Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells

The clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell(PGC) transplantation(PGCT) to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic(MZ) mutants of tcf7l1a(gene essential for head development), pou5f3(gene essential for zygotic genome activation) and chd(gene essential for dorsal development) at F_1 generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1 a and chd, as MZtcf7l1 a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells.     

一种用于提高基因打靶效率的双荧光筛选策略

同源重组介导的基因打靶技术因其准确性高、引入的突变可以预测,已经成为功能基因组研究的重要工具。但是在真核生物体内,天然同源重组的概率极低,对后续的筛选和鉴别造成很大困扰。因此,建立一个广谱性的高效筛选策略极为重要。研究基于"双荧光"的可视化筛选,通过提高筛选效率,从而建立了一种显著提高同源重组打靶效率的新策略。该策略以绿色荧光蛋白基因和新霉素基因为正筛选标记,以红色荧光蛋白基因为负筛选标记。经过G418筛选后,挑取仅表达绿色荧光蛋白的抗性克隆,用于跨界PCR检测。研究尝试运用上述新策略,借助CRISPR/Cas9技术,对猪肌抑素基因进行同源重组介导的基因打靶。在选取的T1和T2两个靶位点处,"双荧光"策略筛选的细胞阳性率分别达到80.5%和86.7%,筛选效率得到显著提高。研究建立的"双荧光"可视化筛选策略具有高效性和广谱性,在动物基因编辑领域具有广阔的应用前景。     

毕赤酵母基因编辑技术研究进展

巴斯德毕赤酵母是一种重要的蛋白表达系统,基因编辑技术作为代谢工程的基本工具,对于毕赤酵母的代谢改造十分重要。近十年基因编辑技术发展迅速,除传统的同源重组和Cre/loxP重组外,相继出现了许多新的基因编辑技术,例如ZFN、TALEN和CRISPR/Cas9等,这些技术的出现使基因编辑更加简便高效。本文对毕赤酵母中传统和新型基因编辑技术的原理应用和研究进展进行了简要综述,并结合相关领域的发展对毕赤酵母基因编辑技术的发展进行了展望。     

Development of a CRISPR/Cas9 system in Entamoeba histolytica: proof of concept

The protozoan parasite Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death on a global scale. The lack of molecular tools for genome editing hinders the study of important biological functions of this parasite. Due to its versatility, the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system has been successfully used to induce site-specific genomic alterations, including in protozoan parasites. In this study, we optimised CRISPR-Cas9 for use as a genetic tool in E. histolytica. We chose a single plasmid approach containing both guide RNA (gRNA) and Cas9 nuclease expression cassettes. The amebic U6 promoter was used to drive the expression of the gRNA and its expression was confirmed by Northern blot analysis. Stable transfectant cell lines were obtained using a destabilising domain of dihydrofolate reductase fused to myc-tagged Cas9 (ddCas9). With this system, we were able to induce ddCas9 expression 16 h following treatment with the small molecule ligand trimethoprim (TMP). Stable cell lines expressing ddCas9 and Luc-gRNA or non-specific (NS)-gRNA were transiently transfected with a plasmid containing a mutated luciferase gene (pDeadLuc) targeted by Luc-gRNA and another plasmid with a truncated luciferase gene (pDonorLuc) to restore luciferase expression and consequent activity. We observed that luminescence signal increased for the cell line expressing Luc-gRNA, suggesting that homologous recombination was facilitated by Cas9 activity. This evidence is supported by the presence of chimeric DNA detected by PCR and confirmed by sequencing of the resulting repaired DNA obtained by homologous recombination. We believe this represents the first report of a CRISPR/Cas9 system use in Entamoeba and provides evidence that this genome editing approach can be useful for genetic studies in this early branching eukaryote.     

