No apparent p53 activation in CRISPR-engineered gene-edited rabbits

Clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9) and base editors (BEs) are revolutionary gene-editing technology that has been widely utilized in biology, biotechnology and medicine. However, recent reports show that CRISPR-Cas9-mediated genome editing can induce a p53-mediated stress response and cell cycle arrest in human cells, while not illustrated in gene-editing animals. In the study, to verify whether there is a phenomenon of p53 activation, by analysing nine gene-edited rabbits using CRISPR-Cas9 and BEs, we provide the first evidence that no apparent p53 expression changes in those rabbits generated by Cas9 or BE-edited, suggesting that p53 may not need to consider for application in gene-edited animals.     

曲古抑菌素A对克隆猪端粒长度的影响

端粒是染色体末端结构, 在细胞分裂时随着DNA复制而缩短, 体细胞核移植能不同程度地延长端粒长度, 但有些克隆动物端粒的长度在体细胞核移植过程中不能有效恢复, 因而这些克隆动物就会表现出早衰现象。文章发现克隆东北民猪以及eGFP、Mx和PGC1α转基因克隆猪的端粒长度与核供体成体成纤维细胞相比显著缩短(P<0.05), 表明体细胞核移植的重编程过程没能延长细胞的“寿命”。曲古抑菌素A(Trichostatin A, TSA)是一种去乙酰化酶抑制剂, 有研究表明其能提高某些物种的体细胞核重编程效率。为了使端粒长度有效恢复, 文章利用40 nmol/L TSA处理1细胞期猪克隆胚胎24 h, 结果发现, 与对照组相比, TSA处理能显著地提高克隆胚胎体外发育的囊胚率(16.35% vs. 2 7.09%, 21.60% vs. 34.90%, P<0.05), 而且囊胚期端粒长度也得到显著延长(P<0.05)。克隆胚胎移植受体后得到了TSA处理组与非处理组的克隆猪, 虽然TSA处理并没有提高克隆效率(1.3% vs. 1.7%, TSA vs. control), 但端粒长度与对照组和供体细胞相比均显著延长(P<0.05)。猪体细胞核移植不能有效恢复端粒长度, 但是TSA处理能有效延长克隆猪端粒长度。     

Targeted Genome Editing by Recombinant Adeno-Associated Virus （rAAV） Vectors for Generating Genetically Modified Pigs

Recombinant adeno-associated virus(rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases.Several advantages,such as simple vector construction,high targeting frequency by homologous recombination,and applicability to many cell types,make rAAV an attractive approach for targeted genome editing.Combined with cloning by somatic cell nuclear transfer(SCNT),this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1,and breast cancer.This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination.We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts,which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.     

Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer

The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2 % of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.     

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9(CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs(sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies.Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.     

Production of Pigs Expressing a Transgene under the Control of a Tetracycline-Inducible System

Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG) pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet)-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP). At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT) was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems.     

Analysis of cell growth and gene expression of porcine adipose tissue‐derived mesenchymal stem cells as nuclear donor cell

In several laboratory animals and humans, adipose tissue‐derived mesenchymal stem cells (ASC) are of considerable interest because they are easy to harvest and can generate a huge proliferation of cells from a small quantity of fat. In this study, we investigated: (i) the expression patterns of reprogramming‐related genes in porcine ASC; and (ii) whether ASC can be a suitable donor cell type for generating cloned pigs. For these experiments, ASC, adult skin fibroblasts (AF) and fetal fibroblasts (FF) were derived from a 4‐year‐old female miniature pig. The ASC expressed cell‐surface markers characteristic of stem cells, and underwent in vitro differentiation when exposed to specific differentiation‐inducing conditions. Expression of DNA methyltransferase (DNMT)1 in ASC was similar to that in AF, but the highest expression of the DNMT3B gene was observed in ASC. The expression of OCT4 was significantly higher in FF and ASC than in AF (P < 0.05), and SOX2 showed significantly higher expression in ASC than in the other two cell types (P < 0.05). After somatic cell nuclear transfer (SCNT), the development rate of cloned embryos derived from ASC was comparable to the development of those derived using FF. Total cell numbers of blastocysts derived using ASC and FF were significantly higher than in embryos made with AF. The results demonstrated that ASC used for SCNT have a potential comparable to those of AF and FF in terms of embryo in vitro development and blastocyst formation.     

Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas

CRISPR-Cas is an efficient method for genome editing in organisms from bacteria to human cells. We describe a transgene-free method for CRISPR-Cas-mediated cleavage in nematodes, enabling RNA-homology-targeted deletions that cause loss of gene function; analysis of whole-genome sequencing indicates that the nuclease activity is highly specific.     

