Generation of α‐1,3‐Galactosyltransferase‐Deficient Porcine Embryonic Fibroblasts by CRISPR/Cas9‐Mediated Knock‐in of a Small Mutated Sequence and a Targeted Toxin‐Based Selection System

The CRISPR/Cas9 system has enabled the editing of mammalian genomes; however, its applicability and efficiency in the pig genome has not been studied in depth. The α‐gal epitope synthesized by α‐1,3‐galactosyltransferase gene (GGTA1) is known as a xenoantigen obtained upon pig‐to‐human xenotransplantation. We here employed the CRISPR/Cas9 system‐mediated knock‐in of endogenous GGTA1 via targeted homologous recombination (HR). Linearized donors with ~800‐bp homology flanking the CRISPR/Cas9 target site [exon 4 (containing ATG) of GGTA1] served as a template for gene targeting by HR. Using a targeted toxin strategy to select clones lacking α‐gal epitope expression, we successfully obtained several knock‐in clones within 3 weeks of initial transfection. These results suggest that the use of CRISPR/Cas9‐mediated HR to knock‐in a mutated fragment at defined loci represents an efficient strategy to achieve the rapid modulation of genes of interest in swine cells and is a promising tool for the creation of KO piglets.     

CRISPR/Cas9-mediated MSTN disruption accelerates the growth of Chinese Bama pigs

CRISPR/Cas9-mediated genome editing technology is a simple and highly efficient and specific genome modification approach with wide applications in the animal industry. CRISPR/Cas9-mediated genome editing combined with somatic cell nuclear transfer rapidly constructs gene-edited somatic cell-cloned pigs for the genetic improvement of traits or simulation of human diseases. Chinese Bama pigs are an excellent indigenous minipig breed from Bama County of China. Research on genome editing of Chinese Bama pigs is of great significance in protecting its genetic resource, improving genetic traits and in creating disease models. This study aimed to address the disadvantages of slow growth and low percentage of lean meat in Chinese Bama pigs and to knock out the myostatin gene (MSTN) by genome editing to promote growth and increase lean meat production. We first used CRISPR/Cas9-mediated genome editing to conduct biallelic knockout of the MSTN, followed by somatic cell nuclear transfer to successfully generate MSTN biallelic knockout Chinese Bama pigs, which was confirmed to have significantly faster growth rate and showed myofibre hyperplasia when they reached sexual maturity. This study lays the foundation for the rapid improvement of production traits of Chinese Bama pigs and the generation of gene-edited disease models in this breed.     

CRISPR/Cas9技术在猪、鸡中的应用研究进展

基因编辑是利用核酸酶在基因组的特定位点产生DNA双链断裂，从而触发细胞自身的DNA损伤修复机制，实现对靶基因序列进行位点特异性修饰。规律成簇间隔短回文重复序列（clustered regularly interspersed short palindromic repeat，CRISPR）及其相关蛋白9（Cas9）系统作为第3代基因编辑技术在农业和基因治疗研究中发挥着重要作用，与传统的锌指核酶和转录激活因子样效应物核酶基因编辑方法相比，它可以靶向基因的任何位点引起DNA双链断裂，实现目标基因的精准敲除或插入外源片段，并能快速、高效地修饰基因组，包括基因敲除、敲入、抑制、激活等，是应用最广泛的基因编辑工具。CRISPR/Cas9技术的出现彻底改变了生命科学、医学和遗传学研究现状。近年来，利用CRISPR/Cas9技术对动物基因组进行修饰，开启了畜禽分子育种的新纪元，不仅极大地推动了现代畜禽养殖技术的发展，而且为人类医学研究做出了特殊贡献，特别是在猪和鸡上。作者以猪和鸡为对象，综述了CRISPR/Cas9技术在异体器官移植供体、人类疾病的生物模型、抗病育种材料、全基因组高通量筛选等方面的研究进展，以及如何利用CRISPR技术快速又简单地生产出基因编辑猪、鸡，为推动CRISPR技术制备其他基因编辑动物提供参考依据。     

CRISPR/Cas9介导的绵羊胎儿成纤维细胞肌肉生长抑制素基因敲除研究

肌肉生长抑制素(myostatin,MSTN)基因突变可引起动物出现"双肌"性状,提高产肉性能。利用CRISPR/Cas9技术制备MSTN基因敲除的绵羊胎儿成纤维细胞,为制备MSTN基因敲除羊提供材料。设计构建4个靶向MSTN基因的CRISPR/Cas9载体,脂质体转染细胞后,通过SURVEYOR分析和测序等方法对敲除效率进行检测,采用极限稀释法挑选稳定敲除的细胞系。试验成功构建了4个靶向MSTN基因的CRISPR/Cas9载体,细胞转染后,测序结果显示pX330-target 1和pX330-target 4载体作用的靶位点处出现突变,SURVEYOR分析检测其在靶位点产生切割的效率分别为24.20%和10.18%。通过极限稀释法,获得12个MSTN基因突变的细胞克隆,其中1个为纯合突变。序列比对发现靶位点处有小片段碱基插入或缺失突变,部分会出现移码突变。成功利用CRISPR/Cas9系统实现了绵羊MSTN基因敲除,证明该系统可有效应用于绵羊基因编辑,产生的突变细胞系为制备MSTN基因敲除羊提供了材料。     

