CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing

Labun, Kornel; Montague, Tessa G.; Krause, Maximilian; Cleuren, Yamila N. Torres; Tjeldnes, Håkon; Valen, Eivind

doi:10.1093/nar/gkz365

Cited by 1,296 publications

(1,064 citation statements)

References 33 publications

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“…CHOPCHOP Labun et al, 2019) was used to predict three guide RNAs targeting the ski7-specific exon 5: et al, 2014). In-house produced Cas9 protein and pooled gRNAs were co-injected in the cell of one-cell TLAB zebrafish embryos.…”

Section: Generation Of Ski7 -/Mutant Fishmentioning

confidence: 99%

Zebrafish Ski7 tunes RNA levels during the oocyte-to-embryo transition

Quio

Schleiffer

Mechtler

et al. 2020

Preprint

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Post-transcriptional mechanisms are crucial for the regulation of gene expression.These mechanisms are particularly important during rapid developmental transitions such as the oocyte-to-embryo transition, which is characterized by dramatic changes to the developmental program in the absence of nuclear transcription. Under these conditions, changes to the RNA content are solely dependent on RNA degradation.Although several mechanisms that promote RNA decay during embryogenesis have been identified, it remains unclear which cellular machineries contribute to remodeling the maternal transcriptome during the oocyte-to-embryo transition. Here, we focused on the auxiliary 3'-to-5' degradation factor Ski7 in zebrafish as its mRNA peaks during this time frame. Homozygous ski7 mutant fish were viable and developed into morphologically normal adults, yet they had decreased fertility. Consistent with the idea that Ski7 participates in remodeling the transcriptome during the oocyte-toembryo transition, transcriptome profiling identified stage-specific mRNA targets of Ski7. Genes upregulated in ski7 mutants were generally lowly expressed in wild type, suggesting that Ski7 maintains low transcript levels for this subset of genes. GO enrichment analyses of genes mis-regulated in ski7 mutants implicated Ski7 in the regulation of redox processes. This was confirmed experimentally by an increased resistance of ski7 mutant embryos to reductive stress. Overall, our results provide first insights into the physiological role of vertebrate Ski7 as an important posttranscriptional regulator during the oocyte-to-embryo transition.

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Section: Generation Of Ski7 -/Mutant Fishmentioning

confidence: 99%

Zebrafish Ski7 tunes RNA levels during the oocyte-to-embryo transition

Quio

Schleiffer

Mechtler

et al. 2020

Preprint

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“…sgRNA oligos were designed using CHOPCHOP (Labun et al, 2019) and cloned into expression Plasmids as described previously (Sanjana et al, 2014;Schmid-Burgk et al, 2014). BLaER1 cells were electroporated in OptiMEM with 5 µg of plasmids driving expression of Cas9 and an sgRNA on a BioRad GenePulser XCell as described previously .…”

Section: Genome Editing and Overexpressionmentioning

confidence: 99%

Priming enables a NEK7-independent route of NLRP3 activation

Gaidt

Szymanska

et al. 2019

Preprint

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The NLRP3 inflammasome plays a central role in antimicrobial defense, as well as in sterile inflammatory conditions. NLRP3 activity is governed by two independent signals. The first signal primes NLRP3, allowing it to respond to its activation signal. In the murine system, the mitotic spindle kinase NEK7 has been identified as a crucial factor in relaying the activation signal to NLRP3. Here we show that the requirement for NEK7 can be bypassed by TAK1-dependent post-translational priming. Under pro-inflammatory conditions that activate TAK1, NEK7 was dispensable for NLRP3 inflammasome formation in human and murine cells. Intriguingly, dissecting the NEK7 requirement in iPSC-derived primary human macrophages revealed that this NEK7-independent mechanism constitutes the predominant NLRP3 priming pathway in these cells. In summary, our results suggest that NEK7 functions as an NLRP3 primingrather than activationfactor that can work in synergy or redundancy with other priming pathways to accelerate inflammasome activation.

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“…Gene editing was performed in HEK293T (originally purchased from ATCC) and the NDUFV3 KO NDUFV3 FLAG cell line published previously [38]. Constructs for CRISPR-Cas9 genome editing were designed using the CHOPCHOP software [60] and oligonucleotides encoding gRNA sequences cloned into the pSpCas9(BB)-2A-GFP (PX458) plasmid (a gift from F. Zhang; Addgene, plasmid 48138; [61]) as previously described [38]. The gRNA sequences used were 5'-ATGTCAGTCAAACTTACCAA for HIGD1A and 5'-ACTTACCTATGGGTACCACC for HIGD2A.…”

Section: Tissue Culture and Generation Of Cell Linesmentioning

confidence: 99%

“…To verify the CRISPR/Cas9 induced insertions and deletions (indels) genomic DNA was isolated from candidate clones using the Quick-DNA kit (Zymo Research) according to manufacturer's specifications. Oligonucleotides provided by the CHOPCHOP software [60] were used for amplification of target regions and cloning into pGEM4Z [62] for M13 primed Sanger sequencing of individual alleles as we have done previously [38]. HIGD1A KO chamber was incubated at 37°C for 3 or 24 hours as indicated.…”

Section: Tissue Culture and Generation Of Cell Linesmentioning

confidence: 99%

HIGD2A is required for assembly of the COX3 module of human mitochondrial complex IV

Hock

Reljić

et al. 2019

Preprint

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AbstractAssembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. A number of assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. While in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.

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CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing

Cited by 1,296 publications

References 33 publications

Zebrafish Ski7 tunes RNA levels during the oocyte-to-embryo transition

Zebrafish Ski7 tunes RNA levels during the oocyte-to-embryo transition

Priming enables a NEK7-independent route of NLRP3 activation

HIGD2A is required for assembly of the COX3 module of human mitochondrial complex IV

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