The analysis of telomere length and telomerase activity in cloned pigs and cows

Inefficiency in the production of cloned animals is most likely due to epigenetic reprogramming errors after somatic cell nuclear transfer (SCNT). In order to investigate whether nuclear reprogramming restores cellular age of donor cells after SCNT, we measured telomere length and telomerase activity in cloned pigs and cattle. In normal pigs and cattle, the mean telomere length was decreased with biological aging. In cloned or transgenic cloned piglets, the mean telomere length was elongated compared to nuclear donor fetal fibroblasts and age-matched normal piglets. In cloned cattle, no increases in mean telomere length were observed compared to nuclear donor adult fibroblasts. In terms of telomerase activity, significant activity was observed in nuclear donor cells and normal tissues from adult or new-born pigs and cattle, with relatively higher activity in the porcine tissues compared to the bovine tissues. Cloned calves and piglets showed the same level of telomerase activity as their respective donor cells. In addition, no difference in telomerase activity was observed between normal and transgenic cloned piglets. However, increased telomerase activity was observed in porcine SCNT blastocysts compared to nuclear donor cells and in vitro fertilization (IVF)-derived blastocysts, suggesting that the elongation of telomere lengths observed in cloned piglets could be due to the presence of higher telomerase activity in SCNT blastocysts. In conclusion, gathering from the comparative studies with cattle, we were able to demonstrate that telomere length in cloned piglets was rebuilt or elongated with the use of cultured donor fetal fibroblasts.     

Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration.     

CRISPR-Cas系统及anti-CRISPR蛋白在微生物中的研究进展

CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISP) and CRISPR associated proteins)系统是细菌用来防御病毒、质粒等外源核酸入侵的一种获得性免疫防御系统。随着研究的深入,CRISPR-Cas系统已发展为一种重要的基因编辑工具,并成功应用于动物、植物和微生物的基因改造中。但该基因编辑方法有时存在基因脱靶效应,从而限制了其推广应用。最近,通过将1种新发现的抗CRISPR蛋白(Anti-CRISPR protein,ACP)与CRISPR-Cas系统相结合,已成功开发出可控制基因脱靶效率的CRISPR-Cas基因编辑工具。本文首先对CRISPR-Cas系统及ACP进行了简要介绍,然后就CRISPR-Cas基因编辑工具及ACP在微生物基因改造的应用现状进行了综述,并对ACP介导的CRISPR-Cas基因编辑方法(ACP-CRISPR-Cas)在微生物基因编辑中的应用前景进行了讨论。     

Detailed Phenotypic and Molecular Analyses of Genetically Modified Mice Generated by CRISPR-Cas9-Mediated Editing

The bacterial CRISPR-Cas9 system has been adapted for use as a genome editing tool. While several recent reports have indicated that successful genome editing of mice can be achieved, detailed phenotypic and molecular analyses of the mutant animals are limited. Following pronuclear micro-injection of fertilized eggs with either wild-type Cas9 or the nickase mutant (D10A) and single or paired guide RNA (sgRNA) for targeting of the tyrosinase (Tyr) gene, we assessed genome editing in mice using rapid phenotypic readouts (eye and coat color). Mutant mice with insertions or deletions (indels) in Tyr were efficiently generated without detectable off-target cleavage events. Gene correction of a single nucleotide by homologous recombination (HR) could only occur when the sgRNA recognition sites in the donor DNA were modified. Gene repair did not occur if the donor DNA was not modified because Cas9 catalytic activity was completely inhibited. Our results indicate that allelic mosaicism can occur following -Cas9-mediated editing in mice and appears to correlate with sgRNA cleavage efficiency at the single-cell stage. We also show that larger than expected deletions may be overlooked based on the screening strategy employed. An unbiased analysis of all the deleted nucleotides in our experiments revealed that the highest frequencies of nucleotide deletions were clustered around the predicted Cas9 cleavage sites, with slightly broader distributions than expected. Finally, additional analysis of founder mice and their offspring indicate that their general health, fertility, and the transmission of genetic changes were not compromised. These results provide the foundation to interpret and predict the diverse outcomes following CRISPR-Cas9-mediated genome editing experiments in mice.     

The Application of CRISPR-Cas9 Genome Editing in Caenorhabditis elegans

Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unparalleled efficacy and facility for mo fying genomes in a wide variety of organisms. Caenorhabditis elegans is one of the most convenient multicellular organisms for gene analysis, and the application of this novel genome editing technique to this organism promises to revolutionize analysis of gene funct in the future. CRISPR-Cas9 has been successfully used to generate imprecise insertions and deletions via non-homologous end-join mechanisms and to create precise mutations by homology-directed repair from donor templates. Key variables are the methods used deliver the Cas9 endonuclease and the efficiency of the single guide RNAs. CRISPR-Cas9-mediated editing appears to be highly spec in C. elegans, with no reported off-target effects. In this review, I briefly summarize recent progress in CRISPR-Cas9-based geno editing in C. elegans, highlighting technical improvements in mutagenesis and mutation detection, and discuss potential future app cations of this technique.     

