title: [A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos].
abstract: Recent years have witnessed the rapid development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas9)system. In order to realize gene knockout with high efficiency and specificity in zebrafish, several labs have synthesized distinct Cas9 cDNA sequences which were cloned into different vectors. In this study, we chose two commonly used zebrafish-codon-optimized Cas9 coding sequences (zCas9_bz, zCas9_wc) from two different labs, and utilized them to knockout seven genes in zebrafish embryos, including the exogenous egfp and six endogenous genes (chd, hbegfa, th, eef1a1b, tyr and tcf7l1a). We compared the knockout efficiencies resulting from the two zCas9 coding sequences, by direct sequencing of PCR products, colony sequencing and phenotypic analysis. The results showed that the knockout efficiency of zCas9_wc was higher than that of zCas9_bz in all conditions.
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Feng-hua
authors.initials: FH
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Hou-peng
authors.initials: HP
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Si-yu
authors.initials: SY
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Xiong
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Zuo-yan
authors.initials: ZY
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Yong-hua
authors.initials: Y
authors.lastname: Sun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D003062:Codon / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000378:metabolism*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D003062:Codon; D012333:RNA, Messenger; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins