文献ID 生物種 種名 用いた技術 ターゲット遺伝子 雑誌名 TI 年 巻・号・ページ 著者 所属機関（国） PMID DOI 抄録 species2 1 animal chicken CRISPR;Cas9; cadherin-like and
PC-esterase domain containing 1
Bioscience reports
Cped1 promotes chicken SSCs formation with the aid of histone acetylation and transcription factor Sox2.
2018 38(5) [Zhang C et al.] Yangzhou University, Yangzhou, China.
30038055 10.1042/BSR20 180707
Spermatogonial stem cells (SSCs) may apply to gene therapy, regenerative medicine in place of embryonic stem cells (ESCs). However, the application of SSCs was severely limited by the low induction efficiency and the lack of thorough analysis of the regulatory mechanisms of SSCs formation. Current evidences have demonstrated multiple marker genes of germ cells, while genes that specifically regulate the formation of SSCs have not been explored. In our study, cadherin-like and PC-esterase domain containing 1 (Cped1) expressed specifically in SSCs based on RNA-seq data analysis.
To study the function of Cped1 in the formation of SSCs, we successfully established a CRISPR/Cas9knockout system. The gene disruption frequency is 37% in DF1 and 25%
in ESCs without off-target effects. Knockout of Cped1 could significantly inhibit the formation of SSCs in vivo and in vitro The fragment of -1050 to -1 bp had the activity as Cped1 gene promoter. Histone acetylation could regulate the expression of Cped1.
We added 5-azaeytidi (DNA methylationinhibitors) and TSA (histone deacetylase inhibitors) respectively during the cultivation of SSCs. TSA was validated to promote the transcription of Cped1. Dual-luciferase reporter assay revealed that active control area of thechickenCped1 gene is -296 to -1 bp. There are Cebpb, Sp1, and Sox2 transcription factor binding sites in this region. Point-mutation experiment results showed that Sox2 negatively regulates the transcription of Cped1. Above results demonstrated that Cped1 is a key gene that regulates the formation of SSCs. Histone
chicken
2 animal chicken CRISPR;Cas9; transcription factors; enhancer
Development Genome and epigenome engineering CRISPR toolkit for in vivo modulation of cis-regulatory interactions and gene expression in the chicken embryo.
2018 145(4) [Williams RM et al.]
University of Oxford, Oxford, UK.
29386245 10.1242/dev.16 0333
CRISPR/Cas9genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present an optimised, adaptable toolkit enabling genome and epigenome engineering in thechicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise novel efficient guide-RNA mini expression vectors utilising chick U6 promoters, provide a strategy for rapid somatic gene knockout and establish a protocol for evaluation of mutational penetrance by targeted next-generation sequencing. We show thatCRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficientCas9(dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity inchickenembryos, facilitating high-resolution analysis of gene regulatory
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3 animal chicken CRISPR;Cas9; GAPDH F1000Research Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line.
2018 7:238 [Antonova E et
al.]
Moscow Institute of Physics and Technology, Moscow Region, Russian Federation.
29946437 10.12688/f1000 research.13457.
2
Background:CRISPR/Cas9system is becoming the dominant genome editing tool in a variety of organisms.CRISPR/Cas9mediated knock out has been demonstrated both in chickencell lines and inchickengerm cells that served to generate genetically modified birds. However, there is limited data aboutCRISPR/Cas9dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci ofchickengenome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken3'-untranslated region of GAPDH gene.Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along withCRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of thechickenGAPDH promoter.
Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce
chicken
4 animal chicken CRISPR;Cas9; TANK-binding kinase 1 (TBK-1)
Frontiers in immunology
CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING- Mediated IFN-beta Induction in Chicken Cells.
2018 9:3010 [Cheng Y et al.] Shanghai Jiao Tong University, Shanghai, China.
30662438 10.3389/fimmu.
2018.03010
TANK-binding kinase 1 (TBK1) is involved in innate immunity, prompting transcriptional induction of type I interferons in response to pathogenic infection. Many studies have focused on mammals but the function of TBK1 inchickens remains poorly defined.
CRISPR/Cas9system has made gene-knockout easy to accomplish. Although CRISPR/Cas9has been used inchickencells, low mutation efficiency limits its wide application inchickens. In this study, an effective gene-knockout system was developed based on theCRISPR/Cas9system inchickenembryonic fibroblast DF-1.
TwoCRISPR/Cas9plasmids were constructed, TBK1-g1 and TBK1-g2, which express gRNAs targeting different sequences of thechickenTBK1 gene. After transfection and enrichment with puromycin screening, the mutation rates as assessed via T7E1 assay were 88.05 and 89.55%, respectively, and subsequent sequence analysis showed mutation efficiencies of 86.67 and 93.33%. With the limiting-dilution method, a chTBK1 gene-deficiency monoclonal cell line was obtained and was named DF-1-TBK1-C3.
