Zygote injection of RNA encoding Cre recombinase results in efficient removal of LoxP flanked neomycin cassettes in pigs

Whitworth, Kristin M.; Cecil, Raissa F.; Benne, Joshua A.; Redel, Bethany K.; Spate, Lee D.; Samuel, Melissa; Prather, Randall S.; Wells, Kevin D.

doi:10.1007/s11248-018-0064-3

Cited by 7 publications

(7 citation statements)

References 44 publications

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“…S1e, f). When reconstructed embryos were cultured for 6 days, almost no difference of blastocyst rates between Dox-treated (19.5%, 40/205) and Dox-untreated (20.4%, 23/113) groups was observed, while the previous report showed that zygote injection of RNA encoding Cre recombinase resulted in reduction of blastocyst rate [ 26 ], indicating that Dox-dependent SpCas9 expression may have less negative impact on porcine embryo development than Cre recombinase expression. These results suggested that the established DIC system in pigs could result in Dox-dependent SpCas9 expression at the cellular and embryonic level.…”

Section: Resultsmentioning

confidence: 87%

“…Additionally, genomic damages [ 20 , 21 ], off-target effects [ 22 , 23 ], and immunological clearance responses [ 24 ] caused by uncontrollable Cas9 expression, will hinder the application of animal models with this constitutively Cas9-expressing system, which is also not avoidable for inducible expression systems based on the Cre-recombinase system, in which sustainable constitutive Cas9 expression exist after activation [ 16 , 25 ]. Furthermore, Cre-recombinase was confirmed to negatively affect porcine embryonic development [ 26 ] and to be potentially genotoxic with pseudo recombination sites in mammalian genomes [ 27 ].…”

Section: Introductionmentioning

confidence: 99%

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Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation

et al. 2023

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Background CRISPR-based toolkits have dramatically increased the ease of genome and epigenome editing. SpCas9 is the most widely used nuclease. However, the difficulty of delivering SpCas9 and inability to modulate its expression in vivo hinder its widespread adoption in large animals. Results Here, to circumvent these obstacles, a doxycycline-inducible SpCas9-expressing (DIC) pig model was generated by precise knock-in of the binary tetracycline-inducible expression elements into the Rosa26 and Hipp11 loci, respectively. With this pig model, in vivo and/or in vitro genome and epigenome editing could be easily realized. On the basis of the DIC system, a convenient Cas9-based conditional knockout strategy was devised through controlling the expression of rtTA component by tissue-specific promoter, which allows the one-step generation of germline-inherited pigs enabling in vivo spatiotemporal control of gene function under simple chemical induction. To validate the feasibility of in vivo gene mutation with DIC pigs, primary and metastatic pancreatic ductal adenocarcinoma was developed by delivering a single AAV6 vector containing TP53-sgRNA, LKB1-sgRNA, and mutant human KRAS gene into the adult pancreases. Conclusions Together, these results suggest that DIC pig resources will provide a powerful tool for conditional in vivo genome and epigenome modification for fundamental and applied research.

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Section: Resultsmentioning

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation

et al. 2023

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“…Cre recombinase expression in the germ line of mice would result in abnormal arrangement of chromosomes and infertility, which might be due to the unexpected interactions of pre‐existing pseudo loxP sequences in the presence of Cre in vivo [85]. Microinjection of Cre mRNA into pig embryos at a high dose could lead to embryonic toxicity, which may indicate the presence of pre‐existing pseudo loxP sequences in the swine genome [86].…”

Section: Swine Models With Recombinase‐specific Recognition Elementsmentioning

confidence: 99%

Genome editing pig models with elements for controllable gene expression

Jin,

Shi,

Liu

et al. 2023

Animal Research and One Health

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Conditional gene regulation systems can control gene expression in predefined tissues or organs at a desired time. Site‐specific recombinase systems and chemically induced gene expression systems are the two most widely used approaches for creating genetically modified (GM) animals with conditional regulation of gene expression. Generation of GM pigs with controllable elements, usually involving multiple gene editing, used to be a major challenge due to a lack of germ line‐competent pluripotent stem cells. With the emergence of artificial endonuclease‐mediated gene editors, a variety of GM pigs with recombinase‐specific recognition elements or chemically induced elements for conditional regulation of gene expression have been generated by the combination of site‐directed knock‐in of somatic cells and somatic cell nuclear transfer technology, allowing conditional deletion of endogenous genes or overexpression of exogenous genes in pigs. These inducible tool pig models will greatly facilitate the production of GM pigs and broaden the applications of transgenic pigs in biomedicine and agriculture fields. In this paper, we review the progress in the construction and application of pigs with controllable elements using gene editing techniques.

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“…By 2018, Whitworth et al had developed a method that utilized the CRISPR/Cas9 technology to remove a loxP flanked neomycin cassette by direct zygote injection of RNA encoding Cre recombinase. This new technique can be used to efficiently remove selectable markers in genetically engineered animals without the need for long-term cell culture and subsequent somatic cell nuclear transfer (SCNT) [67]. Almost certainly, it has a very promising future for transgenic pigs with the advantages of enhancing body growth and minimizing environmental pollution that would be created by the CRISRP/Cas9 method.…”

Section: Applications In Transgenesis and Beyondmentioning

confidence: 99%

Application and Development of CRISPR/Cas9 Technology in Pig Research

Lin¹,

Deng²,

Li³

et al. 2019

Gene Editing - Technologies and Applications

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Pigs provide valuable meat sources, disease models, and research materials for humans. However, traditional methods no longer meet the developing needs of pig production. More recently, advanced biotechnologies such as SCNT and genome editing are enabling researchers to manipulate genomic DNA molecules. Such methods have greatly promoted the advancement of pig research. Three gene editing platforms including ZFNs, TALENs, and CRISPR/Cas are becoming increasingly prevalent in life science research, with CRISPR/Cas9 now being the most widely used. CRISPR/Cas9, a part of the defense mechanism against viral infection, was discovered in prokaryotes and has now developed as a powerful and effective genome editing tool that can introduce and enhance modifications to the eukaryotic genomes in a range of animals including insects, amphibians, fish, and mammals in a predictable manner. Given its excellent characteristics that are superior to other tailored endonucleases systems, CRISPR/Cas9 is suitable for conducting pig-related studies. In this review, we briefly discuss the historical perspectives of CRISPR/Cas9 technology and highlight the applications and developments for using CRISPR/Cas9-based methods in pig research. We will also review the choices for delivering genome editing elements and the merits and drawbacks of utilizing the CRISPR/Cas9 technology for pig research, as well as the future prospects.

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Zygote injection of RNA encoding Cre recombinase results in efficient removal of LoxP flanked neomycin cassettes in pigs

Cited by 7 publications

References 44 publications

Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation

Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation

Genome editing pig models with elements for controllable gene expression

Application and Development of CRISPR/Cas9 Technology in Pig Research

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