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.

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 Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo

2018 ◽  
Vol 115 (38) ◽  
pp. 9351-9358 ◽  
Author(s):  
Robert M. Yarrington ◽  
Surbhi Verma ◽  
Shaina Schwartz ◽  
Jonathan K. Trautman ◽  
Dana Carroll
Keyword(s):  
Genome Editing ◽  
Zinc Finger Nucleases ◽  
Yeast Genome ◽  
Target Sequence ◽  
Practical Applications ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Wide Range

Genome editing with CRISPR-Cas nucleases has been applied successfully to a wide range of cells and organisms. There is, however, considerable variation in the efficiency of cleavage and outcomes at different genomic targets, even within the same cell type. Some of this variability is likely due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin structure, particularly the presence or absence of nucleosomes, on cleavage by the Streptococcus pyogenes Cas9 protein. At multiple target sequences in two promoters in the yeast genome, we find that Cas9 cleavage is strongly inhibited when the DNA target is within a nucleosome. This inhibition is relieved when nucleosomes are depleted. Remarkably, the same is not true of zinc-finger nucleases (ZFNs), which cleave equally well at nucleosome-occupied and nucleosome-depleted sites. These results have implications for the choice of specific targets for genome editing, both in research and in clinical and other practical applications.

A Simple Heat Treatment Increases SpCas9-Mediated Mutation Efficiency in Arabidopsis

2021 ◽  
Author(s):  
Shuta Kurokawa ◽  
Hafizur Rhaman ◽  
Naoshi Yamanaka ◽  
Chisato Ishizaki ◽  
Shaikhul Islam ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Genome Editing ◽  
Target Sequence ◽  
Simple Method ◽  
Guide Rna ◽  
Highly Active ◽  
Seed Population ◽  
Target Sites ◽  
Mutation Efficiency ◽  
Rna Genes

Abstract The CRISPR/Cas9 system is now commonly employed for genome editing in various plants such as Arabidopsis, rice, and tobacco. In general, in genome editing of the Arabidopsis genome, the SpCas9 and guide RNA genes are introduced into the genome by the floral dip method. Mutations induced in the target sequence by SpCas9 are confirmed after selecting transformants by screening the T1 seed population. The advantage of this method is that genome-edited plants can be isolated easily. However, mutation efficiency in Arabidopsis using SpCas9 is not as high as that achieved in rice and tobacco, which are subjected to a tissue culture step. In this study, we compared four promoters and found that the parsley UBIQITIN promoter is highly active in Arabidopsis meristem tissue. Furthermore, we examined whether a simple heat treatment could improve mutation efficiency in Arabidopsis. Just one heat treatment at 37 °C for 24 hours increased the mutation efficiency at all four target sites from 3% to 42%, 43% to 62%, 54% to 75%, and 89 to 91%, respectively, without detectable off-target mutations. We recommend heat treatment of plate-grown plants at 37 °C for 24 hours as a simple method to increase the efficiency of CRISPR/Cas9-mediated mutagenesis in Arabidopsis.

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.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

Insulin Growth Factor-I Promotes Functional Recovery After a Focal Lesion in the Dentate Gyrus

2012 ◽  
Vol 11 (7) ◽  
pp. 818-828 ◽  
Author(s):  
Adhemar Liquitaya-Montiel ◽  
Andrea Aguilar-Arredondo ◽  
Clorinda Arias ◽  
Angelica Zepeda
Keyword(s):  
Growth Factor ◽  
Dentate Gyrus ◽  
Functional Recovery ◽  
Focal Lesion ◽  
Insulin Growth Factor ◽  
Factor I ◽  
Growth Factor I

Normal age-dependent values of serum insulin growth factor-I: Results from a healthy Italian population

2008 ◽  
Vol 31 (5) ◽  
pp. 445-449 ◽  
Author(s):  
G. Aimaretti ◽  
M. Boschetti ◽  
G. Corneli ◽  
V. Gasco ◽  
D. Valle ◽  
...  
Keyword(s):  
Growth Factor ◽  
Serum Insulin ◽  
Italian Population ◽  
Insulin Growth Factor ◽  
Factor I ◽  
Age Dependent ◽  
Growth Factor I

Development of a mito-CRISPR system for generating mitochondrial DNA-deleted strain in Saccharomyces cerevisiae

2020 ◽  
Vol 85 (4) ◽  
pp. 895-901
Author(s):  
Takamitsu Amai ◽  
Tomoka Tsuji ◽  
Mitsuyoshi Ueda ◽  
Kouichi Kuroda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Nuclear Genome ◽  
Target Sequence ◽  
Guide Rna ◽  
Crispr System ◽  
Mitochondrial Target ◽  
Gene Treatment ◽  
Mtdna Deletion

ABSTRACT Mitochondrial dysfunction can occur in a variety of ways, most often due to the deletion or mutation of mitochondrial DNA (mtDNA). The easy generation of yeasts with mtDNA deletion is attractive for analyzing the functions of the mtDNA gene. Treatment of yeasts with ethidium bromide is a well-known method for generating ρ° cells with complete deletion of mtDNA from Saccharomyces cerevisiae. However, the mutagenic effects of ethidium bromide on the nuclear genome cannot be excluded. In this study, we developed a “mito-CRISPR system” that specifically generates ρ° cells of yeasts. This system enabled the specific cleavage of mtDNA by introducing Cas9 fused with the mitochondrial target sequence at the N-terminus and guide RNA into mitochondria, resulting in the specific generation of ρ° cells in yeasts. The mito-CRISPR system provides a concise technology for deleting mtDNA in yeasts.

scholarly journals Application of CRISPR/Cas9 technology in wild apple (Malus sieverii) for paired sites gene editing

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yan Zhang ◽  
Ping Zhou ◽  
Tohir A. Bozorov ◽  
Daoyuan Zhang
Keyword(s):  
Abiotic Stress ◽  
Human Activities ◽  
Genetic Improvement ◽  
Gene Editing ◽  
New Technologies ◽  
Tianshan Mountains ◽  
Biotic And Abiotic Stress ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Albino Phenotype

Abstract Background Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. It is necessary to use new technologies to research its genomic function and molecular improvement. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability varies depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Results In this study, we used 2 systems of vectors with paired sgRNAs targeting to MsPDS. As expected, we successfully induced the albino phenotype of calli and buds in both systems. Conclusions We conclude that CRISPR/Cas9 is a powerful system for editing the wild apple genome and expands the range of plants available for gene editing.

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