Advances in CRISPR-Based Functional Genomics and Nucleic Acid Detection in Pigs

Ruan, Jinxue; Zhang, Xuying; ShuHong, Zhao; Xie, Shengsong

doi:10.3389/fgene.2022.891098

Cited by 3 publications

(2 citation statements)

References 75 publications

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“…PCR and RPA primers were designed using Primer 5.0 software (Premier Biosoft, Palo Alto, CA, USA) and synthesized by TSINGKE (China). Previously constructed plasmids and primers to the porcine ZFX and ASFV p72 genes were used. , The CD163 gene (702 bp) and the porcine LF knock-in gene (543 bp) were amplified by PCR using the wild-type pig genome, and the pLF-KI pig genome, respectively . The sequences are marked in Table S6.…”

Section: Methodsmentioning

confidence: 99%

“…30,41 The CD163 gene (702 bp) and the porcine LF knock-in gene (543 bp) were amplified by PCR using the wild-type pig genome, and the pLF-KI pig genome, respectively. 42 The sequences are marked in Table S6. The pMD18T-CD163 and pMD18T-LF plasmids were constructed by cloning CD163 and LF amplification fragments into the pMD18T vectors (TaKaRa, Japan).…”

Section: Methodsmentioning

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: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

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.

Self Cite

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Expanding the CRISPR toolbox by engineering Cas12a orthologs of metagenomic discovery

Tao,

Xu,

et al. 2024

Preprint

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Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility in genome editing and molecular diagnostic applications. However, the widespread adoption of CRISPR-Cas12a nucleases and their variants has been hindered by the requirement for a specific protospacer adjacent motif (PAM), relatively low CRISPR RNA (crRNA) activity and the inability to multiplex nucleic acid detection alone. To overcome these limitations, we employed a comprehensive framework combined with AlphaFold2 to de novo mine 1,261 previously unexploited Cas12a orthologs from the global microbiome. Following experimental analysis, we identified the most promising 21 Cas12a nuclease orthologs and designated them "Genie scissor 12" (Gs12). Our analysis uncovered two exceptional variants among these newly identified orthologs: Gs12-10, a first natural PAM-less Cas12a ortholog, which can recognize 52 distinct PAM types, representing a significant 1.8-fold expansion in recognition range compared to the relative LbCas12a PAM; and Gs12-7MAX, an engineered variant of Gs12-7 that exhibited 1.27-fold higher editing efficiency than enAsCas12a-HF. Furthermore, we harnessed Gs12-1, Gs12-4, Gs12-9, and Gs12-18, along with their corresponding engineered crRNAs, to develop a powerful four-channel multiplexed CRISPR-based nucleic acid detection system. The discovery of diverse functions in Cas12a offers a deeper understanding of the CRISPR/Cas12a family. Also, it holds great promise for expanding its applications and uncovering the untapped potential of other CRISPR/Cas systems.

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Deciphering the Role of the SREBF1 Gene in the Transcriptional Regulation of Porcine Adipogenesis Using CRISPR/Cas9 Editing

Aksoy,

Bilinska,

Stachowiak

et al. 2024

IJMS

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Sterol regulatory element-binding protein 1 (SREBP1) is an important transcription factor that controls lipid metabolism and adipogenesis. Two isoforms, SREBP1a and SREBP1c, are generated by alternative splicing of the first exon of the SREBF1 gene. The porcine SREBF1 gene has mainly been studied for its role in lipid metabolism in adipose tissues, but little is known about its involvement, and the role of its two isoforms, in adipogenesis. The aim of the present study was to introduce a deletion in the 5′-regulatory region of the SREBF1c gene, considered crucial for adipogenesis, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. This approach allows for the evaluation of how inhibiting SREBF1c transcription affects the expression of other genes essential for adipocyte differentiation, particularly PPARG, CEBPA, CEBPB, CEBPD, GATA2, and FABP4. It was observed that disrupting the SREBF1c isoform had no effect on the GATA2 gene but did result in a decrease in the expression of the CEBPA and CEBPD genes, an increase in the expression of CEBPB, and an inhibition in the expression of the PPARG and FABP4 genes. These changes in gene expression blocked adipogenesis, as could be seen by the failure of lipid droplets to accumulate. Our results provide evidence highlighting the pivotal role of the SREBP1c isoform in the regulation of porcine adipogenesis.

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Advances in CRISPR-Based Functional Genomics and Nucleic Acid Detection in Pigs

Cited by 3 publications

References 75 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

Expanding the CRISPR toolbox by engineering Cas12a orthologs of metagenomic discovery

Deciphering the Role of the SREBF1 Gene in the Transcriptional Regulation of Porcine Adipogenesis Using CRISPR/Cas9 Editing

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