Production of second-generation cloned cats by somatic cell nuclear transfer

We successfully produced second-generation cloned cats by somatic cell nuclear transfer (SCNT) using skin cells from a cloned cat. Skin cells from an odd-eyed, all-white male cat (G0 donor cat) were used to generate a cloned cat (G1 cloned cat). At 6 months of age, skin cells from the G1 cloned cat were used for SCNT to produce second-generation cloned cats. We compared the in vitro and in vivo development of SCNT embryos that were derived from the G0 donor and G1 cloned donor cat's skin fibroblasts. The nuclei from the G0 donor and G1 cloned donor cat's skin fibroblasts fused with enucleated oocytes with equal rates of fusion (60.7% vs. 58.8%, respectively) and cleavage (66.3% vs. 63.4%). The 2-4-cell SCNT embryos were then transferred into recipients. One of the five recipients of G0 donor derived NT embryos (20%) delivered one live male cloned kitten, whereas 4 of 15 recipients of the G1 cloned donor cat derived NT embryos (26%) delivered a total of seven male second-generation cloned kittens (four live kittens from one surrogate, plus two stillborn kittens, and one live kitten that died 2d after birth from three other surrogate mothers). The four second-generation cloned kittens from the same surrogate all had a white coat color; three of the four second-generation cloned kittens had two blue eyes, and one of the second-generation cloned kittens had an odd-eye color. Despite low cloning efficiency, cloned cats can be used as donor cats to produce second-generation cloned cats.     

Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc^−/−) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.     

Efficiency of human lactoferrin transgenic donor cell preparation for SCNT

The combination of somatic cell nuclear transfer (SCNT) and transgenic technology leads to the production of transgenic cloned animals, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. The purpose of this study was to establish an efficient procedure to prepare human lactoferrin (hLTF) transgenic donor cells for SCNT. Thus, two cell culture systems were employed: caprine mammary epithelial cells (for evaluation of the hTLF transgenic expression in vitro), and fetal-derived fibroblast cells (for identification of competent transgenic donor cells). Induced by hormonal signals, recombinant hLTF was detected in the supernatant of transfected mammary epithelial cells by Western blot. Reliable hLTF transgenic fibroblast cell clones were identified by screening with multiple PCR amplification, EGFP fluorescence, and chromosomal counting (32.5+/-2.3%). This study may provide an effective upstream system to prepare SCNT donor cells for the production of human recombinant pharmaceuticals from the milk of transgenic animals.     

Generation of goats by nuclear transfer: a retrospective analysis of a commercial operation (1998–2010)

At LFB USA, Inc., the ultimate use for transgenic cloned goats is for the production of recombinant human protein therapeutics in their milk. This retrospective analysis of the Somatic Cell Nuclear Transfer (SCNT) program, spanning from 1998 to 2010, examined parameters potentially affecting the outcomes and efficiencies in this commercial operation. Over 37,000?+?ova were utilized in the SCNT protocol producing a total of 203 cloned goats. Fifty one (51) clones were produced from non-transfected (transgenic and non-transgenic animal donor) cell lines and 152 clones were produced from transfected cell lines. Comparisons and summaries of (a) transfected versus non-transfected cell lines, (b) relationship of SCNT parameters to offspring produced, (c) skin versus fetal cells, (d) fresh versus cryopreserved cells, (e) parameters from all cell lines used versus those producing SCNT offspring, (f) variation among cell sources, (g) methods of SCNT parturition management and effects on live offspring, and lastly (h) SCNT variation by program are reported. Findings indicate that (a) non-transfected cell lines were more efficient versus transfected cell lines in generating viable cloned offspring on a per reconstructed embryo transferred basis, (b) transfected fetal fibroblasts had improved efficiency versus transfected skin fibroblasts, (c) the percentage of non-transfected cell lines that produced offspring was statistically higher than transfected cell lines, (d) and induction of parturition improved the percentage of viable offspring. In summary, this retrospective analysis on the SCNT process has identified certain parameters for improved efficiency in producing viable cloned goats in a commercial setting.

     

Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing

Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.Supplementary InformationThe online version of this article (10.1007/s13238-021-00838-7) contains supplementary material, which is available to authorized users.     

Cas9HF1 enhanced specificity in Ustilago maydis

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely used as a tool to precisely manipulate genomic sequence targeted by sgRNA (single guide RNA) and is adapted in different species for genome editing. One of the major concerns of CRISPR-Cas9 is the possibility of off-target effects, which can be remedied by the deployment of high fidelity Cas9 variants. Ustilago maydis is a maize fungal pathogen, which has served as a model organism for biotrophic pathogens for decades. The successful adaption of CRISPR-Cas9 in U. maydis greatly facilitated effector biology studies. Here, we constructed an U. maydis reporter strain that allows in vivo quantification of efficiency and target specificity of three high fidelity Cas9 variants, Cas9HF1, Cas9esp1.1 and Cas9hypa. This approach identified Cas9HF1 as most specific Cas9 variant in U. maydis. Furthermore, whole genome sequencing showed absence of off-target effects in U. maydis by CRISPR-Cas9 editing.