Knockout of Myostatin Gene by CRISPR/Cas9 System in Ovine Fetal Fibroblasts

Mutation in myostatin (MSTN) gene resulted in double muscle effect,generating more mutton.To knock out MSTN gene in sheep fetal fibroblast by CRISPR/Cas9 system and obtain MSTN gene knockout cell lines,four plasmids were designed and constructed to target MSTN gene,and confirmed correctly by sequencing.The correct plasmids were delivered into the fetal fibroblast cells.The targeting efficiency was detected using SURVEYOR assay Kit.The stable transfected cell colonies were obtained via limiting dilution procedure.The sequence results demonstrated that the pX330-target 1 and pX330-target 4 plasmids could successfully knockout MSTN gene,and the targeting efficiency were 24.20% and 10.18%,respectively.Twelve MSTN gene knockout cell colonies were obtained via limiting dilution,and one of them was homozygous mutation.Several indel mutations were discovered at specific site,and some of them were frame-shift mutation.Therefore,we concluded that the CRISPR/Cas9 system could apply to the gene editing of sheep efficiently,and the gene knockout cell clones had potential application in generating MSTN gene knockout sheep.     

Generation of PDX‐1 mutant porcine blastocysts by introducing CRISPR/Cas9‐system into porcine zygotes via electroporation

Recently, we established the GEEP (“gene editing by electroporation of Cas9 protein”) method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome‐editing efficiency. To produce an animal model for diabetes, we targeted PDX‐1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX‐1 exon 1 was introduced into in vitro‐fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX‐1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX‐1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.     

CRISPR/Cas9基因组编辑技术及其在草类植物中的应用

CRISPR/Cas9系统作为新一代基因组编辑技术,一经问世就受到广泛关注。CRISPR/Cas9系统具有对特定基因位点进行精确修饰(包括敲除、插入、替换等)的强大功能,同时具有操作简单、效率高、适应性广等技术优势,目前已在微生物、植物、动物以及人类基因治疗等整个生命科学领域得到应用。本文简要介绍了CRISPR/Cas9技术的发展历程、作用机理、技术优势、功能及应用领域,总结该技术在植物领域的基因组定向编辑中的研究进展。并提出了这项技术在草类植物基因功能分析、基因代谢调控途径与遗传改良等方面的应用前景,为相关领域的研究工作奠定了基础。     

CRISPR/Cas9技术在猪常见病毒性疾病防控中的应用

CRISPR/Cas系统是一种存在于古生菌和细菌中重要的适应性免疫系统,CRISPR/Cas9技术可以实现对特定基因序列进行编辑,目前已经成为应用最广泛的基因编辑工具,利用其可以实现对病毒进行基因改造或解析宿主与病毒相互作用,优化疫苗生产和抗病毒动物分子育种等.猪作为我国最重要的食品动物,在国民经济和社会稳定中发挥着重...     

CRISPR/Cas9技术在畜禽育种中的研究进展

利用传统方法在畜禽特定基因座上进行基因组修饰时,只能通过在体细胞中进行同源重组再经细胞核移植实现。传统同源重组方法的困难性和低效性阻碍了基因修饰在畜禽遗传育种中的广泛应用。近年来,位点特异性核酸内切酶的发现为靶向基因修饰提供了一条更直接的途径,主要由于这些酶能直接在DNA序列上进行一步式的基因编辑。成簇规律间隔短回文重复序列/Cas关联蛋白9(clustered regularly interspersed short palindromic repeat/CRISPR associated protein 9,CRISPR/Cas9)是一种RNA导向的DNA内切酶,精准定位于特定的靶位点,高效完成RNA导向的DNA识别及编辑。CRISPR/Cas9技术作为精准而强大的第3代基因组编辑工具,已经成功应用于猪、牛、山羊、绵羊和鸡上,这些CRISPR/Cas9基因编辑畜禽可作为研究人或畜禽生理和病理的生物模型、生产功能性蛋白质的生物反应器或器官移植的供体。特别是在畜禽生产方面,CRISPR/Cas9基因编辑可用于改善生产遗传特性及畜产品质量,提高畜禽对疾病的抵抗力。作者对当前畜禽中特定位点基因组修饰的CRISPR/Cas9技术的原理及基因组编辑在畜禽育种中应用的最新进展进行了综述,以期为推进CRISPR/Cas9技术在畜禽育种中的研究提供参考。     

