Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers

Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non‐homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ～20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25‐fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR‐Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.     

同源重组法构建枯草芽孢杆菌转酮酶缺失突变菌株

采用同源重组法高效构建枯草芽孢杆菌转酮酶(tkt)缺失突变株。以大肠杆菌(E.coliDH5α)质粒pBlUSKM为框架,构建出基于枯草杆菌(B.S104)tkt基因位点的整合载体pb-Trs-n,将此载体重组到Bacillus subtilis104中,从新霉素抗性平板上挑取转化子,整合载体pb-Trs-n的测序结果与Kunst.F报道的tkt基因高度同源(98.9%)。同源重组后,B.S104染色体上的tkt基因(2 004bp)部分与载体pb-Trs-n的neo基因(1 197bp)发生了同源交换,确定了该转化子为枯草芽孢杆菌转酮酶缺失突变株(tkt-,neo),该方法构建枯草芽孢杆菌转酮酶(tkt)缺失突变株是可行的,为D-核糖工程菌的研究奠定了基础。     

枯草芽孢杆菌cdd基因敲除及对胞苷发酵的影响

目的:通过敲除出发菌株上的胞苷脱氨酶基因,阻断嘧啶代谢通量由胞苷流向尿苷和尿嘧啶,选育胞苷产生菌。方法:采用同源重组的方法敲除枯草芽孢杆菌TS8的胞苷脱氨酶基因cdd,并通过遗传稳定性实验验证其缺失标记和胞苷产量,通过摇瓶发酵实验对比出发菌株和缺失株的产苷水平。结果:cdd基因缺失菌株TSb发酵72h,发酵液中胞苷产量达到1.72g/L,与原始菌株相比提高了44.19%,且遗传性状稳定。结论:cdd基因的缺失可有效阻断嘧啶代谢通量由胞苷流向尿苷和尿嘧啶,提高胞苷产量。     

A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis phages

Here, we present pRH030, a new CRISPR-Cas9 tool for the genetic engineering of Bacillus phages and beyond. It is based on the Streptococcus pyogenes cas9 with its native constitutive promoter, tracrRNA, and a gRNA precursor. The constitutive expression of Cas9 was conducive to the inactivation of viral attackers and enhanced phage mutagenesis efficiency up to 100%. The gRNA precursor can be built up to an artificial CRISPR array with up to 5 spacers (target sequences) assembled from ordinary oligonucleotides and directly cloned into pRH030. Required time and resources remain comparable to a single gRNA cloning. These properties make pRH030 an attractive new system for the modification of Bacillus phages and qualify it for research beyond genetic construction.     

CRISPR/Cas9介导的同源重组技术构建猪肌抑素基因敲除细胞系

肌抑素是肌肉增生的负调控因子,为改良家畜产肉性状的重要候选基因。本研究设计了Cas9/sgRNA表达载体与供体DNA,通过电转染方法将其导入猪PK15细胞,经G418抗性筛选和荧光蛋白标记甄别,筛选到带阳性标记的细胞克隆。通过跨界PCR、长距离PCR、Western印迹、Southern印迹及PCR产物测序,证明了猪肌抑素的第3外显子序列特异性同源重组事件的发生。在猪肌抑素的第3外显子区域找到了1个有效的CRISPR/Cas9打靶位点,带阳性标记的细胞克隆经过多次筛选分离,获得了肌抑素单等位基因失活的稳定细胞系,为深入研究肌抑素的功能提供了重要的实验材料。     

Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing

The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.     

Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.     

基于CRISPR/Cas系统的噬菌体基因组编辑

对原核生物获得性免疫系统CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR- associated genes)的研究促进了新一代基因组编辑工具的产生和发展。噬菌体既是原核生物CRISPR阵列(CRISPR array)进化的原动力,又是CRISPR/Cas系统防御的对象。噬菌体功能基因组学研究的速率却落后于发现新噬菌体和测定基因组序列的速率。基于CRISPR/Cas系统的噬菌体基因组编辑,可为噬菌体功能基因组学研究提供新手段。本文评述了基于CRISPR/Cas系统编辑噬菌体基因组的几例开创性研究,并且比较了多种操作程序的异同点和优缺点。同时,进一步构建了联合使用CRISPR/Cas系统与噬菌体重组系统开展噬菌体基因组编辑的新方案,讨论了新方案的潜在局限性,并对如何选择不同方案给予了建议。     

Targeting the Bacillus subtilis genome: an efficient and clean method for gene disruption