The DF-1-TBK1-C3 cells exhibited normal morphology and maintained stable proliferation ability compared to wild-type cells. The gene-overexpression system and luciferase reporter assay showed that IFN-beta induction induced by chSTING was almost completely blocked in DF-1-TBK1-C3 cells. With quantitative real-time PCR, we further confirmed the essential role of chTBK1 in the chSTING-mediated IFN-beta induction. At last, the study demonstrated that the chTBK1 knockout system is also applicable in primary chick embryo fibroblasts (CEFs). In this study, an effective gene- knockout system was applied inchickens, a TBK1 gene-deleted DF-1 cell line was successfully created using this system, and with the chTBK1 knockout cells, chTBK1 was revealed to be indispensable in STING-mediated IFN-beta activation inchicken
chicken
表３ 諸外国でのゲノム編集技術を用いた研究動向調査
5 animal chicken Cas9 Nanos2 Journal of cellular biochemistry
Nanos2 promotes differentiation of chicken (Gallus gallus) embryonic stem cells to male germ cells.
2018 119(6):4435- 4446
[Zhang W et al.] Yangzhou University, Yangzhou, Jiangsu, China.
29143989 10.1002/jcb.265 28
Nanos2 is an evolutionarily conserved RNA-binding protein containing 2 CCHC-type zincfingermotives. Here, we report that Nanos2 is strongly expressed in the testis compared to other tissues inchicken(Gallus gallus). Overexpression and knockout plasmid vectors were constructed, and in-vitroCas9/gRNA digestion and T7 endonuclease I (T7E1) assay indicated that Nanos2-g1 possessed the highest knockout activity. In vitro and in vivo, Nanos2 overexpression accelerated the production of embryoid bodies (EBs) and SSC-like cells and promoted cvh, c-kit, and integrin alpha6 expression. Immunofluorescence staining, periodic acid schiff (PAS) and flow cytometry (FCM) assay showed that primordial germ cells (PGCs) and spermatogonial stem cells (SSCs) formation were significantly promoted. On the contrary, Nanos2 knockout delayed the production of EBs and SSC-like cells and correspondingly reduced cvh, c- kit, and integrin alpha6 expression. Simultaneously, the quantity of PGCs and SSCs was blocked. Collectively, these results uncovered a novel function of Nanos2 involved in chickenmale germ cell differentiation, where it acts as a facilitator.
chicken
6 animal chicken CRISPR;Cas9; transient receptor potential canonical channel (TRPC5)
Journal of neurophysiolog y
TRPC5 is required for the NO-dependent increase in dendritic Ca(2+) and GABA release from chick retinal amacrine cells.
2018 119(1):262-273 [Maddox JW et al.]
Louisiana State University , Baton Rouge, La, USA.
28978766 10.1152/jn.0050 0.2017
GABAergic signaling from amacrine cells (ACs) is a fundamental aspect of visual signal processing in the inner retina. We have previously shown that nitric oxide (NO) can elicit release of GABA independently from activation of voltage-gated Ca(2+) channels in cultured retinal ACs. This voltage-independent quantal GABA release relies on a Ca(2+) influx mechanism with pharmacological characteristics consistent with the involvement of the transient receptor potential canonical (TRPC) channels TRPC4 and/or TRPC5. To determine the identity of these channels, we evaluated the ability of NO to elevate dendritic Ca(2+) and to stimulate GABA release from cultured ACs under conditions known to alter the function of TRPC4 and 5. We found that these effects of NO are phospholipase C dependent, have a biphasic dependence on La(3+), and are unaffected by moderate concentrations of the TRPC4-selective antagonist ML204. Together, these results suggest that NO promotes GABA release by activating TRPC5 channels in AC dendrites. To confirm a role for TRPC5, we knocked down the expression of TRPC5 usingCRISPR/Cas9-mediated gene knockdown and found that both the NO-dependent Ca(2+) elevations and increase in GABA release are dependent on the expression of TRPC5. These results demonstrate a novel NO- dependent mechanism for regulating neurotransmitter output from retinal ACs. NEW &
NOTEWORTHY Elucidating the mechanisms regulating GABAergic synaptic transmission in the inner retina is key to understanding the flexibility of retinal ganglion cell output. Here, we demonstrate that nitric oxide (NO) can activate a transient receptor potential canonical 5 (TRPC5)-mediated Ca(2+) influx, which is sufficient to drive vesicular GABA release from retinal amacrine cells. This NO-dependent mechanism can bypass the need for depolarization and may have an important role in
chicken
7 animal chicken CRISPR;Cas9; Scientific
reports
High fidelity CRISPR/Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells.
2018 8(1):15126 [Idoko-Akoh A et al.]
University of Edinburgh, Midlothian, UK.
