scholarly journals In vitro Electroporation in the Presence of CRISPR/Cas9 Reagents as a Safe and Effective Method for Producing Biallelic Knock-Out Porcine Embryos

2021 ◽  
Vol 5 (1) ◽  
Author(s):  
Masahiro Sato ◽  
◽  
Hansol Jin ◽  
Eri Akasaka ◽  
Kazuchika Miyoshi
Keyword(s):  
Viral Infection ◽  
Target Gene ◽  
Direct Injection ◽  
Porcine Genome ◽  
Knock Out ◽  
Guide Rna ◽  
Insertion And Deletion ◽  
Cas9 Protein ◽  
Cytoplasmic Injection

The production of genetically modified (GM) pigs is considered valuable in biomedical research for the development of model animals for various diseases and pigs with resistance against viral infection. The porcine genome may be modified using several methods, such as somatic cell nuclear transfer (SCNT) using GM cells as the SCNT donor, direct injection of the transgene or the genome editing components (GEC) into fertilized eggs referred to as zygotes, the in vitro electroporation (EP) of the zygotes in the presence of GECs, viral infection using retroviruses, injection of the GECs into the SCNT-treated embryos, and the in vitro EP of the SCNT-treated embryos in the presence of GECs. In our previous study, we administered a cytoplasmic injection of CRISPR/Cas9-based GEC into parthenogenetically-activated porcine oocytes (referred to as parthenotes) and observed that these oocytes comprised a mixture of genome-edited and genome-unedited cells, referred to as the “mosaic”. In contrast, when in vitro EP of the SCNT-treated embryos in the presence of GEC was performed, bi-allelic knock out (KO) of the target gene was detected in most oocytes (82%; 9/11). The production of bi-allelic KO piglets is particularly beneficial for investigating GM domestic animals as it does not require further breeding trials to obtain bi-allelic KO individuals, which would otherwise be a time-consuming and laborious task. In this context, the present study was aimed to confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos without multiple breeding trials, for which parthenotes were subjected to EP in the presence of a ribonucleoprotein containing Cas9 protein and single-guide RNA (targeted toward GGTA1). The treated embryos were cultured until they transformed into blastocysts. The genomic DNA isolated from these blastocysts was used for molecular biology analysis to detect the possible insertion and deletion of sequences (indels) at the GGTA1 locus. Among the 32 blastocysts obtained, 21 (66%) were observed to be the bi-allelic KO ones. The remaining embryos either had a normal phenotype (25%; 8/32) or mosaic mutations (9%; 3/32). These findings confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos.

scholarly journals In vitro Electroporation in the Presence of CRISPR/Cas9 Reagents as a Safe and Effective Method for Producing Biallelic Knock-Out Porcine Embryos

2021 ◽  
Vol 5 (1) ◽  
Author(s):  
Masahiro Sato ◽  
◽  
Hansol Jin ◽  
Eri Akasaka ◽  
Kazuchika Miyoshi
Keyword(s):  
Viral Infection ◽  
Target Gene ◽  
Direct Injection ◽  
Porcine Genome ◽  
Knock Out ◽  
Guide Rna ◽  
Insertion And Deletion ◽  
Cas9 Protein ◽  
Cytoplasmic Injection