基于新的CRISPR/Cas9技术实现斑马鱼LHX9基因的快速编辑及对F0突变体性腺发育的初筛

目的:CRISPR/Cas9系统在斑马鱼的反向遗传学中的到了广泛应用,但突变基因的表型观察往往需要在突变鱼系的F1中进行,费时较长。LHX9作为LIM家族的一种转录因子,在胚胎早期的泌尿生殖嵴中有广泛分布;且LHX9基因敲除的小鼠存在性腺发育不良。本研究拟通过一种新的CRISPR/Cas9基因编辑技术,采用四条sgRNA对LHX9基因进行VASA转基因斑马鱼的基因敲除,以观察该基因缺陷对斑马鱼性腺发育的影响。方法:利用新的CRISPR/Cas9技术,设计四条针对斑马鱼LHX9基因3号外显子的20bp的sgRNA,通过非克隆体外转录得到靶位点的四条sgRNA。将以上靶位点的四条sgRNA与Cas9核酸酶蛋白同时注射入单细胞期的斑马鱼胚胎内,利用PCR鉴定突变型类型和突变比例。通过对LHX9基因突变体的VASA转基因斑马鱼进行荧光观察,发现LHX9基因缺陷的斑马鱼性腺发育的情况。结果:靶向Exon 3的四条sgRNA可成功编辑斑马鱼LHX9基因,敲除效率高达82%,Sanger测序发现产生10种不同的移码突变类型。通过该方法对VASA转基因斑马鱼的LHX9基因进行编辑,发现LHX9基因突变导致dph6的的斑马鱼原始生殖细胞增殖和迁移受到影响。结论:基于4条sgRNA注射的CRISPR/Cas9技术,可以快速地产生具有突变表型的G0斑马鱼,具有应用潜力。LHX9基因敲除导致原始生殖细胞的发育和迁移受到影响,提示该基因参与了斑马鱼早期性腺的发育。     

Defining endogenous barcoding sites for CRISPR/Cas9-based cell lineage tracing in zebrafish

There is a growing interest in developing experimental methods for tracking the developmental cell lineages of a complex organism.The recently developed CRISPR/Cas9-based barcoding method is,although highly promising,difficult to scale up because it relies on exogenous barcoding sequences that are engineered into the genome.In this study,we characterized 78 high-quality endogenous sites in the zebrafish genome that can be used as CRISPR/Cas9-based barcoding sites.The 78 sites are all highly expressed in most of the cell types according to single-cell RNA sequencing(scRNA-seq)data.Hence,the barcoding information of the 78 endogenous sites is recovered by the available scRNA-seq platforms,enabling simultaneous characterization of cell type and cell lineage information.     

Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.     

基于CRISPR/Cas9基因编辑技术的EGFP基因定点整合表达

目的:利用CRISPR/Cas9基因编辑技术,实现EGFP基因在CHO细胞ACTB基因座位置定点整合和表达,建立基于CRISPR/Cas9技术的外源基因定点整合和表达技术。方法:根据CHO细胞β-actin(ACTB)基因起始密码子区基因序列,设计相应CRISPR/Cas9系统,同时构建含有ACTB同源臂和EGFP基因的同源供体载体(donor vector),通过脂质体转染法同时转染CRISPR/Cas9和供体载体,流式分选EGFP阳性细胞,分析基因编辑技术在EGFP基因定点整合和表达方面的可行性。结果:构建了能有效切割CHO细胞ACTB基因的CRISPR/Cas9系统,筛选到EGFP定点整合至ACTB基因座并有效表达的细胞,ACTB基因缺失后由于γ-actin代偿性表达增强,ACTB缺失细胞形态和生长未受影响。结论:单纯依靠基因编辑技术可以实现1 kb以内的基因同源置换,但效率较低,如实现更大片段的外源基因置换,需借助其它实验技术。     