30310080 10.1038/s41598 -018-33244-x
Primordial germ cells (PGCs), the embryonic precursors of the sperm and egg, are used for the introduction of genetic modifications into avian genome. Introduction of small defined sequences using genome editing has not been demonstrated in bird species. Here, we compared oligonucleotide-mediated HDR using wild type SpCas9 (SpCas9-WT) and high fidelity SpCas9-HF1 in PGCs and show that many loci in chickenPGCs can be precise edited using donors containingCRISPR/Cas9-blocking mutations positioned in the protospacer adjacent motif (PAM). However, targeting was more efficient using SpCas9-HF1 when mutations were introduced only into the gRNA target sequence. We subsequently employed an eGFP-to-BFP conversion assay, to directly compare HDR mediated by SpCas9-WT and SpCas9-HF1 and discovered that SpCas9-HF1 increases HDR while reducing INDEL formation. Furthermore, SpCas9- HF1 increases the frequency of single allele editing in comparison to SpCas9-WT. We used SpCas9-HF1 to demonstrate the introduction of monoallelic and biallelic point mutations into the FGF20 gene and generate clonal populations of edited PGCs with defined homozygous and heterozygous genotypes. Our results demonstrate the use of oligonucleotide donors and high fidelityCRISPR/Cas9variants to perform precise
chicken
8 animal chicken CRISPR;Cas9; human interferon beta
Scientific reports
Efficient production of human interferon beta in the white of eggs from ovalbumin gene-targeted hens.
2018 8(1):10203 [Oishi I et al.] National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka, Japan.
29976933 10.1038/s41598 -018-28438-2
Transgenicchickens could potentially serve as bioreactors for commercial production of recombinant proteins in egg white. Many transgenicchickens have been generated by randomly integrating viral vectors into their genomes, but transgene expression has proved insufficient and/or limited to the initial cohort. Herein, we demonstrate the feasibility of integrating human interferon beta (hIFN-beta) into thechickenovalbumin locus and producing hIFN-beta in egg white. We knocked in hIFN-beta into primordial germ cells using aCRISPR/Cas9protocol and then generated germline chimeric roosters by cell transplantation into recipient embryos. Two generation-zero founder roosters produced hIFN-beta knock-in offspring, and all knock-in female offspring produced abundant egg-white hIFN-beta (~3.5 mg/ml). Although female offspring of the first generation were sterile, their male counterparts were fertile and produced a second generation of knock-in hens, for which egg-white hIFN-beta production was comparable with that of the first generation. The hIFN-beta bioactivity represented only ~5% of total egg-white hIFN-beta, but unfolding and refolding of hIFN-beta in the egg white fully recovered the bioactivity. These results suggest that transgene insertion at thechickenovalbumin locus can result in abundant and stable expression of an exogenous protein deposited into egg white and should be amenable to industrial
chicken
9 animal chicken CRISPR;Cas9; IFN-induced proteins with tetratricopeptide repeats 5
Scientific reports
Chicken Interferon-induced Protein with Tetratricopeptide Repeats 5 Antagonizes Replication of RNA Viruses.
2018 8(1):6794 [Santhakumar D et al.]
Lancaster University, Lancaster, UK.
29717152 10.1038/s41598 -018-24905-y
The intracellular actions of interferon (IFN)-regulated proteins, including IFN-induced proteins with tetratricopeptide repeats (IFITs), attribute a major component of the protective antiviral host defense. Here we applied genomics approaches to annotate thechickenIFIT locus and currently identified a single IFIT (chIFIT5) gene. The profound transcriptional level of this effector of innate immunity was mapped within its unique cis-acting elements. This highly virus- and IFN-responsive chIFIT5 protein interacted with negative sense viral RNA structures that carried a triphosphate group on its 5' terminus (ppp-RNA). This interaction reduced the replication of RNA viruses in lentivirus-mediated IFIT5-stablechickenfibroblasts whereasCRISPR/Cas9-edited chIFIT5 gene knockout fibroblasts supported the replication of RNA viruses. Finally, we generated mosaic transgenicchickenembryos stably expressing chIFIT5 protein or knocked-down for endogenous chIFIT5 gene. Replication kinetics of RNA viruses in these transgenicchickenembryos demonstrated the antiviral potential of chIFIT5 in ovo. Taken together, these findings propose that IFIT5 specifically antagonize RNA viruses by sequestering viral nucleic acids inchickens, which are unique in innate immune sensing and responses to viruses of both poultry and human health
chicken
10 animal chicken CRISPR;Cas9; tva; tvc; tvj Viruses Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells.
2018 10(11) [Koslova A et al.] Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic.
30400152 10.3390/v10110 605
Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We usedCRISPR/Cas9genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate thatCRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistantchickens.
chicken
11 animal chicken CRISPR;Cas9; ovalbumin 3 Biotech Efficient knock-in at the chicken ovalbumin locus using adenovirus as a CRISPR/Cas9 delivery system.
2019 9(12):454 [Qin X et al.] Guangxi University, Guangxi, China.
31832301 10.1007/s13205 -019-1966-3
In this study, efficient knock-in (KI) of human epidermal growth factor (hEGF) cDNA at the ovalbumin (OV) locus in culturedchickencells was achieved using adenovirus as a delivery forCRISPR/Cas9elements and optimizing donor vector construction. The strategy of recruiting donor DNA to the insertion site further improved the KI efficiency. The inserted hEGF cDNA can expressed in primary oviduct cells and secreted hEGF promoted proliferation of Hela cells. Moreover, we achieved efficient KI in blastoderm cells without altering their induction in vitro and obtained germline chimeric KIchickenembryos by transplanting KI blastoderm cells as well as injecting adenovirus directly, in vivo. Our results provided an efficient KI method forchicken cells and embryos, and lay the foundation for more convenient production of KIchicken at the OV locus, which will promote the development of oviduct-specific bioreactor.
chicken
12 animal chicken CRISPR;Cas9; HMG-box protein 1
Biochemical and biophysical research communication s
Transcription factor HBP1: A regulator of senescence and apoptosis of preadipocytes.
