The Application of NHEJ-CRISPR/Cas9 and Cre-Lox System in the Generation of Bivalent Duck Enteritis Virus Vaccine against Avian Influenza Virus.
The Application of NHEJ-CRISPR/Cas9 and Cre-Lox System in the Generation of Bivalent Duck Enteritis Virus Vaccine against Avian Influenza Virus.
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DOI：
10.3390/v10020081
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发表时间：
2018-02-13
期刊：
影响因子：
--
通讯作者：
Iqbal M
中科院分区：
文献类型：
--
作者：
Chang P;Yao Y;Tang N;Sadeyen JR;Sealy J;Clements A;Bhat S;Munir M;Bryant JE;Iqbal M
在鸭流行高致病性禽流感（HPAI）的地区，迫切需要针对鸭的疫苗来预防禽流感，以帮助流感疾病的控制工作。鸭肠炎病毒（DEV）基因组大，宿主范围窄，是一种很有希望的候选病毒载体，可用于开发针对鸭的疫苗。成簇规则间隔回文重复序列（CRISPR）/Cas9系统是一种通用的基因编辑工具，已被证明有利于基因修饰和重组DNA病毒载体疫苗的构建。目前，有两种常用的基因插入方法：非同源末端连接（NHEJ）和同源定向修复（HDR）。由于其在效率和独立于同源臂的分子要求方面的优势，我们利用NHEJ依赖性CRISPR/Cas9将流感血凝素（HA）抗原表达盒插入DEV基因组中。插入物最初用报告绿色荧光蛋白（GFP）标记，随后使用Cre-Lox系统去除GFP基因插入物。此外，通过引入不依赖于病毒基因组的双诱饵序列，建立了通用供体质粒系统。总之，我们提供了使用整合的NHEJ-CRISPR/Cas9和Cre-Lox系统产生针对流感病毒的重组DEV病毒载体疫苗的原理证明。
Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP), and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.
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