动物基因组编辑中提升CRISPR/Cas9介导的同源重组效率研究进展*

基因组编辑技术可以对DNA或RNA进行精准改造,极大地促进了生命科学的发展。CRISPR/Cas9系统在靶位点诱导DNA发生双链或单链损伤,细胞对损伤部位采用无供体模板的非同源末端连接(non-homologous end joining,NHEJ)或有供体模板的同源重组(homologous recombination,HR)修复。基于HR的基因组编辑策略通常被用于获得DNA的精准改造,而NHEJ在动物DNA损伤修复中起主导作用。为了提升HR效率,研究人员设计了多种方案,包括CRISPR/Cas9系统优化和DNA修复通路调控等。从DNA损伤修复途径、Cas9变体选择、sgRNA设计、供体模板设计、DNA修复途径相关蛋白功能调控、供体模板募集效率提升、细胞周期调控及编辑细胞生存效率提升等方面详细综述了相关研究成果,发现尚未开发出放之四海而皆准的HR提升策略,基于HR的基因组编辑需要针对具体案例制定个体化策略。旨在为动物基因组编辑中提升CRISPR/Cas9介导的HR效率研究提供理论参考,为动物基因功能分析、基因治疗和经济动物基因编辑育种提供帮助。     

Viral recombination systems limit CRISPR-Cas targeting through the generation of escape mutations

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应用Surrogate 报告载体提高RISPR/Cas9 对TMEM215基因的打靶效率*

目的:构建Surrogate报告载体,并利用Surrogate报告载体提高CRISPR/Cas9对HEK293T细胞TMEM215基因打靶效率。方法:构建针对人TMEM215的CRISPR/Cas9表达载体及相应Surrogate报告载体,两者共转HEK293T细胞,通过流式分析、T7EI检测、TA克隆测序等明确Surrogate报告载体对不同sgRNA打靶效率的检测及对基因修饰细胞的筛选富集作用。结果:流式分析结果表明,Surrogate报告载体成功检测出不同sgRNA的打靶效率,并筛选出高效率sgRNA;T7EI检测及TA克隆测序显示,外加嘌呤霉素抗性筛选时,Surrogate报告载体可有效富集基因修饰细胞。结论:成功构建Surrogate报告载体,并利用Surrogate报告载体提高CRISPR/Cas9对HEK293T细胞TMEM215基因的打靶效率。     

Efficient and specific generation of knockout mice using Campylobacter jejuni CRISPR/Cas9 system

The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) system is now widely utilized to generate genome engineered mice; however, some studies raised issues related to off-target mutations with this system. Herein, we utilized the Campylobacter jejuni Cas9 (CjCas9) system to generate knockout mice. We designed sgRNAs targeting mouse Tyr or Foxn1 and microinjected into zygotes along with CjCas9 mRNA. We obtained newborn mice from the microinjected embryos and confirmed that 50% (Tyr) and 38.5% (Foxn1) of the newborn mice have biallelic mutation on the intended target sequences, indicating efficient genome targeting by CjCas9. In addition, we analyzed off-target mutations in founder mutant mice by targeted deep sequencing and whole genome sequencing. Both analyses revealed no off-target mutations at potential off-target sites predicted in silico and no unexpected random mutations in analyzed founder animals. In conclusion, the CjCas9 system can be utilized to generate genome edited mice in a precise manner.     

应用RGS双荧光替代性报告载体提高CRISPR/Cas9对猪BMP15基因的打靶效率

我国是家猪养殖和消费大国,提高母猪的繁殖力对于促进我国生猪产业的发展具有重要的作用。排卵率和产仔数是影响家畜繁殖力的关键因素,其中BMP15 (bone morphogenetic protein 15)基因已被鉴定是控制绵羊排卵数和多胎性状的一个主效基因,然而目前在家猪BMP15基因中尚未发现类似绵羊多胎品系的天然突变。基于高等哺乳动物基因功能的保守性和CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)等基因组编辑技术对动物基因组定点修饰的高效性,应用CRISPR/Cas9技术对家猪BMP15基因进行精确的遗传修饰,使家猪获得类似多胎绵羊的天然突变,对于研究该基因对家猪繁殖力的影响以及培育高繁殖力家猪新品系具有重要的意义。本研究通过CRISPR/Cas9对长白猪胎儿成纤维(porcine embryonic fibroblasts, PEF)细胞中BMP15基因进行打靶,T7E1分析显示打靶效率仅有5%。随后通过共转染RGS双荧光替代性报告载体(RFP-GFP surrogate reporter),并应用流式细胞术分选出双荧光细胞,富集到基因组被CRISPR/Cas9修饰的细胞,使基因打靶效率提高至18%。本研究结果表明,应用RGS双荧光替代性报告载体可以有效提高CRISPR/Cas9在PEF细胞中对BMP15基因的打靶效率,为今后通过体细胞核移植技术培育BMP15基因编辑猪进行了有效的探索。     