2019 517(2):216-220 [Chen H et al.] Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Harbin, China.
31331641 10.1016/j.bbrc.2 019.07.048
BACKGROUND: /aim: HMG-box protein 1 (HBP1) plays an important role in the senescence and apoptosis of mammalian cells, but its role inchickencells remains unclear. The aim of this study was to investigate the effects of HBP1 on senescence and apoptosis ofchickenpreadipocytes. METHODS: The immortalizedchicken preadipocyte cell line (ICP2) was used as a cell model.ChickenHBP1 knockout and overexpressing preadipocyte cell lines were established usingCRISPR/Cas9gene editing technology and lentiviral infection. Western blotting was used to detect the protein expression of HBP1 and senescence markers p16 and p53. Cell senescence was measured by Sa-beta-Gal staining and apoptosis was detected by flow cytometry.
RESULTS: HBP1 was highly expressed in senescent ICP2 cells compared with young ICP2 cells. After the deletion of HBP1, the degree of senescence, the apoptosis rate and the protein expression levels of p16 and p53 were significantly reduced. After the overexpression of HBP1, the degree of senescence, the apoptosis rate and the protein expression levels of p16 and p53 were significantly increased. CONCLUSION: HBP1 promotes the senescence and apoptosis ofchickenpreadipocytes.
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13 animal chicken CRISPR;Cas9; exogenous genes of donor plasmids into Z chromosomes
FASEB journal Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.
2019 33(7):8519-8529 [Lee HJ et al.] Seoul National University, Seoul, South Korea.
30951374 10.1096/fj.2018 02671R
Clustered regularly interspaced short palindromic repeats (CRISPR) andCRISPR- associated protein 9 (Cas9) have facilitated the production of genome-edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chickenmodels using theCRISPR/Cas9-mediated nonhomologous end joining repair pathway inchickenprimordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences andCRISPR/Cas9plasmids intochickenPGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenicchickens was accomplished through subsequent transplantation of the Z chromosome-targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to thechickensex Z chromosome without detected off-target effects. The genome-modifiedchickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome-editing method could be used to develop manychickenmodels and should significantly expand the application of genome-modified avians.-Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome ofchicken
chicken
14 animal chicken CRISPR;Cas9; G0/G1 switch gene 2 (g0s2)
FASEB journal Disruption of G0/G1 switch gene 2 ( G0S2) reduced abdominal fat deposition and altered fatty acid composition in chicken.
2019 33(1):1188-1198 [Park TS et al.] Seoul National University, Pyeongchang-gun, Gangwon- do, Korea.
30085885 10.1096/fj.2018 00784R
Chickenas a food source is one of the most widespread domestic animals, and it has been used extensively as a research model. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system is the most efficient and reliable tool for precise genome-targeted modification and has generated considerable excitement for industrial applications, as well as biologic science. Unlike in mammals, germline-transmittable primordial germ cells (PGCs) inchickenwere used as an alternative strategy for the production of genetically alteredchickens. Here, by combining theCRISPR-Cas9platform and germ cell-mediated germline transmission, we generated G0/G1 switch gene 2 ( G0S2) knockout (KO)chickens, and G0S2 null KO chickens showed a dramatic reduction of abdominal fat deposition without affecting other economic traits. Additionally, G0S2 null KOchickens had altered fatty acid compositions in their blood and abdominal fat compared with wild-typechickens under normal dietary conditions. The global mRNA sequencing data showed that G0S2 disruption inchickens would activate the adipose tissue-specific peroxisomal oxidation pathway, and enoyl-coenzyme A (CoA), hydratase/3-hydroxyacyl CoA dehydrogenase might be a target molecule in metabolic homeostasis in thechickenadipose tissue. Our results demonstrate that theCRISPR-Cas9system withchickenPGCs can facilitate the production of specific genome-editedchickens for practical applications, as well as basic research.-Park, T. S., Park, J., Lee, J. H., Park, J.-W., Park, B.-C. Disruption of G0/G1 switch gene 2 ( G0S2) reduced abdominal fat deposition and altered fatty acid
chicken
15 animal chicken CRISPR;Cas9; CXCR4 Frontiers in immunology
Blocking of the CXCR4-CXCL12 Interaction Inhibits the Migration of Chicken B Cells Into the Bursa of Fabricius.
2019 10:3057 [Laparidou M et al.]
Reproductive Biotechnology, School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany.
31998323 10.3389/fimmu.
2019.03057
B cells have first been described inchickens as antibody producing cells and were named after the Bursa of Fabricius, a unique organ supporting their development.
