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"Animal genetic engineering."
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GMOs
An overview of genetic modification and how this biotechnology affects food, humans, animals, and medicine. Includes a discussion of the ethical concerns of genetic biotechnology.
Book
Frequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryos
Background
The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting
RAG2
,
IL2RG
,
SCD5
, and
Ig Heavy chain
were examined.
Results
First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt
Ig Heavy chain
in pigs; frequency of off-targeting was 80 and 70% on
AR
and
RBFOX1
locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5′ side of
AR
, suggesting that off-targeting activities are systematic events. However, the mismatches on
RBFOX1
were not limited to the 5′ side, indicating unpredictability of the events.
Conclusions
The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.
Journal Article
Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set
Background
Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency.
Results
109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring.
Conclusion
Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.
Journal Article
Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research
Background
Genetic engineering technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides a powerful tool for developing disease models and determining gene functions. Recent interests in canine cancer models have highlighted the necessity of developing genetic engineering tools for dogs. In this study, we attempted to generate optimized CRISPR/Cas9 system to target canine tumor protein 53 (
TP53
), one of the most crucial tumor suppressor genes, to establish TP53 knockout canine cells for canine cancer research.
Results
We constructed CRISPR/Cas9 vectors using each of three
TP53
gene-targeting guide RNAs (gRNAs) with minimal off-target potential. After transfection, we obtained several clones of
TP53
knockout cells containing “indel” mutations in the targeted locus which had infinite cellular life span, resistance to genotoxicity, and unstable genomic status in contrast to normal cells. Of the established TP53 knockout cells, TP53KO#30 cells targeted by TP53 gRNA #30 showed non-cancerous phenotypes without oncogenic activation both in vitro and in vivo. More importantly, no off-target alteration was detected in TP53KO#30 cells. We also tested the developmental capacity of TP53 knockout cells after application of the somatic cell nuclear transfer technique.
Conclusions
Our results indicated that
TP53
in canine cells was effectively and specifically targeted by our CRISPR/Cas9 system. Thus, we suggest our CRISPR/Cas9-derived canine
TP53
knockout cells as a useful platform to reveal novel oncogenic functions and effects of developing anti-cancer therapeutics.
Journal Article
Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins
Background
Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient.
Results
Here we describe
Easi-
CRISPR (
E
fficient
a
dditions with
s
sDNA
i
nserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that
Easi
-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring.
Conclusions
Easi-
CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus,
Easi-
CRISPR offers a comprehensive means of building large-scale Cre-
LoxP
animal resources.
Journal Article