The production of genetically modified (GM) pigs is considered valuable in biomedical research for the development of model animals for various diseases and pigs with resistance against viral infection. The porcine genome may be modified using several methods, such as somatic cell nuclear transfer (SCNT) using GM cells as the SCNT donor, direct injection of the transgene or the genome editing components (GEC) into fertilized eggs referred to as zygotes, the in vitro electroporation (EP) of the zygotes in the presence of GECs, viral infection using retroviruses, injection of the GECs into the SCNT-treated embryos, and the in vitro EP of the SCNT-treated embryos in the presence of GECs. In our previous study, we administered a cytoplasmic injection of CRISPR/Cas9-based GEC into parthenogenetically-activated porcine oocytes (referred to as parthenotes) and observed that these oocytes comprised a mixture of genome-edited and genome-unedited cells, referred to as the “mosaic”. In contrast, when in vitro EP of the SCNT-treated embryos in the presence of GEC was performed, bi-allelic knock out (KO) of the target gene was detected in most oocytes (82%; 9/11). The production of bi-allelic KO piglets is particularly beneficial for investigating GM domestic animals as it does not require further breeding trials to obtain bi-allelic KO individuals, which would otherwise be a time-consuming and laborious task. In this context, the present study was aimed to confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos without multiple breeding trials, for which parthenotes were subjected to EP in the presence of a ribonucleoprotein containing Cas9 protein and single-guide RNA (targeted toward GGTA1). The treated embryos were cultured until they transformed into blastocysts. The genomic DNA isolated from these blastocysts was used for molecular biology analysis to detect the possible insertion and deletion of sequences (indels) at the GGTA1 locus. Among the 32 blastocysts obtained, 21 (66%) were observed to be the bi-allelic KO ones. The remaining embryos either had a normal phenotype (25%; 8/32) or mosaic mutations (9%; 3/32). These findings confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos.

scholarly journals Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex

2021 ◽  
Vol 22 (6) ◽  
pp. 2937
Author(s):  
Monika Halat ◽  
Magdalena Klimek-Chodacka ◽  
Jagoda Orleanska ◽  
Malgorzata Baranska ◽  
Rafal Baranski
Keyword(s):  
Circular Dichroism ◽  
Conformational Changes ◽  
Structural Changes ◽  
Electronic Circular Dichroism ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Rnp Complex ◽  
Characteristic Features ◽  
Circular Dichroïsm

The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.

scholarly journals Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels

2017 ◽  
Vol 114 (31) ◽  
pp. 8283-8288 ◽  
Author(s):  
Jun Liu ◽  
Yong Juan Zhao ◽  
Wan Hua Li ◽  
Yun Nan Hou ◽  
Ting Li ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Bimolecular Fluorescence Complementation ◽  
Knock Out ◽  
Guide Rna ◽  
Type Iii ◽  
Cyclic Adp Ribose ◽  
Human Multiple Myeloma ◽  
Hybrid Screening

CD38 catalyzes the synthesis of the Ca2+ messenger, cyclic ADP-ribose (cADPR). It is generally considered to be a type II protein with the catalytic domain facing outside. How it can catalyze the synthesis of intracellular cADPR that targets the endoplasmic Ca2+ stores has not been resolved. We have proposed that CD38 can also exist in an opposite type III orientation with its catalytic domain facing the cytosol. Here, we developed a method using specific nanobodies to immunotarget two different epitopes simultaneously on the catalytic domain of the type III CD38 and firmly established that it is naturally occurring in human multiple myeloma cells. Because type III CD38 is topologically amenable to cytosolic regulation, we used yeast-two-hybrid screening to identify cytosolic Ca2+ and integrin-binding protein 1 (CIB1), as its interacting partner. The results from immunoprecipitation, ELISA, and bimolecular fluorescence complementation confirmed that CIB1 binds specifically to the catalytic domain of CD38, in vivo and in vitro. Mutational studies established that the N terminus of CIB1 is the interacting domain. Using shRNA to knock down and Cas9/guide RNA to knock out CIB1, a direct correlation between the cellular cADPR and CIB1 levels was demonstrated. The results indicate that the type III CD38 is functionally active in producing cellular cADPR and that the activity is specifically modulated through interaction with cytosolic CIB1.

scholarly journals RNAi in Dictyostelium: The Role of RNA-directed RNA Polymerases and Double-stranded RNase

2002 ◽  
Vol 13 (2) ◽  
pp. 445-453 ◽  
Author(s):  
Henrik Martens ◽  
Jindrich Novotny ◽  
Jürgen Oberstrass ◽  
Theodore L. Steck ◽  
Pamela Postlethwait ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Target Gene ◽  
Helicase Domain ◽  
Limited Capacity ◽  
Hairpin Rna ◽  
Knock Out ◽  
Endogenous Gene ◽  
The One