Understanding different factors mediating the early migration of B cells into the bursa of Fabricius is crucial for the study of B cell biology. While CXCL12 (stromal derived factor 1) was found to play an important role in B lymphocyte trafficking in mammals, its role in thechickenis still unknown. Previous studies indicated thatchickenCXCL12 and its receptor CXCR4 are simultaneously expressed during bursal development. In this study, we investigated whether the CXCR4/CXCL12 interaction mediates B cell migration inchickenembryo. We used theCRISPR/Cas9system to induce a CXCR4 knockout inchickenB cells which led to chemotaxis inhibition toward CXCL12. This was confirmed by adoptive cell transfer and inhibition of the CXCR4/CXCL12 interaction by blocking with the small inhibitor AMD3100. In addition, we found that the chickenexhibits similarities to mice when it comes to CXCR4 being dependent on B cell receptor expression. B cells lacking the B cell receptor failed to migrate toward CXCL12 and showed no response upon CXCL12 stimulation. Overall, we demonstrated the significance of CXCR4/CXCL12 inchickenB cell development in vivo and the
chicken
16 animal chicken CRISPR;Cas9; methyl binding domain protein 4 (mbd4)
Frontiers in immunology
Chicken MBD4 Regulates Immunoglobulin Diversification by Somatic Hypermutation.
2019 10:2540 [Costello R et al.]
University of Illinois at Chicago, Chicago, IL, USA.
31736964 10.3389/fimmu.
2019.02540
Immunoglobulin (Ig) diversification occurs via somatic hypermutation (SHM) and class switch recombination (CSR), and is initiated by activation-induced deaminase (AID), which converts cytosine to uracil. Variable (V) region genes undergo SHM to create amino acid substitutions that produce antibodies with higher affinity for antigen. The conversion of cytosine to uracil in DNA promotes mutagenesis. Two distinct DNA repair mechanisms regulate uracil processing in Ig genes. The first involves base removal by the uracil DNA glycosylase (UNG), and the second detects uracil via the mismatch repair (MMR) complex. Methyl binding domain protein 4 (MBD4) is a uracil glycosylase and an intriguing candidate for involvement in somatic hypermutation because of its interaction with the MMR MutL homolog 1 (MLH1). We found that the DNA uracil glycosylase domain of MBD4 is highly conserved among mammals, birds, shark, and insects. Conservation of the human andchickenMBD4 uracil glycosylase domain structure is striking. Here we examined the function of MBD4 inchickenDT40 B cells which undergo constitutive SHM. We constructed structural variants of MBD4 DT40 cells usingCRISPR/Cas9genome editing. Disruption of the MBD4 uracil glycosylase catalytic region increased SHM frequency in IgM loss assays. We propose
chicken
17 animal chicken CRISPR;Cas9; Atg5 In vitro cellular
&
developmental biology. Animal
Knockout of Atg5 inhibits proliferation and promotes apoptosis of DF-1 cells.
2019 55(5):341-348 [Liao Z et al.] South China Agricultural University, Guangzhou, China.
31025250 10.1007/s11626 -019-00342-7
Atg5, as a switch of cell autophagy and apoptosis, plays an important regulatory role in the occurrence and development of autophagy. Atg5 has been reported to involve the autophagy process but little in the apoptotic process. Here, we constructed an Atg5(- /-) DF-1 cell line using theCRISPR/Cas9assay and confirmed the significant difference in growth kinetics between Atg5(-/-) DF-1 cells and wild-type DF-1 cells.
Importantly, we found that Atg5 suppresses the cellular proliferation and induce the apoptosis in DF-1 cells by Hoechst's staining, flow cytometry, and caspase activity assay. All these findings indicated that Atg5 plays an important role in the proliferation of DF-1 cells. On the other hand, we compared the expression of autophagy key proteins LC3 and P62 in Atg5 knockout cells and wild-type cells, and detected the aggregation point distribution of LC3 protein in cells by laser confocal technique; our results showed that Atg5 knockout inhibited autophagy compared with wild-type cells.
The present findings further help to resolve the molecular mechanisms regulating Atg5 chicken
18 animal chicken CRISPR;Cas9; ALV subgroup A (tva)
Journal of animal science and biotechnology
Sequential disruption of ALV host receptor genes reveals no sharing of receptors between ALV subgroups A, B, and J.
2019 10:23 [Lee HJ et al.] Seoul National University, Seoul, Korea.
30976416 10.1186/s40104 -019-0333-x
Background: Previously, we showed that targeted disruption of viral receptor genes in avian leukosis virus (ALV) subgroups using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9))-based genome editing confers resistance to ALV subgroups B and J. Here, we used the same strategy to target the receptor expressed by ALV subgroup A (TVA) and generatechickencells resistant to infection by this virus. Results:CRISPR/Cas9-based disruption of exon 2 within the tva gene of DF-1 fibroblasts conferred resistance to infection by ALV subgroup A regardless of whether frameshift mutations were introduced during editing.
