Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

Liu, Hui; Liu, Chang; Zhao, Yuhang; Han, Xuejie; Zhou, Zhengwei; Wang, Chen; Li, Rongfeng; Li, Xueling

doi:10.1016/s2095-3119(17)61748-9

Cited by 16 publications

(8 citation statements)

References 28 publications

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“…CRISPR/Cas is much simpler to implement than the other reported systems such as ZFN and TALEN [ 51 , 52 ]. High-efficiency editing can be achieved even in large and complex plant genomes at each of the multiple targeted genomic loci [ 53 , 54 ].…”

Section: Discussionmentioning

confidence: 99%

Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don)

et al. 2021

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Background To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies. Results We have demonstrated CRISPR/Cas9 genome editing in P. radiata targeting a single-copy cell wall gene GUX1 in somatic embryogenic tissue and produced plantlets from the edited tissue. We generated biallelic INDELs with an efficiency of 15 % using a single gRNA. 12 % of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33 %, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic. Conclusions We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using plasmid expressed Cas9 gRNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.

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Section: Discussionmentioning

confidence: 99%

Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don)

et al. 2021

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“…The CRISPR/Cas9 system has advantages compared with other genome editing technologies such as transcription activator-like effector nuclease and zinc finger nuclease approaches, including ease of use, high efficiency, and adaptability to different organisms [33,34].…”

Section: Discussionmentioning

confidence: 99%

Knockout of butyrophilin subfamily 1 member A1 (BTN1A1) alters lipid droplet formation and phospholipid composition in bovine mammary epithelial cells

Han

Zhang

Xing

et al. 2020

J Animal Sci Biotechnol

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Background: Milk lipids originate from cytoplasmic lipid droplets (LD) that are synthesized and secreted from mammary epithelial cells by a unique membrane-envelopment process. Butyrophilin 1A1 (BTN1A1) is one of the membrane proteins that surrounds LD, but its role in bovine mammary lipid droplet synthesis and secretion is not well known. Methods: The objective was to knockout BTN1A1 in bovine mammary epithelial cells (BMEC) via the CRISPR/Cas9 system and evaluate LD formation, abundance of lipogenic enzymes, and content of cell membrane phospholipid (PL) species. Average LD diameter was determined via Oil Red O staining, and profiling of cell membrane phospholipid species via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Lentivirus-mediated infection of the Cas9/sgRNA expression vector into BMEC resulted in production of a homozygous clone BTN1A1 (−/−). The LD size and content decreased following BTN1A1 gene knockout. The mRNA abundance of fatty acid synthase (FASN) and peroxisome proliferator-activated receptor-gamma (PPARG) was downregulated in the BTN1A1 (−/−) clone. Subcellular analyses indicated that BTN1A1 and LD were co-localized in the cytoplasm. BTN1A1 gene knockout increased the percentage of phosphatidylethanolamine (PE) and decreased phosphatidylcholine (PC), which resulted in a lower PC/PE ratio. Conclusions: Results suggest that BTN1A1 plays an important role in regulating LD synthesis via a mechanism involving membrane phospholipid composition.

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“…We also tested a novel U6 promoter isolated from Douglas r. The lack of signi cant differences in the transcriptional e ciencies among the 3 U6 promoters demonstrates similar functionality in their respective species. CRISPR/Cas is much simpler to implement than the other reported systems such as ZFN and TALEN [54,55]. High-e ciency editing can be achieved even in large and complex plant genomes at each of the multiple targeted genomic loci [56,57].…”

Section: Discussionmentioning

confidence: 99%

Genome Editing With CRISPR/Cas9 in Pinus Radiata (D. Don)

Poovaiah

Phillips

Geddes

et al. 2020

Preprint

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BackgroundTo meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity from planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase and genome complexity. Genetic modification (GM) offers the opportunity to make transformational changes in shorter time frames but is challenged by current genetically modified organism (GMO) regulations. Genome editing, which can be used to generate site-specific mutations, offers the opportunity to rapidly implement targeted improvements and is globally regulated in a less restrictive way than GM technologies.ResultsWe evaluated U6 snRNA promoters from three different species that were able to drive expression of guide RNA (gRNA) for CRISPR/Cas9 genome editing in P. radiata. Using a single-copy cell wall gene GUX1 as a target, we have demonstrated genome editing using CRISPR/Cas9 in somatic embryogenic tissue and plantlets derived from the edited tissue. We generated biallelic INDELs with an efficiency of 15% using a single gRNA. Twelve percent of the transgenic embryogenic tissue was edited when two gRNAs were used and deletions of up to 1.3 kb were identified. However, the regenerated plants did not contain large deletions but had single nucleotide insertions at one of the target sites. We also assessed the use of CRISPR/Cas9 ribonucleoproteins (RNPs) for their ability to accomplish DNA-free genome editing in P. radiata. We chose a hybrid approach, with RNPs co-delivered with a plasmid-based selectable marker. A two-gRNA strategy was used which produced an editing efficiency of 33%, and generated INDELs, including large deletions. Using the RNP approach, deletions found in embryogenic tissue were also present in the plantlets. But, all plants produced using the RNP strategy were monoallelic.Conclusion We have demonstrated the generation of biallelic and monoallelic INDELs in the coniferous tree P. radiata with the CRISPR/Cas9 system using DNA and RNPs respectively. This opens the opportunity to apply genome editing in conifers to rapidly modify key traits of interest.

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Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

Cited by 16 publications

References 28 publications

Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don)

Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don)

Knockout of butyrophilin subfamily 1 member A1 (BTN1A1) alters lipid droplet formation and phospholipid composition in bovine mammary epithelial cells

Genome Editing With CRISPR/Cas9 in Pinus Radiata (D. Don)

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Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

Cited by 16 publications

References 28 publications

Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don)

Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don)

Knockout of butyrophilin subfamily 1 member A1 (BTN1A1) alters lipid droplet formation and phospholipid composition in bovine mammary epithelial cells

Genome Editing With CRISPR/Cas9&nbsp;in Pinus Radiata (D. Don)

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Genome Editing With CRISPR/Cas9 in Pinus Radiata (D. Don)