A method to disrupt multiple Bacillus subtilis genes is described. A resistance cassette is used to interrupt an amplified target sequence from the B. subtilis chromosome. The cassette is composed of a gene conferring resistance to chloramphenicol (Cm) or spectinomycin (Sp) flanked by two directly oriented β cognate sites (six site) (SCS or SSS, respectively). The linearized construct is used to transform B. subtilis competent cells with selection for Cm or Sp resistance. Transformants with the desired gene disrupted by the SCS or SSS cassette, integrated by a double cross-over event, were confirmed by PCR analysis. A segregationally unstable plasmid-borne β site-specific recombinase is transferred into the background. Protein β catalyzes excision of the intervening sequence between the two six sites leading to a target gene disrupted only by a six site. This site has an internal promoter capable of reading downstream genes. To generate multiple disruptions, the cycle can be repeated many times provided that two six sites are separated by about a 70-kb interval.     

枯草芽孢杆菌整合载体研究进展

芽孢杆菌质粒经常在复制时出现不稳定的单链(ssDNA)形式,从而导致质粒载体的丢失,而采用整合载体将克隆基因整合到宿主染色体,是克服枯草杆菌质粒不稳定性的一个有效途径。本综述了芽孢杆菌整合载体的研究历程、整合机理、整合类型及其应用和前景。     

枯草芽孢杆菌无标记遗传操作技术研究进展

枯草芽孢杆菌是革兰氏阳性菌的模式生物,长期以来在代谢工程和工业微生物领域扮演重要角色。枯草芽孢杆菌无标记遗传操作技术对后基因组时代的基因功能研究和菌株生理特性的改造起着关键作用。综述枯草芽孢杆菌无标记遗传操作所使用的负筛选标记基因,总结目前主流的无标记遗传操作的策略,并提出枯草芽孢杆菌无标记遗传操作技术面临的主要问题和发展方向。     

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.     

动物基因组定点整合转基因技术研究进展

传统转基因技术,如显微注射、转座子、慢病毒转染等将目的基因插入基因组内的整合方式是随机的,这些随机整合对后期转基因动物品系组建和育种带来诸多不利,因此有研究人员提出了定点整合转基因技术。目前该技术的定点整合效率非常低,主要取决于两个方面：一是靶位点产生DNA双链断裂(double-strand break, DSB)的效率;二是断裂后的靶位点与携带同源臂及外源基因的供体质粒发生同源重组的效率,其中同源重组修复(homologous recombination repair, HDR)是基因组定点整合最为依赖的修复机制。靶位点产生DSB后,机体的DNA修复既可能发生HDR,也可能发生非同源末端连接(nonhomologous end joining, NHEJ),并且两者之间存在竞争关系,因此激活HDR或抑制NEHJ都可提高定点整合转基因的效率。本文结合影响定点整合的因素,对提高定点整合效率最新探索方面进行了综述。     

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.     

Toxoplasma CRISPR/Cas9 constructs are functional for gene disruption in Neospora caninum

Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite.     

新一代基因组编辑系统CRISPR/Cpf1

CRISPR/Cas系统几乎存在于所有的细菌和古菌中,是用来抵御外来病毒和噬菌体入侵的获得性免疫防御机制。2012年起CRISPR/Cas9被改造为基因编辑工具,并衍生出一系列高效、便捷的基因编辑工具,迅速在基础理论、基因诊断和临床治疗等研究领域中得到广泛应用。然而,CRISPR/Cas9也存在细胞毒性、脱靶效应和基因插入困难等一些亟待解决的问题,在一定程度上限制了CRISPR/Cas9的应用。Cpf1是2015年报道的一种新型CRISPR效应蛋白,具有许多与Cas9不同的特性,有利于克服CRISPR/Cas9应用中的一些限制。本文综述了近两年来对CRISPR/Cpf1的研究进展和应用,并对其应用前景和发展方向进行了展望。     

Locus-specific integration of extrachromosomal transgenes in C. elegans with the CRISPR/Cas9 system

We established a method to generate integration from extrachromosomal arrays with the CRISPR/Cas9 system. Multi-copy transgenes were integrated into the defined loci of chromosomes by this method, while a multi-copy transgene is integrated into random loci by previous methods, such as UV- and gamma-irradiation. The effects of a combination of sgRNAs, which define the cleavage sites in extrachromosomes and chromosomes, and the copy number of potential cleavable sequences were examined. The relative copy number of cleavable sequences in extrachromosomes affects the frequency of fertile F1 transgenic animals. The expression levels of the reporter gene were almost proportional to the copy numbers of the integrated sequences at the same integration site. The technique is applicable to the transgenic strains abundantly stored and shared among the C. elegans community, particularly when researchers use sgRNAs against common plasmid sequences such as β-lactamase.