Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs

Kim, Brian H; Zhang, Guangjun

doi:10.1534/g3.119.401035

Cited by 16 publications

(12 citation statements)

References 34 publications

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“…Second, large indels may disrupt the sequencing PCR primers' binding sites, preventing amplification of such alleles. Third, the pooled RNPs may also lead to deletion of large sequences that span two loci being targeted ( Hoshijima et al, 2019 ; Kim and Zhang, 2020 ; Moreno-Mateos et al, 2015 ; Wu et al, 2018 ). To test the latter possibility, we Sanger sequenced amplicons from regions that span multiple targeted loci within slc24a5 .…”

Section: Resultsmentioning

confidence: 99%

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

Kroll

Powell

Ghosh

et al. 2021

eLife

171

193

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Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

show abstract

Section: Resultsmentioning

confidence: 99%

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

Kroll

Powell

Ghosh

et al. 2021

eLife

171

193

View full text Add to dashboard Cite

show abstract

“…Second, large indels may disrupt the sequencing PCR primers binding sites, preventing amplification of such alleles. Third, the pooled RNPs may also lead to deletion of large sequences that span two loci being targeted (Hoshijima et al, 2019;Kim and Zhang, 2020;Moreno-Mateos et al, 2015;Wu et al, 2018). To test the latter possibility, we Sanger sequenced amplicons from regions that span multiple targeted loci within slc24a5.…”

Section: Sequencing Of Targeted Loci Reveals the Diversity Of Null Almentioning

confidence: 99%

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

Kroll

Powell

Ghosh

et al. 2020

Preprint

View full text Add to dashboard Cite

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts.We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

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“…Moreover, our results also demonstrated that the combination of zebrafish–human comparative cancer genomics and functional genetic studies in zebrafish is a powerful approach for a long‐lasting challenge for identifying novel cancer driver genes on large CNAs/aneuploid chromosomes. Considering the plethora of available zebrafish mutants from large‐scale forward genetic screens and convenient reverse genetics, such as TALEN and CRISPR, 89‐92 a relatively large number of candidate cancer driver genes can be functionally validated in vivo with a similar approach as demonstrated for the smarcad1a gene here.…”

Section: Discussionmentioning

confidence: 99%

Loss of smarcad1a accelerates tumorigenesis of malignant peripheral nerve sheath tumors in zebrafish

Han

Jiang

Kumari

et al. 2021

Genes Chromosomes & Cancer

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Malignant peripheral nerve sheath tumors (MPNSTs) are a type of sarcoma that generally originates from Schwann cells. The prognosis for this type of malignancy is relatively poor due to complicated genetic alterations and the lack of specific targeted therapy. Chromosome fragment 4q22‐23 is frequently deleted in MPNSTs and other human tumors, suggesting tumor suppressor genes may reside in this region. Here, we provide evidence that SMARCAD1, a known chromatin remodeler, is a novel tumor suppressor gene located in 4q22‐23. We identified two human homologous smarcad1 genes (smarcad1a and smarcad1b) in zebrafish, and both genes share overlapping expression patterns during embryonic development. We demonstrated that two smarcad1a loss‐of‐function mutants, sa1299 and p403, can accelerate MPNST tumorigenesis in the tp53 mutant background, suggesting smarcad1a is a bona fide tumor suppressor gene for MPNSTs. Moreover, we found that DNA double‐strand break (DSB) repair might be compromised in both mutants compared to wildtype zebrafish, as indicated by pH2AX, a DNA DSB marker. In addition, both SMARCAD1 gene knockdown and overexpression in human cells were able to inhibit tumor growth and displayed similar DSB repair responses, suggesting proper SMARCAD1 gene expression level or gene dosage is critical for cell growth. Given that mutations of SMARCAD1 sensitize cells to poly ADP ribose polymerase inhibitors in yeast and the human U2OS osteosarcoma cell line, the identification of SMARCAD1 as a novel tumor suppressor gene might contribute to the development of new cancer therapies for MPNSTs.

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Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs

Cited by 16 publications

References 34 publications

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes

Loss of smarcad1a accelerates tumorigenesis of malignant peripheral nerve sheath tumors in zebrafish

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