Conversely, overexpression of the wild-type TVA receptor (wtTVA) by tva-modified DF-1 clones restored susceptibility to ALV subgroup A. The results confirm that exon 2, which contains the low-density lipoprotein receptor class A domain of TVA, is critical for virus entry. Furthermore, we sequentially modified DF-1 cells by editing the tva, tvb, and Na(+)/H(+) exchange 1 (chNHE1) genes, which are the specific receptors for ALV subgroups A, B, and J, respectively. Conclusions: Simultaneous editing of multiple receptors to block infection by different subgroups of ALV confirmed that ALV subgroups A, B, and J do not share host receptors. This strategy could be used to generate cells resistant to multiple viral pathogens that use distinct receptors for cell
chicken
19 animal chicken CRISPR;Cas9; dazl; pou5f3;
ovalbumin
Journal of biological engineering
HMEJ-mediated efficient site-specific gene integration in chicken cells.
2019 13:90 [Xie L et al.] Guangxi University, Nanning, Guangxi, China.
31832093 10.1186/s13036 -019-0217-9
Background: The production of transgenicchickencells holds great promise for several diverse areas, including developmental biology and biomedical research. To this end, site-specific gene integration has been an attractive strategy for generating transgenic chickencell lines and has been successfully adopted for inserting desired genes and regulating specific gene expression patterns. However, optimization of this method is essential for improving the efficiency of genome modification in this species. Results:
Here we compare gene knock-in methods based on homology-independent targeted integration (HITI), homology-directed repair (HDR) and homology mediated end joining (HMEJ) coupled with a clustered regularly interspaced short palindromic repeat associated protein 9 (CRISPR/Cas9) gene editing system inchickenDF-1 cells and primordial germ cells (PGCs). HMEJ was found to be a robust and efficient method for gene knock-in inchickenPGCs. Using this method, we successfully labeled the germ cell specific gene DAZL and the pluripotency-related gene Pou5f3 inchickenPGCs through the insertion of a fluorescent protein in the frame at the 3' end of the gene, allowing us to track cell migration in the embryonic gonad. HMEJ strategy was also successfully used in Ovalbumin, which accounts for more than 60% of proteins in chickeneggs, suggested its good promise for the mass production of protein with pharmaceutical importance using thechickenoviduct system. Conclusions: Taken together, these results demonstrate that HMEJ efficiently mediates site-specific gene integration inchickenPGCs, which holds great potential for the biopharmaceutical
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20 animal chicken CRISPR;Cas9; ovalbumin Journal of bioscience and bioengineering
Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology- independent targeted integration.
2019 [Shi M et al.] Kyushu University, Fukuoka, Japan.
31594694 10.1016/j.jbiosc.
2019.09.011
It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as theCRISPR/Cas9system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9system for the development of transgenicchickens, guide RNA sequences (gRNAs) of theCRISPR/Cas9system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus ofchickenDF-1 and embryonic fibroblasts using theCRISPR/Cas9system mediated by homology- independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9- VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of
chicken
21 animal chicken CRISPR;Cas9; EAV-EP genome Sheng wu gong cheng xue bao
= Chinese journal of biotechnology
[CRISPR/Cas9-mediated foreign gene targeted knock-in into the chicken EAV-HP genome].
2019 35(2):236-243 [Guo M et al.] Shaanxi University of Technology, Hanzhong, Shaanxi, China.
30806053 10.13345/j.cjb.1 80224
The study aims to useCRISPR/Cas9introducing foreign gene targeted knock-in into chickenEAV-HP genome. First, specific primers were designed for amplification of EAV-HP left, right homologous arms and enhanced green fluorescent protein (eGFP) expression cassette. PCR products of homologous arms were ligated to both sides of eGFP by overlap extension PCR, resulting in full-length donor DNA fragment designated as LER. Then LER fragments were cloned into pMD19-T to obtain donor vector pMDT-LER. Subsequently, the donor vector pMDT-LER was transfected into HEK293T cells to verify the expression of eGFP gene. Furthermore, co-transfection of CRISPR/Cas9expression vector and pMDT-LER intochickenDF-1 cells was performed to achieve eGFP transgenic cells. Meanwhile, eGFP expression was observed in cells, and the event of eGFP integration into EAV-HP genome was detectable by amplification of target DNA. Finally, the transgenic DF-1 cells were passaged seven times, and the stable integration and expression of eGFP was checked by PCR and Western blotting. These results demonstrated that eGFP gene was knocked into the EAV-HP genome successfully, which provides a new integration site
chicken
22 animal chicken CRISPR;Cas9; activation-induced cytidine deaminase
Virologica Sinica
A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies.
2019 34(6):641-647 [Wang B et al.] Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
31240617 10.1007/s12250 -019-00142-z
Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. Achickenlymphoma cell line, DT40, is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase (AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line (DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus. The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and
chicken
23 animal cow CRISPR;Cas9; collagen type VIII alpha 1 chain (COL8A1)
Cell biology international
Effects of COL8A1 on the proliferation of muscle-derived satellite cells.
2018 42(9):1132-1140 [Li X et al.] North-east Agricultural University, Harbin, China.
29696735 10.1002/cbin.10 979
Collagen type VIII alpha 1 chain (COL8A1) is a component of the extracellular matrix.