CRISPR/Cas9系统在猪基因编辑中的研究进展

随着基因编辑技术不断发展,从最初的同源重组到现在的CRISPR系统,基因编辑的操作步骤得到简化,编辑效率得到提高且降低了成本。CRISPR/Cas9系统可在RNA的引导下对特定DNA位点进行靶向编辑,CRISPR/Cas9技术操作简单、定位准确,近年来被广泛使用。本文主要介绍CRISPR/Cas9系统,并对该系统在猪遗传改良、抗病育种、构建人类疾病模型及异种器官移植4个方面的应用研究进行综述。     

CRISPR/Cas9基因编辑技术在绵羊中的应用研究进展

随着基因编辑技术的迅速发展,研究者利用基因编辑技术在越来越多的领域中取得了许多突破性的进展。目前,众多的基因编辑工具中CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)系统应用最为广泛。CRISPR/Cas9及其衍生出的碱基编辑器(base editor, BE)和先导编辑器(prime editor, PE)系统使研究者可以在目标基因组中简单、高效地完成碱基的删除、插入和替换等修饰。CRISPR/Cas9相关基因编辑技术在生产具有特定遗传特征的动物方面有巨大的潜在价值。绵羊作为具有许多重要经济性状的家畜,是理想的基因工程研究大动物模型。CRISPR/Cas9相关基因编辑技术已经用于绵羊基因组工程研究,如生产具有优良特性的品种,利用乳腺生产疾病治疗药物,构建用于人类疾病和再生医学研究的动物模型。作者从CRISPR/Cas9基因编辑技术的原理出发,着重阐述了利用CRISPR/Cas9系统进行基因编辑的具体流程,并概述了CRISPR/Cas9系统...     

CRISPR/Cas9基因编辑技术在家畜育种新材料创制中的研究进展

规律性成簇间隔的短回文重复序列/CRISPR相关蛋白（clustered regularly interspaced short palindromic repeats/CRISPR-associated,CRISPR/Cas）系统是最新一代能对细胞或生物体基因组进行精准编辑的基因工程技术,与前两代基因编辑技术ZFN和TALEN相比,CRISPR/Cas具有应用成本低、适用编辑范围广、打靶效率高、操作简单、可支持多位点操作等诸多优点。近年来,CRISPR/Cas系统尤其是Type II类、A型的CRISPR/Cas9系统已经作为最新一代基因编辑技术被广泛应用于提高家畜繁殖效率、生产性能、抗病性以及动物模型构建等研究中,并创制了一批基因编辑牛羊育种新材料。本文就其发展历程、技术改造和优化最新进展以及在家畜繁殖性状、生产性状和抗病性状等方面的研究应用进行综述,重点介绍了该系统在家畜育种学研究中已取得的最新进展,并就CRISPR/Cas9基因编辑技术在家畜育种应用中现存的问题及其应用前景进行简要论述。     

两种不同猪瘟疫苗对育肥猪的免疫效果比较

为比较猪瘟疫苗E2基因工程亚单位疫苗和猪瘟活疫苗对育肥猪免疫效果和生产成绩的影响,以便为规模猪场在选择猪瘟疫苗时提供参考。本试验从汉中某代养场选取了一批健康的断奶仔猪分成三组,A组免疫猪瘟E2基因工程亚单位疫苗（简称E2亚单位疫苗）,B组免疫猪瘟活疫苗（脾淋源）,C组为对照组不做猪瘟疫苗免疫。免疫60 d后各组随机抽取10、15和12份血样用猪瘟病毒抗体检测试剂盒检测猪瘟抗体水平。结果表明接种的两种猪瘟疫苗对育肥猪的免疫效果差异极显著（P<0.01）,其中E2亚单位疫苗相比于猪瘟活疫苗能够明显提高育肥猪抗体阻断率和降低离散度,并且有助于育肥猪的健康生长。     

