New Developments in CRISPR/Cas-based Functional Genomics and their Implications for Research Using Zebrafish

Prykhozhij, Sergey V.; Cáceres, Lucía; Berman, Jason N.

doi:10.2174/1566523217666171121164132

Cited by 15 publications

(11 citation statements)

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“…In the last few years, genome editing technologies, particularly based on Crispr/Cas9 (Liu et al 2019; Prykhozhij et al 2017), have made it relatively simple to generate lines of zebrafish carrying mutations in targeted genes. To date, by far the most commonly used approach in zebrafish has been to generate loss of function alleles through imprecise DNA repair by the non-homologous end-joining pathway.…”

Section: Mutants Morphants or Crispants?mentioning

confidence: 99%

Looking to the future of zebrafish as a model to understand the genetic basis of eye disease

Cavodeassi

Wilson²

2019

Hum Genet

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In this brief commentary, we provide some of our thoughts and opinions on the current and future use of zebrafish to model human eye disease, dissect pathological progression and advance in our understanding of the genetic bases of microphthalmia, andophthalmia and coloboma (MAC) in humans. We provide some background on eye formation in fish and conservation and divergence across vertebrates in this process, discuss different approaches for manipulating gene function and speculate on future research areas where we think research using fish may prove to be particularly effective.

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Section: Mutants Morphants or Crispants?mentioning

confidence: 99%

Looking to the future of zebrafish as a model to understand the genetic basis of eye disease

Cavodeassi

Wilson²

2019

Hum Genet

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“…Microinjection of antisense morpholino oligonucleotides (MOs) into one-cell stage zebrafish embryos allows rapid, simple and specific reverse genetic studies by inhibition of mRNA splicing or translation (Nasevicius and Ekker, 2000; Eisen and Smith, 2008). Efficient transgenesis methods and large-scale mutagenesis screens, mRNA injection, and genomic editing tools such as the CRISPR/cas9 approach are developed in zebrafish (Prykhozhij et al, 2018). Zebrafish embryos are also suitable for large-scale chemical screening to identify novel therapeutic targets and compounds.…”

Section: Zebrafish For Drug Discovery In Tauopathiesmentioning

confidence: 99%

Heparan Sulfate as a Therapeutic Target in Tauopathies: Insights From Zebrafish

Soussi-Yanicostas

2018

Front. Cell Dev. Biol.

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Microtubule-associated protein tau (MAPT) hyperphosphorylation and aggregation, are two hallmarks of a family of neurodegenerative disorders collectively referred to as tauopathies. In many tauopathies, including Alzheimer’s disease (AD), progressive supranuclear palsy (PSP) and Pick’s disease, tau aggregates are found associated with highly sulfated polysaccharides known as heparan sulfates (HSs). In AD, amyloid beta (Aβ) peptide aggregates associated with HS are also characteristic of disease. Heparin, an HS analog, promotes misfolding, hyperphosphorylation and aggregation of tau protein in vitro. HS also provides cell surface receptors for attachment and uptake of tau seeds, enabling their propagation. These findings point to HS-tau interactions as potential therapeutic targets in tauopathies. The zebrafish genome contains genes paralogous to MAPT, genes orthologous to HS biosynthetic and chain modifier enzymes, and other genes implicated in AD. The nervous system in the zebrafish bears anatomical and chemical similarities to that in humans. These homologies, together with numerous technical advantages, make zebrafish a valuable model for investigating basic mechanisms in tauopathies and identifying therapeutic targets. Here, we comprehensively review current knowledge on the role of HSs in tau pathology and HS-targeting therapeutic approaches. We also discuss novel insights from zebrafish suggesting a role for HS 3-O-sulfated motifs in tau pathology and establishing HS antagonists as potential preventive agents or therapies for tauopathies.

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“…However, limitations such as possibility to design an active sgRNA close to the intended site of nucleotide change, unintended indels due to the simultaneous activation of NHEJ, and low efficiency of knockin has made it difficult to apply HDR-based approaches for single-nucleotide substitutions in a high-throughput manner in zebrafish [ 20 , 21 ]. Furthermore, the design of donor DNA is not straightforward and several versions with homology arms of different lengths need to be tested for each site [ 10 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish

Carrington

Weinstein

2020

Cells

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The ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% of human disease-causing mutations. Recently developed base editing nucleases provide an excellent alternate to CRISPR/Cas9-mediated homology dependent repair for generation of zebrafish with point mutations. A new set of cytosine base editors, termed BE4max and AncBE4max, demonstrated improved base editing efficiency in mammalian cells but have not been evaluated in zebrafish. Therefore, we undertook this study to evaluate their efficiency in converting C:G to T:A base pairs in zebrafish by somatic and germline analysis using highly active sgRNAs to twist and ntl genes. Our data demonstrated that these improved BE4max set of plasmids provide desired base substitutions at similar efficiency and without any indels compared to the previously reported BE3 and Target-AID plasmids in zebrafish. Our data also showed that AncBE4max produces fewer incorrect and bystander edits, suggesting that it can be further improved by codon optimization of its components for use in zebrafish.

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New Developments in CRISPR/Cas-based Functional Genomics and their Implications for Research Using Zebrafish

Cited by 15 publications

References 0 publications

Looking to the future of zebrafish as a model to understand the genetic basis of eye disease

Looking to the future of zebrafish as a model to understand the genetic basis of eye disease

Heparan Sulfate as a Therapeutic Target in Tauopathies: Insights From Zebrafish

BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish

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