We show that in Dictyostelium discoideum an endogenous gene as well as a transgene can be silenced by introduction of a gene construct that is transcribed into a hairpin RNA. Gene silencing was accompanied by the appearance of sequence-specific RNA ∼23mers and seemed to have a limited capacity. The threeDictyostelium homologues of the RNA-directed RNA polymerase (RrpA, RrpB, and DosA) all contain an N-terminal helicase domain homologous to the one in the dicer nuclease, suggesting exon shuffling between RNA-directed RNA polymerase and the dicer homologue. Only the knock-out of rrpA resulted in a loss of the hairpin RNA effect and simultaneously in a loss of detectable ∼23mers. However, ∼23mers were still generated by theDictyostelium dsRNase in vitro with extracts from rrpA−, rrpB−, and DosA− cells. Both RrpA and a target gene were required for production of detectable amounts of ∼23mers, suggesting that target sequences are involved in ∼23mer amplification.

scholarly journals One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

2021 ◽  
Vol 22 (5) ◽  
pp. 2249
Author(s):  
Fuminori Tanihara ◽  
Maki Hirata ◽  
Nhien Thi Nguyen ◽  
Osamu Sawamoto ◽  
Takeshi Kikuchi ◽  
...  
Keyword(s):  
Gene Editing ◽  
Multiple Gene ◽  
Knock Out ◽  
Editing Efficiency ◽  
Guide Rnas ◽  
Cas9 Protein ◽  
One Step ◽  
Step Generation ◽  
The One

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

scholarly journals Programmed mutation of liver fluke granulin using CRISPR/Cas9 attenuates virulence of infection-induced hepatobiliary morbidity

2018 ◽  
Author(s):  
Patpicha Arunsan ◽  
Wannaporn Ittiprasert ◽  
Michael J. Smout ◽  
Christina J. Cochran ◽  
Victoria H. Mann ◽  
...  
Keyword(s):  
Biliary Tract ◽  
Liver Fluke ◽  
Knock Out ◽  
Guide Rna ◽  
Food Borne ◽  
Cas9 Nuclease ◽  
Parasitic Flatworm ◽  
Exon 1 ◽  
Juvenile Stages

AbstractInfections with several flatworm parasites represent group 1 biological carcinogens, i.e. definite causes of cancer. Infection with the food-borne liver fluke Opisthorchis viverrini causes cholangiocarcinoma (CCA). Whereas the causative agent for most cancers, including CCA in the West, remains obscure, the principal risk factor for CCA in Thailand is opisthorchiasis. We exploited this established link to explore the role of the secreted parasite growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions of the biliary tract. We targeted the Ov-grn-1 gene for programmed knockout and investigated gene-edited parasites in vitro and in experimentally infected hamsters. Both adult and juvenile stages of the liver fluke were transfected with a plasmid encoding a guide RNA sequence specific for exon 1 of Ov-grn-1 and the Cas9 nuclease. Deep sequencing of amplicon libraries from genomic DNA from gene-edited parasites exhibited programmed, Cas9-catalyzed mutations within the Ov-grn-1 locus, and tandem analyses by RT-PCR and western blot revealed rapid depletion of Ov-grn-1 transcripts and protein. Newly excysted juvenile flukes that had undergone editing of Ov-grn-1 colonized the biliary tract, grew and developed over a period of 60 days, were active and motile, and induced a clinically relevant pathophysiological tissue phenotype of attenuated biliary hyperplasia and fibrosis in comparison to infection with wild type flukes. This is the first report of gene knock-out using CRISPR/Cas9 in a parasitic flatworm, demonstrating the activity and utility of the process for functional genomics in these pathogens. The striking clinical phenotype highlights the role in virulence that liver fluke growth factors play in biliary tract morbidity during chronic opisthorchiasis.