利用CRISPR/Cas9和λ-Red级联技术构建产肠毒素大肠杆菌LT敲除菌株

本研究旨在利用CRISPR/Cas9和λ-Red级联的技术对产肠毒素大肠杆菌（enterotoxigenic Escherichia coli，ETEC）K88的热不稳定性肠毒素（heat-labile toxin，LT）基因进行无痕敲除并获得K88 LT^-缺陷菌株。通过序列比对获取LT两端同源序列，并构建包含LT边界、氯霉素筛选标记、sgRNA和LT同源臂的供体片段；将供体片段转化至ETEC K88，同时分别利用λ-Red同源重组系统和CRISPR/Cas9基因编辑系统，对LT基因进行敲除；通过PCR验证获得了K88 LT^-缺陷菌株，并通过试验测定了敲除菌株的溶血能力和生长曲线。结果显示，λ-Red同源重组系统可成功地将LT基因替换为相应的供体片段，CRISPR/Cas9基因编辑系统可高效地对筛选标记进行删除，最终通过λ-Red和CRISPR/Cas9结合的基因编辑系统可成功对ETEC K88的LT基因进行无痕敲除。体外试验结果表明，K88 LT^-缺陷菌株的溶血能力丧失，并且生长速度比野生型菌株减缓，LT可能和ETEC K88的致病能力和生长性能有关。表明λ-Red和CRISPR/Cas9级联的基因敲除方法可用于LT毒素基因及其他一些大肠杆菌基因的敲除。K88 LT^-缺陷菌株的构建为下一步研究LT毒素的致病机制奠定基础。     

无PAM限制的CRISPR/Cas9系统构建及效率验证

CRISPR/Cas9系统在进行靶向基因敲除、转录激活以及抑制时,需要通过识别Cas9靶位点上的一个原间隔子相邻基序(PAM)序列进行特异性靶向基因编辑。现阶段,被广泛认可的化脓性链球菌SpCas9仅识别比较短的PAM为NGG(N为任意碱基,G为鸟嘌呤)。本实验在野生型SpCas9、PAM为NG的xCas9和SpCas9-NG的基础上,构建预期PAM为NNG的突变体SpCas9NNG、预期PAM为NNN的突变体xCas9NNN和ngCas9NNN,通过双荧光素酶报告基因检测评价其切割活性。结果显示:本次实验构建的ngCas9NNN突变体对PAM为NNN的靶标DNA具有显著的切割活性,为后续拓宽CRISPR/Cas9系统的靶向范围提供重要参考。     

CRISPR/Cas9基因敲除研究进展

功能基因组学的发展促进基因敲除技术的进步,基因敲除技术从载体的构建到细胞筛选再到动物模型都取得长足进步。基因敲除手段种类繁多,其中CRISPR/Cas9作为常用的基因编辑技术,具有高效、便捷、精确等优点,在农业、医学、生物学的模型构建中被广泛运用。随着科研工作的逐渐深入,CRISPR/Cas9技术逐渐渗透到植物和微生物研究领域中。文章综述近年来CRISPR/Cas9的应用,为科研工作的开展提供参考。     

CRISPR/Cas9技术及其在猪基因编辑中的应用

CRISPR/Cas9（clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9）是一种新型基因编辑系统,是由细菌和古细菌对抗入侵病毒及外源DNA的适应性免疫防御系统演变而来的。该系统具有简易、精准、经济和高效的优点,目前已广泛应用于基因编辑及其相关研究中。本文总结了近几年CRISPR/Cas9技术在猪基因组编辑中的研究进展,并展望了其应用前景。     

Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic manipulation

Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy-dependence measured using a CRISPR/Cas9 live-cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO-1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non-essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (D_m) values ranging from 4.6–15.8 μM and 2.1–5.1 μM for measures of anti-proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score with D_m values correlating with VPS34 response (r = 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy-dependent and sensitive to HCQ at pharmacologically-relevant exposures.     

CRISPR/dCas9技术在基因表达调控中的研究进展

CRISPR/dCas9是在CRISPR/Cas9基因编辑系统的基础上改造升级建立起的一种用于调控基因组转录与表观遗传修饰的系统,它不仅继承了CRISPR/Cas9系统的精准性,同时还展现出了良好的作用效果。在该系统中dCas9蛋白保留了Cas9蛋白结合DNA的能力而切割功能不复存在。将dCas9蛋白与不同的激活、抑制效应子域和表观遗传调控酶偶联,可以对基因表达与表观遗传修饰进行精确调控。组蛋白乙酰化、组蛋白甲基化、DNA甲基化等表观遗传修饰过程是基因表达的基础,对整个生命过程作出了巨大的贡献,同时表观遗传与多种疾病和癌症都存在因果关系,因此以CRISPR/dCas9系统为框架的不同表观遗传修饰系统在人类疾病治疗和癌症研究领域具有重要的研究价值。笔者简要介绍了CRISPR/Cas9系统的发现过程以及作用原理,主要总结了以CRISPR/dCas9系统为框架的不同调控系统在基因表达调控和表观遗传调控中的应用以及优化过程,以期为从事相关领域的科研工作者提供一些参考。