for genome editing in zebrafish. However, the low efficiencies of genome editing and germline transmission require a time-intensive and laborious screening work. Here, we reported a method based on in vitro oocyte storage by injecting oocytes in advance and incubating them in oocyte storage medium to significantly improve the efficiencies of genome editing and germline transmission by in vitro fertilization (IVF) in zebrafish. Compared to conventional methods, the prior micro-injection of zebrafish oocytes improved the efficiency of genome editing, especially for the sgRNAs with low targeting efficiency. Due to high throughputs, simplicity and flexible design, this novel strategy will provide an efficient alternative to increase the speed of generating heritable mutants in zebrafish by using CRISPR/Cas9 system.
College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China.|Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 400714, China.
Shao-Lin
SL
Xie
Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 400714, China.
Wan-Ping
WP
Bian
College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China.|Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 400714, China.
Chao
C
Wang
Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 400714, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
Muhammad
M
Junaid
College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China.
Ji-Xing
JX
Zou
Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 400714, China.
De-Sheng
DS
Pei
Nucleic Acids Res. 2015 Dec 15;43(22):e157
26253739
Fungal Genet Biol. 2016 Jan;86:47-57
26701308
Cell Res. 2013 Apr;23(4):465-72
23528705
Sci Rep. 2014 Oct 08;4:6545
25293390
Sci Rep. 2014 Jan 10;4:3632
24407151
Biol Open. 2015 Jan 16;4(2):180-5
25596277
Cell. 2014 Feb 13;156(4):836-43
24486104
Animal. 2015 Mar;9(3):464-70
25391393
Dev Dyn. 1995 Jul;203(3):253-310
8589427
Mol Plant. 2015 Dec 7;8(12 ):1820-3
26524930
Theriogenology. 2016 May;85(8):1499-506
26893166
Nature. 2012 Nov 1;491(7422):114-8
23000899
Biotechniques. 2015 Apr 01;58(4):161-70
25861928
Nat Methods. 2013 Aug;10(8):741-3
23817069
Cell Rep. 2016 Mar 8;14 (9):2263-72
26923600
Nat Biotechnol. 2015 May;33(5):538-42
25798939
Anim Biotechnol. 2015;26(4):279-88
26158460
PLoS One. 2015 May 26;10(5):e0128319
26010089
Fertil Steril. 2015 Dec;104(6):1426-34.e1-8
26353081
ACS Synth Biol. 2016 Apr 15;5(4):356-9
26714206
Theriogenology. 2016 Feb;85(3):509-518.e1
26483307
Int J Biochem Cell Biol. 2014 Oct;55:329-34
25194339
Nat Biotechnol. 2008 Jun;26(6):702-8
18500334
Yi Chuan. 2016 Feb;38(2):144-54
26907778
Nat Biotechnol. 2013 Mar;31(3):227-9
23360964
Hum Reprod. 2016 May;31(5):1024-33
26936884
PLoS One. 2013 Jul 09;8(7):e68708
23874735
Nat Methods. 2011 Jul 17;8(9):753-5
21765410
Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):13904-9
23918387
Genome Res. 2015 Jul;25(7):1030-42
26048245
PLoS One. 2014 May 29;9(5):e98186
24873830
Reproduction. 2008 Mar;135(3):285-92
18299421
Science. 2013 Feb 15;339(6121):819-23
23287718
J Mol Cell Biol. 2015 Dec;7(6):580-3
26459633
G3 (Bethesda). 2014 May 28;4(7):1291-5
24875628
Exp Anim. 2016 Jul 29;65(3):275-83
26972821
Development. 2013 Dec;140(24):4982-7
24257628
Transgenic Res. 2010 Jun;19(3):363-71
19821047
Genome Res. 2014 Jan;24(1):125-31
24253447
Anim Reprod Sci. 2014 Dec 30;151(3-4):262-8
25458322
J Reprod Dev. 2006 Oct;52(5):669-74
16873990
Nat Protoc. 2013 Nov;8(11):2281-308
24157548
Anim Reprod Sci. 2015 Mar;154:86-94
25592860
J Assist Reprod Genet. 2014 Nov;31(11):1553-5
25205204
Dev Dyn. 2013 Jan;242(1):44-52
23074011