scholarly journals Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yasuaki Kohama ◽  
Shuichiro Higo ◽  
Yuki Masumura ◽  
Mikio Shiba ◽  
Takumi Kondo ◽  
...  
Keyword(s):  
Direct Injection ◽  
Adeno Associated Virus ◽  
Systemic Injection ◽  
Guide Rna ◽  
Human Cardiomyocytes ◽  
Wide Range ◽  
Targeted Integration ◽  
Induced Pluripotent

Abstract Post-mitotic cardiomyocytes have been considered to be non-permissive to precise targeted integration including homology-directed repair (HDR) after CRISPR/Cas9 genome editing. Here, we demonstrate that direct delivery of large amounts of transgene encoding guide RNA (gRNA) and repair template DNA via intra-ventricular injection of adeno-associated virus (AAV) promotes precise targeted genome replacement in adult murine cardiomyocytes expressing Cas9. Neither systemic injection of AAV nor direct injection of adenovirus promotes targeted integration, suggesting that high copy numbers of single-stranded transgenes are required in cardiomyocytes. Notably, AAV-mediated targeted integration in cardiomyocytes both in vitro and in vivo depends on the Fanconi anemia pathway, a key component of the single-strand template repair mechanism. In human cardiomyocytes differentiated from induced pluripotent stem cells, AAV-mediated targeted integration fluorescently labeled Mlc2v protein after differentiation, independently of DNA synthesis, and enabled real-time detection of sarcomere contraction in monolayered beating cardiomyocytes. Our findings provide a wide range of applications for targeted genome replacement in non-dividing cardiomyocytes.

scholarly journals Somatic cell reprogramming-free generation of genetically modified pigs

2016 ◽  
Vol 2 (9) ◽  
pp. e1600803 ◽  
Author(s):  
Fuminori Tanihara ◽  
Tatsuya Takemoto ◽  
Eri Kitagawa ◽  
Shengbin Rao ◽  
Lanh Thi Kim Do ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Gene Editing ◽  
Genetically Modified ◽  
Efficient Production ◽  
Simple Method ◽  
Guide Rna ◽  
Somatic Cell Reprogramming ◽  
Cas9 Protein ◽  
Somatic Cell Nucleus

Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.

scholarly journals Selection-free, high frequency genome editing by homologous recombination of human pluripotent stem cells using Cas9 RNP and AAV6

2018 ◽  
Author(s):  
Renata M. Martin ◽  
Kazuya Ikeda ◽  
Nobuko Uchida ◽  
Kyle Cromer ◽  
Toshi Nishimura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Guide Rna ◽  
Large Gene ◽  
Marker Selection ◽  
Cas9 Protein

AbstractCombination of genome editing and human pluripotent stem cells (hPSCs) offers a platform for in vitro disease modeling, drug discovery and personalized stem cell therapeutics. However, incorporation of large modifications using CRISPR/Cas9-based genome editing in hPSCs typically requires the use of selection markers due to low editing efficiencies. Here we report a novel editing technology in hPSCs using Cas9 protein complexed with chemically modified single guide RNA (sgRNA) and recombinant AAV6 (rAAV6) vectors for donor delivery without marker selection. With these components, we demonstrate targeted integration of a 2.2 kb DNA expression cassette in hPSCs at frequencies up to 94% and 67% at the HBB and MYD88 loci, respectively. We used this protocol to correct the homozygous sickle cell disease (SCD) mutation in an iPSC line derived from a SCD patient with a frequency of 63%. This Cas9/AAV6 system allows for both the integration of large gene cassettes and the creation of single nucleotide changes in hPSCs at high frequencies, eliminating the need for multiple editing steps and marker selection, thus increasing the potential of editing human pluripotent cells for both research and translational applications.