High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10‐fold enhancements in GT frequencies have been achieved using DNA virus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV‐based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.     

应用SSA报告载体提高ZFN和CRISPR/Cas9对猪IGF2基因的打靶效率

IGF2(Insulin-like growth factor 2)基因作为最复杂多样的生长因子之一,对猪胎儿发育以及出生后生长发育和肌肉生成起着非常重要的作用。通过基因组编辑技术对我国本地猪种的IGF2基因作精确的遗传修饰,对于提高本地猪种的瘦肉率具有重要的育种意义。文章在蓝塘猪胎儿成纤维细胞(Porcine fetal fibroblasts, PEF)中检测了锌指核酸酶(Zinc finger nucleases, ZFN)和CRISPR/Cas9对IGF2基因的打靶效率,结果表明CRISPR/Cas9对IGF2基因的切割效率最高可达9.2%,显著高于ZFN的切割效率(<1%),但两者均未达到作为体细胞核移植(Somatic nuclear transfer, SCNT)供体细胞所需的打靶效率。应用SSA (Single-strand annealing)报告载体筛选技术来富集IGF2基因被ZFN和CRISPR/Cas9修饰过的PEF细胞,结果表明,该技术可使CRISPR/Cas9的打靶效率提高5倍左右,对ZFN的打靶效率具有更大的增强作用。     

Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system

The mammalian zygote-mediated CRISPR/Cas system can efficiently generate targeted genome-modified animals. However, this system is limited by the risk of off-target mutations. Here we show that offset-nicking by Cas9 nickase and paired gRNAs allows us to generate region deleted mice and targeted knock-in mice without off-target mutations.     

An evolved AAV variant enables efficient genetic engineering of murine T cells

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利用CRISPR/Cas9系统构建Tiki1基因修饰猪模型

Tiki1基因是哈佛大学儿童医学院贺熹教授实验室发现的一个对蛙头部的诱导起到决定性作用的新基因,但Tiki1基因在小鼠等啮齿类动物中缺失,因此无法利用小鼠等小动物来研究其在哺乳动物中的作用.本文利用CRISPR/Cas9系统结合体细胞克隆技术构建Tiki1基因修饰猪模型,研究Tiki1基因在猪发育中的作用.我们利用贺熹教授团队提供的人Tiki1基因序列,在猪的基因组数据库中比对出与其同源性最高的一段序列设计2个靶位点(g1和g2).以设计的靶位点构建打靶质粒转染猪胎儿成纤维细胞,经细胞筛选、PCR扩增及测序共鉴定了52个单细胞克隆株.最终选择靶位点g1为纯合双敲的5个单细胞克隆株和靶位点g2为纯合双敲的3个单细胞克隆株作为构建Tiki1基因敲除猪的核供体.我们共计构建了720个重组胚胎,分别植入3头代孕母猪,其中有1头经B超检测成功怀孕并妊娠到期产下13头发育正常的克隆猪,经测序鉴定其中12头为Tiki1基因双敲除猪模型,Tiki1基因敲除克隆猪健康存活至今.结果表明Tiki1基因对于猪早期发育的作用机理不同于蛙,其在猪早期发育的过程中的具体作用机理有待后续进一步的深入研究.