High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis

Ramlee, Muhammad Khairul; Yan, Tingdong; Cheung, Alice; Chuah, Charles; Li, Shang

doi:10.1038/srep15587

Cited by 76 publications

(51 citation statements)

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“…Fluorescent PCR coupled with capillary gel electrophoresis was employed to detect insertion/deletion (indel) mutations in targeted cells following published method [ 13 ]. This technique can accurately and efficiently predict the number of nucleotide(s) inserted or deleted in a high throughput manner, and more importantly it can distinguish heterozygous mutants from homozygous mutants.…”

Section: Resultsmentioning

confidence: 99%

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IFNAR2-dependent gene expression profile induced by IFN-α in Pteropus alecto bat cells and impact of IFNAR2 knockout on virus infection

et al. 2017

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Bats are important reservoirs of many viruses, which are capable of infecting the host without inducing obvious clinical diseases. Interferon and the downstream interferon regulated genes (IRGs) are known to act as the first line of defense against viral infections. Little is known about the transcriptional profile of genes being induced by interferon in bats and their role in controlling virus infection. In this study, we constructed IFNAR2 knockout bat cell lines using CRISPR technology and further characterized gene expression profiles induced by the most abundant IFN-α (IFN-α3). Firstly, we demonstrated that the CRISPR/Cas9 system is applicable for bat cells as this represents the first CRIPSR knockout cell line for bats. Our results showed the pleiotropic effect of IFN-α3 on the bat kidney cell line, PaKiT03. As expected, we confirmed that IFNAR2 is indispensable for IFN-a signaling pathway and plays an important role in antiviral immunity. Unexpectedly, we also identified novel IFNAR2-dependent IRGs which are enriched in pathways related to cancer. To our knowledge, this seems to be bat-specific as no such observation has been reported for other mammalian species. This study expands our knowledge about bat immunology and the cell line established can provide a powerful tool for future study into virus-bat interaction and cancer biology.

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Section: Resultsmentioning

confidence: 99%

“…The HEX-labelled primer was used to amplify parental wild-type gDNA as control, while 6-FAM-labelled primer was used for targeted clones with potential indel mutations. PCR was conducted with Taq DNA polymerase (QIAGEN, Germany) and PCR products were analyzed using capillary gel electrophoresis as described previously [ 13 ].…”

Section: Methodsmentioning

confidence: 99%

IFNAR2-dependent gene expression profile induced by IFN-α in Pteropus alecto bat cells and impact of IFNAR2 knockout on virus infection

et al. 2017

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“…Detection of indels using PCR and capillary electrophoresis is used here and elsewhere (Ramlee et al 2015; Yang et al 2015); in comparison to the more generally used DNA sequencing method (Bortesi and Fischer 2015) it has a much shorter time span from sampling to identification of mutants. Furthermore, screening polyploid plants by sequencing, and verifying coverage of all alleles might lead to analysis of many replicates.…”

Section: Resultsmentioning

confidence: 99%

Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts

et al. 2016

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Key message Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts. AbstractSite-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines. Three different regions of the gene encoding granule-bound starch synthase (GBSS) were targeted under different experimental setups, resulting in mutations in at least one allele in 2–12 % of regenerated shoots, with multiple alleles mutated in up to 67 % of confirmed mutated lines. Most mutations resulted in small indels of 1–10 bp, but also vector DNA inserts of 34–236 bp were found in 10 % of analysed lines. No mutations were found in an allele diverging one bp from a used guide sequence, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential. To meet the challenge of screening large numbers of lines, a PCR-based high-resolution fragment analysis method (HRFA) was used, enabling identification of multiple mutated alleles with a resolution limit of 1 bp. Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. One remaining wild-type (WT) allele was shown sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-016-2062-3) contains supplementary material, which is available to authorized users.

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“…This method achieved an editing efficiency of nearly 100% in HEK293T cells and roughly 85% in HepG2 cells (Figure 3 -figure supplement 1B). Monoclonal populations of edited cells were sorted using FACS, screened, and the final edited sequence was determined using TOPO TA cloning (Ramlee et al, 2018).…”

Section: Cell Line Generationmentioning

confidence: 99%

Repression of ferritin light chain translation by human eIF3

Pulos-Holmes¹,

Srole²,

Lee

et al. 2018

Preprint

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16Iron is essential to life, but excess iron is detrimental as it catalyzes reactive hydroxyl radical 17 production. To prevent iron toxicity, intracellular iron homeostasis is regulated by ferritin, a 18 protein complex composed of a mixture of two subunits: ferritin light chain (FTL) and ferritin 19 heavy chain (FTH). Ferritin expression is regulated post-transcriptionally by the iron response 20 proteins (IRPs), which bind an RNA hairpin -the iron responsive element (IRE) -located in 21 the FTL and FTH mRNA 5ʹ-untranslated region (5ʹ-UTR). Here, we show FTL translation is also 22 regulated in an IRP-independent manner by eukaryotic translation initiation factor 3 (eIF3). We 23 find that eIF3 represses FTL translation by interacting with a region of the 5ʹ-UTR immediately 24 downstream of the IRE. Furthermore, we find that loss of eIF3-mediated regulation of FTL 25 translation likely contributes to a subset of clinically-observed hyperferritinemia cases. 26 Introduction 27Iron is essential for a spectrum of metabolic pathways and cellular growth. However, if 28 not properly managed, excess iron catalyzes the production of harmful radicals that lead to lipid 29 peroxidation, protein and DNA damage, and ultimately cell death (Palmer, Roberts, and Bero 30 1994). To safeguard against these damaging effects, cells ubiquitously express ferritin, an iron-31 sequestering protein complex. Ferritin is composed of a dynamic mixture of 24 individual 32 subunits of two types, the ferritin heavy chain (FTH) and the ferritin light chain (FTL), which 33 assemble into a hollow shell that encapsulates and stores up to 4,500 Fe 3+ atoms (Harrison and 34 Arosio 1996) (Anderson and McLaren 2012). Though the two subunits that make up this 35 complex are structurally similar, they are not functionally interchangeable (Harrison and Arosio 36 1996). The FTH subunit catalyzes the oxidation of Fe 2+ to Fe 3+ , while the FTL subunit lacks 37 catalytic activity and instead serves as a nucleation site for mineralization, increases complex 38All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ribosomal subunit recruitment and consequently inhibits translation. This inhibition prevents the 50 presence of excess ferritin and ensures sufficient levels of accessible iron. In response to an 51 increase in the labile iron pool (LIP) or under high iron conditions, IRP1 is loaded with an iron 52 sulfur cluster produced by mitochondria that prevents IRE binding, and IRP2 is targeted for 53 proteasomal degradation (Fig. 1A,B) (Wilkinson and Pantopoulos 2014). These processes 54 relieve the transcript from IRP/IRE generated steric hindrances, allowing for translation to 55 proceed and for the excess iron to be deposited into newly formed ferritin complexes. IRP-IRE 56 interactions are known to be disrupted in clinically relevant cases of hyperferritinemia, in which 57 single nucleotide mutations throughout the IRE ...

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High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis

Cited by 76 publications

References 28 publications

IFNAR2-dependent gene expression profile induced by IFN-α in Pteropus alecto bat cells and impact of IFNAR2 knockout on virus infection

IFNAR2-dependent gene expression profile induced by IFN-α in Pteropus alecto bat cells and impact of IFNAR2 knockout on virus infection

Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts

Repression of ferritin light chain translation by human eIF3

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