3 Embryo knockout efficiency improved when targeting ovine suppressor of cytokine signalling 2 with 2 small guide RNA

2019 ◽  
Vol 31 (1) ◽  
pp. 127 ◽  
Author(s):  
A. K. Mahdi ◽  
J. F. Medrano ◽  
P. J. Ross
Keyword(s):  
Growth Factor ◽  
Direct Injection ◽  
High Growth ◽  
Cumulus Cells ◽  
Target Sequence ◽  
Guide Rna ◽  
Insulin Growth Factor ◽  
Cas9 Protein ◽  
And Control ◽  
Mutation Efficiency

Suppressor of cytokine signalling (SOCS2) is a key regulator of growth. Growth hormone (GH) and insulin growth factor-1 induce SOCS2 expression, which acts as a suppressor of GH and insulin growth factor-1 signalling by preventing the activation of the signal transducer and activator of transcription proteins. In mice, SOCS2 knockout results in a gigantic phenotype with elevated GH levels, longer bones, enlargement of internal organs, and 30 to 40% heavier body weight in comparison with wild-type animals. High growth was also noted in naturally occurring SOCS2 mutant pigs. The aim of this project was to produce high-growth sheep by introducing sense mutations to SOCS2 by direct injection of CRISPR/Cas9 in ovine embryos. In this study, we compared the editing efficiency of 2 small guide RNA (sgRNA), which were designed to target the first exon of SOCS2. Ovine ovaries were collected from a local slaughterhouse and cumulus-oocyte complexes were aspirated from 2- to 6-mm antral follicles, matured invitro for 22h, and then denuded from the surrounding cumulus cells. Approximately 6 pL of CRISPR/Cas9 solution (80ng µL−1 of sgRNA, 80ng µL−1 of Cas9 mRNA, and 160ng µL−1 of Cas9 protein in low-TE buffer) was microinjected into the cytoplasm of metaphase II oocytes using a laser-assisted microinjection technique (Bogliotti et al. 2016 J. Vis. Exp. 116, e54465). Groups of 50 oocytes were injected with the CRISPR/Cas9 mix containing sgRNA1 (group G1), sgRNA2 (group G2), sgRNA1 and sgRNA2 (group G1/2), or low-TE buffer alone (control). After microinjection, IVF was carried out using fresh semen, and oocytes were co-incubated with the sperm for 16h. Embryos were cultured in groups of 25 in 50-µL drops of KSOM (Evolve, Zenith Technologies, Cork, Ireland) with 4mg mL−1 of BSA under oil at 38.5°C, 5% CO2, 5% O2, and 90% N2. Blastocysts were collected on Day 7 post-fertilization to assess mutation efficiency. The CRISPR/Cas9 target sequence was amplified using nested PCR from single-blastocyst whole lysates and analysed by Sanger sequencing. Mutations in each embryo were identified by visual inspection of the sequence traces aided by trace deconvolution software (SnapGene, GSL Biotech, Chicago, IL, USA). The experiment was replicated 3 times. Blastocyst rates were 22.6, 21.3, 21.3, and 29.3% for G1, G2, G1/2, and control, respectively. Targeting the exon with 2 sgRNA (G1/2) resulted in the highest mutation rate (87.3%), with an average indel size of 55.6bp, leading to 100% nonfunctional SOCS2, and with 87.6% of the sequenced embryos presenting bi-allelic mutations. Using a single sgRNA caused mutations in 47.3 and 38.6% of embryos for G1 and G2, respectively, with averages indel size of 8.2bp in G1 and 13.8bp in G2. In G1, 88.6% of the mutations were biallelic and 90% would result in nonfunctional SOCS2. In G2, 50% of the mutations were biallelic and 40% translated to nonfunctional SOCS2. Based on these results, the percentage of SOCS2 knockout embryos was 37.7, 7.7, and 76.4% in G1, G2, and G1/2, respectively. In conclusion, the knockout efficiency improved after targeting ovine SOCS2 with 2 sgRNA.

Sign in / Sign up

Export Citation Format

Share Document