The advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research

Zhang, Jinfu; Khazalwa, Emmanuel Mulaya; Abkallo, Hussein M.; Zhou, Yuan; Nie, Xiongwei; Ruan, Jinxue; Zhao, Changzhi; Wang, Jieru; Xu, Jing; Li, Xinyun; Zhao, Shuhong; Zuo, Erwei; Steinaa, Lucilla; Xie, Shengsong

doi:10.1016/j.jgg.2021.03.015

Cited by 12 publications

(7 citation statements)

References 158 publications

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“…The wide application of CRISPR genome editing technology in pig germplasm improvement will result in more multiple gene-edited pigs with numerous disease-resistant traits. , The nucleic acid detection of genetically modified pigs is critical. The developed multiplex RAVI-CRISPR technology can facilitate PoC testing for various applications, such as the simultaneous detection of coinfections and multiple gene editions in pigs.…”

Section: Discussionmentioning

confidence: 99%

“…34−36 The outstanding advantage of this multiplex CRISPR nucleic acid assay compared to other CRISPR colorimetric/fluorescence assays is the use of modified green-red-yellow, fluorescent signal conversion ssDNA-FQ and ssRNA-FQ reporters combinations suitable for visualization with the naked eye, eliminating the need for expensive microplate readers and specialized hand-held devices for visualization. 27−29 The wide application of CRISPR genome editing technology in pig germplasm improvement 37 will result in more multiple gene-edited pigs with numerous disease-resistant traits. 38,39 The nucleic acid detection of genetically modified pigs is critical.…”

Section: Discussionmentioning

confidence: 99%

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Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine

Wang

Tao

et al. 2023

ACS Synth. Biol.

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The Rapid Visual CRISPR (RAVI-CRISPR) assay employs Cas12a and Cas13a enzymes for precise gene detection in a sample. However, RAVI-CRISPR is limited in single-tube multiplex detection applications due to the lack of specific single-strand (ss) DNA-fluorescently quenched (ssDNA-FQ) and RNA-fluorescently quenched (ssRNA-FQ) reporter cleavage mechanisms. We report the development of a sensitive and specific dual-gene Cas12a and Cas13a diagnostic system. To optimize the application for field testing, we designed a portable multiplex fluorescence imaging assay that could distinguish test results with the naked eye. Herein, dual gene amplified products from multiplex recombinase polymerase amplification (RPA) were simultaneously detected in a single tube using Cas12a and Cas13a enzymes. The resulting orthogonal DNA and RNA collateral cleavage specifically distinguishes individual and mixed ssDNA-FQ and ssRNA-FQ reporters using the green–red–yellow, fluorescent signal conversion reaction system, detectable with portable blue and ultraviolet (UV) light transilluminators. As a proof-of-concept, reliable multiplex RAVI-CRISPR detection of genome-edited pigs was demonstrated, exhibiting 100% sensitivity and specificity for the analysis of CD163 knockout, lactoferrin (LF) knock-in, and wild-type pig samples. This portable naked-eye multiplex RAVI-CRISPR detection platform can provide accurate point-of-care screening of genetically modified animals and infectious diseases in resource-limited settings.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine

Wang

Tao

et al. 2023

ACS Synth. Biol.

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“…Therefore, production of conditional and reversible gene knockout large animals remains a challenge due to serial gene targeting and serial rounds of SCNT are needed. As a large animal, pigs are ideal animal models to explore gene function because pigs are closer to humans than rodents in embryonic development, anatomy, physiological systems, and metabolic regulation (Fan and Lai, 2013Zhang et al, 2021). With the ReCOIN system, we successfully generated a pig model, in which endogenous porcine TP53 could be conditionally and reversibly knocked out.…”

Section: Discussionmentioning

confidence: 99%

CIRKO: A chemical-induced reversible gene knockout system for studying gene function in situ

Shi

Qiu

Chen

et al. 2022

Preprint

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Conditional loss and restoration of function are becoming important approaches for investigating gene function. Given that reversible conditional gene knockouts in cells required complicated manipulation, conditional inactivation and reactivation of a gene in primary somatic cells with limited proliferative capacity and in animal models remain difficult to achieve. Here, we first developed a reportable and reversible conditional intronic cassette (ReCOIN), wherein inactivation and reactivation of the gene are mediated via sequential expression of Cre and Flp recombinases, respectively. The expression pattern of the target gene can be monitored by direct visualization. To simply and tightly control temporal expression of the recombinases, on the basis of ReCOIN, we further presented a dual chemical-induced reversible gene knockout system (CIRKO) by insertion of reverse tetracycline transcriptional activator (rtTA) and tetracycline response element (TRE)-controlled Cre and FlpoERT2 recombinases cassettes into Rosa26 and Hipp11 loci of cells, respectively, in which transcription termination of the target gene can be induced at a specific stage in the presence of doxycycline, while gene restoration is achieved in the presence of doxycycline and tamoxifen simultaneously. This system provides a simple, rapid, and flexible gene switch for studying gene function in situ both in vitro and in vivo.

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“…There are three methods available for GE, namely, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9, and their application in pigs, particularly CRISPR/Cas9, has been recently reviewed [ 2 , 3 ]. The GE vectors or tools create genomic DNA double strains breakage (DSB) that requires immediate repair via nonhomologous end joining (NHEJ) [ 4 ] and/or microhomology-mediated end joining [ 5 ] to sustain cell survival with the risks of functional loss of gene editing.…”

Section: The Pathways Of Generation Of Gene-edited Pigs From Laborato...mentioning

confidence: 99%

— Invited Review — Reproductive technologies needed for the generation of precise gene-edited pigs in the pathways from laboratory to farm

Peng

Chuang

et al. 2023

Anim Biosci

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Gene editing (GE) offers a new breeding technique (NBT) of sustainable value to animalagriculture. There are 3 GE working sites covering 5 feasible pathways to generate GE pigs along with the crucial intervals of GE/genotyping, microinjection/electroporation, induced pluripotent stem cells (iPSCs), somatic cell nuclear transfer (SCNT), cryopreservation, and nonsurgical embryo transfer (NSET). The extension of NBT in the new era of pig breeding depends on the synergistic effect of GE and reproductive biotechnologies; the outcome relies not only on scientific due diligence and operational excellence but also on the feasibility of application on farms to improve sustainability.

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The advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research

Cited by 12 publications

References 158 publications

Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine

Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine

CIRKO: A chemical-induced reversible gene knockout system for studying gene function in situ

— Invited Review — Reproductive technologies needed for the generation of precise gene-edited pigs in the pathways from laboratory to farm

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