Our previous studies suggested that COL8A1 is associated with the proliferation of muscle-derived satellite cells (MDSCs). Additionally, it has been demonstrated that COL8A1 promotes the proliferation of smooth muscle cells and liver cancer cells.
Therefore, we predicted that COL8A1 is associated with the proliferation ofbovine MDSCs, which have potential applications in research. In this study, we constructed vectors to activate and repress COL8A1 inbovineMDSCs using theCRISPR/Cas9 technique and determined the effects of COL8A1 modulation by EdU labeling, Western blotting, and dual-luciferase reporter assays. The results showed that activation of COL8A1 increased the number of EdU-positive cells and expression of the proliferation markers cyclin B1 (CCNB1) and P-AKT. The expression of P-Akt was unchanged after addition of LY294002 (a protein kinase inhibitor capable of blocking the signal transduction pathway of the phosphoinositide 3-kinase). In contrast, repression of COL8A1 reduced the number of EdU-positive cells and expression of CCNB1 and P- AKT. We also observed upregulation and downregulation of COL8A1 following the overexpression and repression of EGR1, respectively. The dual-luciferase reporter assay revealed that EGR1 regulates the promoter activity of COL8A1. To our knowledge, this is the first study demonstrating that EGR1 positively regulates the expression of COL8A1, which in turn promotes the proliferation ofbovineMDSCs via
cow
24 animal cow CRISPR;Cas9; extracellular
matrix protein 2 Cell biology international
Effect of ECM2 expression on bovine skeletal muscle-derived satellite cell differentiation.
2018 42(5):525-532 [Liu C et al.] Northeast Agricultural University, Harbin, Heilongjiang, China.
29274297 10.1002/cbin.10 927
Extracellular matrix components have important regulatory functions during cell proliferation and differentiation. In recent study, extracellular matrix were shown to have a strong effect on skeletal muscle differentiation. Here, we aimed to elucidate the effects of extracellular matrix protein 2 (ECM2), an extracellular matrix component, on the differentiation ofbovineskeletal muscle-derived satellite cells (MDSCs). Western blot and immunofluorescence analyses were used to elucidate the ECM2 expression pattern inbovineMDSCs during differentiation in vitro.CRISPR/Cas9technology was used to activate or inhibit ECM2 expression to study its effects on the in vitro differentiation ofbovineMDSCs. ECM2 expression was shown to increase gradually duringbovineMDSC differentiation, and the levels of this protein were higher in more highly differentiated myotubes. ECM2 activation promoted MDSC differentiation, whereas its suppression inhibited the differentiation of these cells. Here, for the first time, we demonstrated the importance of ECM2 expression duringbovineMDSC differentiation; these results could lead to treatments that help to increase beefcattle
cow
25 animal cow CRISPR; apoptosis- associated speck- like protein containing a caspase recruitment domain
Frontiers in microbiology
Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Activation of NLRP3 and NLRC4 Inflammasomes With Differential Requirement for ASC.
2018 9:1661 [Wu Q et al.] China Agricultural University, Beijing, China.
30087667 10.3389/fmicb.2 018.01661
Escherichia coli is a common cause of mastitis in dairycows. The adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) synergizes with caspase-1 to regulate inflammasome activation during pathogen infection. Here, the ASC gene was knocked out inbovinemammary epithelial (MAC-T) cells using clustered, regularly interspaced, short palindromic repeat
(CRISPR)/CRISPR-associated (Cas)-9 technology. MAC-T cells were pre-incubated with and without Lactobacillus rhamnosus GR-1 and then exposed to E. coli. Western blot analysis demonstrated increased expression of NLRP3 and NLRC4 following E. coli infection, but this increase was attenuated by pre-incubation with L. rhamnosus GR-1, regardless of ASC knockout. Western blot and immunofluorescence analyses revealed that pre-incubation with L. rhamnosus GR-1 decreased E. coli-induced caspase-1 activation at 6 h after E. coli infection, as also observed in ASC-knockout MAC-T cells. The E. coli-induced increase in caspase-4 mRNA expression was inhibited by pre-incubation with L. rhamnosus GR-1. ASC knockout diminished, but did not completely prevent, increased production of IL-1beta and IL-18 and cell pyroptosis associated with E. coli infection, whereas pre-incubation with L. rhamnosus GR-1 inhibited this increase. Our data indicate that L. rhamnosus GR-1 suppresses activation of ASC-dependent NLRP3 and NLRC4 inflammasomes and production of downstream IL-lbeta and IL-18 during E. coli infection. L. rhamnosus GR-1 also inhibited E. coli-induced cell pyroptosis, in part through attenuation of NLRC4 and non-
cow
26 animal cow TALENs; bta-miR-192 Genes &
genomics
Selected microRNA-192 mutant indicates association with several function genes in bovine cells.
2018 40(4):361-371 [Zi C et al.] Nanjing Agricultural University, Nanjing, China.
