Fast and sensitive detection of indels induced by precise gene targeting

Yang, Zhipeng; Steentoft, Catharina; Hauge, Camilla; Hansen, Lars; Thomsen, Allan Lind; Niola, Francesco; Vester-Christensen, Malene Bech; Frödin, Morten; Clausen, Henrik; Wandall, Hans H.; Bennett, Eric

doi:10.1093/nar/gkv126

Cited by 152 publications

(143 citation statements)

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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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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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“…GFP ϩ /DsRed2 ϩ cells were enriched by fluorescenceactivated cell sorting (FACS) and single-cell cloned by limited dilution. Indels at the respective target sites were characterized by Indel Detection by Amplicon Analysis (IDAA) (106), and indels identified in individual cell clones that were selected were confirmed by Sanger sequencing.…”

Section: Zfn Knockout Gene Targetingmentioning

confidence: 99%

De novo expression of human polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6) in colon adenocarcinoma inhibits the differentiation of colonic epithelium

Lavrsen

Dabelsteen

Vakhrushev

et al. 2018

Journal of Biological Chemistry

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Edited by Eric R. FearonAberrant expression of O-glycans is a hallmark of epithelial cancers. Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) that target different proteins and are differentially expressed in cells and organs. Here, we investigated the expression patterns of all of the GalNAc-Ts in colon cancer by analyzing transcriptomic data. We found that GalNAc-T6 was highly up-regulated in colon adenocarcinomas but absent in normal-appearing adjacent colon tissue. These results were verified by immunohistochemistry, suggesting that GalNAc-T6 plays a role in colon carcinogenesis. To investigate the function of GalNAc-T6 in colon cancer, we used precise gene targeting to produce isogenic colon cancer cell lines with a knockout/rescue system for GALNT6. GalNAc-T6 expression was associated with a cancer-like, dysplastic growth pattern, whereas GALNT6 knockout cells showed a more normal differentiation pattern, reduced proliferation, normalized cell-cell adhesion, and formation of crypts in tissue cultures. O-Glycoproteomic analysis of the engineered cell lines identified a small set of GalNAc-T6 -specific targets, suggesting that this isoform has unique cellular functions. In support of this notion, the genetically and functionally closely related GalNAc-T3 homolog did not show compensatory functionality for effects observed for GalNAc-T6. Taken together, these data strongly suggest that aberrant GalNAc-T6 expression and site-specific glycosylation is involved in oncogenic transformation.

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“…Gene targeting was performed in the CHO ZN-GS Ϫ/Ϫ (glutamate synthase) (Sigma) cells or HEK293 (ATCC) using GFP/ Crimson-tagged zinc finger nucleases and transcription activator-like effector nucleases (TALENs) or GFP-tagged clustered and regularly interspaced short palindromic repeats (CRISPR)-Cas9s (supplemental Table S1) with our recently developed screening strategy (60). CHO-GS cells were maintained as suspension cultures in EX-CELL CHO CD Fusion serum-free media, supplemented with 4 mM L-glutamine.…”

Section: Precise Gene Targeting Of Glycogenes In Cho and Hek293 Cellsmentioning

confidence: 99%

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

Larsen

Narimatsu

Joshi

et al. 2017

Journal of Biological Chemistry

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Protein O-mannosylation is found in yeast and metazoans, and a family of conserved orthologous protein O-mannosyltransferases is believed to initiate this important post-translational modification. We recently discovered that the cadherin superfamily carries O-linked mannose (O-Man) glycans at highly conserved residues in specific extracellular cadherin domains, and it was suggested that the function of E-cadherin was dependent on the O-Man glycans. Deficiencies in enzymes catalyzing O-Man biosynthesis, including the two human protein O-mannosyltransferases, POMT1 and POMT2, underlie a subgroup of congenital muscular dystrophies designated ␣-dystroglycanopathies, because deficient O-Man glycosylation of ␣-dystroglycan disrupts laminin interaction with ␣-dystroglycan and the extracellular matrix. To explore the functions of O-Man glycans on cadherins and protocadherins, we used a combinatorial gene-editing strategy in multiple cell lines to evaluate the role of the two POMTs initiating O-Man glycosylation and the major enzyme elongating O-Man glycans, the protein O-mannose ␤-1,2-N-acetylglucosaminyltransferase, POMGnT1. Surprisingly, O-mannosylation of cadherins and protocadherins does not require POMT1 and/or POMT2 in contrast to ␣-dystroglycan, and moreover, the O-Man glycans on cadherins are not elongated. Thus, the classical and evolutionarily conserved POMT O-mannosylation pathway is essentially dedicated to ␣-dystroglycan and a few other proteins, whereas a novel O-mannosylation process in mammalian cells is predicted to serve the large cadherin superfamily and other proteins.Protein O-glycosylation of the O-mannose type was originally thought to be found only in yeast and fungi, but studies over the last 30 years have identified O-Man 2 glycans and specific glycoproteins carrying O-Man glycans in human and rodents (1-9). The basement membrane glycoprotein ␣-dystroglycan (␣-DG) was for some time the only well characterized O-mannosylated protein known in mammals despite evidence that O-Man glycans constitute a major part of the total O-glycans in the brain (1,2,8,9). O-Mannosylation of ␣-DG is essential for assembly and function of the dystrophin-glycoprotein complex that links the cytoskeleton with the extracellular matrix, and deficiencies in all of the enzymes involved in the O-Man glycosylation underlie a subgroup of congenital muscular dystrophies (10 -12). More recently, the human O-Man glycoproteome was characterized, and it was found that the large superfamily of cadherins (cdhs) and protocadherins (pcdhs) are also decorated with ␣-linked O-Man glycans on extracellular cadherin (EC) domains. The attachment sites of these O-Man glycans appear to be highly conserved throughout evolution (13,14); moreover, O-mannosylation of E-cadherin was suggested to be crucial for E-cadherin-mediated cell adhesion (15,16).O-Man glycosylation in metazoans is initiated in the endoplasmic reticulum (ER) by transfer of mannose from dolichol monophosphate-activated mannose to serine and threonine by the POMT1 and POMT2 p...

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Fast and sensitive detection of indels induced by precise gene targeting

Cited by 152 publications

References 23 publications

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

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

De novo expression of human polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6) in colon adenocarcinoma inhibits the differentiation of colonic epithelium

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

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