Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish.
Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish.
复制标题
斑马鱼基因组编辑中各种核定位信号融合 Cas9 蛋白和 Cas9 mRNA 的比较
DOI：
10.1534/g3.117.300359
复制
发表时间：
2018-03-02
期刊：
影响因子：
--
通讯作者：
Zu Y
中科院分区：
文献类型：
--
作者：
Hu P;Zhao X;Zhang Q;Li W;Zu Y
聚类规则间隔短回文重复序列(CRISPR)/Cas9系统已在多种生物中被证明是一种高效、精确的基因组编辑技术。然而，细胞核定位信号（NLS）融合到不同末端和Cas9 mRNA的Cas9蛋白的基因编辑效率尚未得到系统的比较。在这里，我们比较了带有NLS的Cas9蛋白与斑马鱼色素沉着相关的N-、C-或N-和C-端以及N-NLS-Cas9-NLS-C mRNA融合的能力，以靶向tyr基因和gol基因中的两个位点。表型分析显示，所有类型的Cas9导致注射胚胎中相似比例的色素沉着降低。T7核酸内切酶I （T7E1）基因组分析表明，所有类型的Cas9在4个靶点上相似地诱导突变。测序结果进一步证实，在所有四种类型的cas9注射胚胎中，靶位点的indel突变频率较高（tyr1 > 66%, tyr2 > 73%, gol1 > 50%, gol2 > 35%），并且indel突变的类型多种多样（超过6种）。此外，所有类型的Cas9在多重基因组编辑上都表现出高效的靶向突变，从而同时产生多种表型。总之，我们得出结论，各种nls融合的Cas9蛋白和Cas9 mrna在靶向单个或多个基因方面具有相似的基因组编辑效率，这表明CRISPR/Cas9基因组编辑的效率高度依赖于引导rna （gRNAs）和基因位点。这些发现可能有助于简化使用CRISPR/Cas9系统进行基因编辑的Cas9的选择。
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites (tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system.