29892841 10.1007/s13258 -017-0635-3
MicroRNAs are implicated in many cellular processes such as cell differentiation and development, tumorigenesis, and immune regulation. In this study, miR192 was detected using quantitative real-time polymerase chain reaction (qRT-PCR) when MDBK cells were exposed to Escherichia coli. Cells with malfunction of bta-miR-192 were established usingtranscription activator-like effector nuclease(TALEN) technology. Finally, bta-miR-192 mutant cells were screened for differentially expressed genes using RNA-sequencing (RNA-seq). The results showed that miR192 significantly decreased in cells exposed to E. coli F18ac and E. coli K88ac. The RNA- seq results showed that 1673 differentially expressed transcripts were identified; 890 genes were upregulated and 775 genes were downregulated. With the gene ontology enrichment analysis, 431 differentially expressed genes (DEGs) were classified into 937 gene ontology terms. The pathway enrichment analysis showed that 535 genes were involved in 254 pathway terms. Interestingly, most of these DEGs were associated with the pathways in cancers or infectious diseases. When the selected DEGs (n = 162) in these pathways were intersected with 120 differential transcripts, 11 DEGs were identified. Subsequently, several genes associated with regulation, cancers, or viral infections, such as LEF1, AXIN2, MX1, and FCGR2B, were identified among the DEGs using functional analysis. Furthermore, associations between bta-miR-192 and DEGs were detected by intersecting the bta-miR-192's target genes with the DEGs, indicating that three genes including CBL, DICER1 and TRERF1 were involved in this relationship. These findings provided useful guidance for investigating the role played
cow
27 animal cow CRISPR; phosphodiesterase
2A
Journal of cellular physiology
MiR-139 promotes differentiation of bovine skeletal muscle-derived satellite cells by regulating DHFR gene expression.
2018 234(1):632-641 [Zhou S et al.] Northeast Agricultural University, Harbin, Heilongjiang, China.
30078180 10.1002/jcp.268 17
MicroRNAs play an important regulatory role in the proliferation and differentiation of skeletal muscle-derived satellite cells (MDSCs). In particular, miR-139 can inhibit tumor cell proliferation and invasion, and its expression is down-regulated during C2C12 myoblast differentiation. The aim of this study was thus to examine the effect and potential mechanism of miR-139 inbovineMDSCs. The expression of miR-139 was found to be significantly increased duringbovineMDSC differentiation by stem-loop reverse transcription-polymerase chain reaction amplification. Statistical analysis of the myotube fusion rate was done through immunofluorescence detection of desmin, and western blotting was used to measure the change in protein expression of the muscle differentiation marker genes MYOG and MYH3. The results showed that the miR-139 mimic could enhance the differentiation ofbovineMDSCs, whereas the inhibitor had the opposite effect. By using the dual-luciferase reporter system, miR- 139 was found to target the 3'-untranslated region of the dihydrofolate reductase (DHFR) gene and regulate its expression. In addition, the expression of miR-139 was found to be regulated by its host gene phosphodiesterase 2A (PDE2A) via inhibition of the latter byCRISPRinterference (CRISPRi). Overall, our findings indicate that miR- 139 plays an important role in regulating the differentiation ofbovineMDSCs.
cow
28 animal cow CRISPR;Cas9; OCT4 Proceedings of
the National Academy of Sciences of the United States of America
OCT4/POU5F1 is required for NANOG expression in bovine blastocysts.
2018 115(11):2770- 2775
[Simmet K et al.]
Ludwig-Maximilians-Universitat Munchen, Munich, Germany.
29483258 10.1073/pnas.1 718833115
Mammalian preimplantation development involves two lineage specifications: first, the CDX2-expressing trophectoderm (TE) and a pluripotent inner cell mass (ICM) are separated during blastocyst formation. Second, the pluripotent epiblast (EPI; expressing NANOG) and the differentiated primitive endoderm (PrE; expressing GATA6) diverge within the ICM. Studies in mice revealed that OCT4/POU5F1 is at the center of a pluripotency regulatory network. To study the role of OCT4 inbovinepreimplantation development, we generated OCT4 knockout (KO) fibroblasts byCRISPR-Cas9and produced embryos by somatic cell nuclear transfer (SCNT). SCNT embryos from nontransfected fibroblasts and embryos produced by in vitro fertilization served as controls. In OCT4 KO morulae (day 5), approximately 70% of the nuclei were OCT4 positive, indicating that maternal OCT4 mRNA partially maintains OCT4 protein expression during early development. In contrast, OCT4 KO blastocysts (day 7) lacked OCT4 protein entirely. CDX2 was detected only in TE cells; OCT4 is thus not required to suppress CDX2 in the ICM. Control blastocysts showed a typical salt-and-pepper distribution of NANOG- and GATA6-positive cells in the ICM. In contrast, NANOG was absent or very faint in the ICM of OCT4 KO blastocysts, and no cells expressing exclusively NANOG were observed. This mimics findings in OCT4-deficient human blastocysts but is in sharp contrast to Oct4-null mouse blastocysts, where NANOG persists and PrE development fails. Our study supportsbovineembryogenesis as a model for early human development and exemplifies a general strategy for studying the
cow
