Efficient targeted integration directed by short homology in zebrafish and mammalian cells

Wierson, Wesley A.; Welker, Jordan M.; Almeida, Maira P.; Mann, Carla M.; Webster, Dennis A.; Torrie, Melanie E.; Weiss, Trevor; Sekhar, Kondapalli Venkata Gowri Chandra; Vollbrecht, Macy; Lan, Merrina; McKeighan, Kenna C.; Levey, Jacklyn; Ming, Zhitao; Wehmeier, Alec; Mikelson, Christopher S.; Haltom, Jeffrey A.; Kwan, Kristen M.; Chien, Chi Bin; Balciunas, Darius; Ekker, Stephen C.; Clark, Karl J.; Webber, Beau R.; Moriarity, Branden S.; Solin, Stacy L.; Carlson, Daniel F.; Dobbs, Drena; McGrail, Maura; Essner, Jeffrey J.

doi:10.7554/elife.53968

Cited by 122 publications

(180 citation statements)

References 38 publications

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“…The 2A-Cre targeting vector used for integration is part of our pPRISM vector series that contains cassettes with linked fluorescent secondary marker for allele tracking (Welker et al, in preparation). Recovery of precise 2A-Cre integration alleles was highly efficient, with frequencies ranging from 10-100%, similar to other cargos as reported previously (Wierson et al, 2020). As expected, the expression of functional Cre recombinase was restricted to cell populations defined by ascl1b, olig2 and neurod1.…”

Section: Introductionsupporting

confidence: 87%

“…To generate proneural Cre drivers we integrated a 2A-Cre recombinase cDNA cassette in frame into a coding exon of the zebrafish ascl1b, olig2 and neurod1 genes ( Figure 1). Our strategy for CRISPR/Cas9 precision targeted integration uses short homology arms flanking a targeting cassette to likely drive integration by homology mediated end joining (HMEJ) (Wierson et al, 2020). The 2A-Cre targeting construct contains a secondary fluorescent marker cassette 6 with the g-crystallin (g-cry) promoter driving EGFP expression ( Figure 1A).…”

Section: Crispr/cas9 Mediated Knock-in Of 2a-cre Into Ascl1b Olig2 mentioning

confidence: 99%

“…To streamline isolation of zebrafish Cre drivers for specific cell lineages and cell types, we applied our recently reported method for CRISPR/Cas9 short homology directed targeted integration (Wierson et al, 2020) to generate Cre transgenic lines. As proof of principle we targeted a 2A-Cre cassette in frame into a coding exon in the proneural transcription factor genes ascl1, olig2 and neurod1 that define neural stem, progenitor and post-mitotic cell lineages (Aprea et al, 2014;Bertrand et al, 2002;Hevner et al, 2006;Kim et al, 2011;Takebayashi et al, 2002;Wilkinson et al, 2013;Zhou and Anderson, 2002).…”

Section: Introductionmentioning

confidence: 99%

“…The pattern of switching induced by the neurod1-2A-Cre line extended throughout neurons in the forebrain, midbrain, hindbrain, retina, and the PNS (Figure 3 D), as expected, given neurod1 is expressed in committed neural progenitors and early post mitotic neurons throughout the nervous system (Rauch, 2003). Together with the in situ In summary, in this study we applied our CRISPR/Cas9 short homology directed targeted integration strategy (Wierson et al, 2020) to generate a set of zebrafish proneural Cre drivers that express functional Cre recombinase in the pattern of the endogenous targeted ascl1b, olig2, and neurod1 genes. Using 48 bp homology arms in the universal gRNA targeting vector we were able to efficiently recover precise in-frame 2A-Cre recombinase integration alleles after screening a maximum of only 20 adult F0 fish.…”

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confidence: 99%

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Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

Almeida

Welker

Ekker³

et al. 2020

Preprint

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The Cre/lox recombinase system has been widely used for spatiotemporal control of gene expression in animal model systems, however, efficient methods to isolate zebrafish Cre drivers that reliably recapitulate endogenous gene expression patterns are needed. Here, we apply CRISPR/Cas9 targeting to integrate a 2A-Cre recombinase transgene with 48bp homology arms into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10%-100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase in the expected proneural cell populations. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre drivers whose spatial and temporal restricted expression mimics endogenous genes, surmounting the challenges associated with promoter BAC cloning and transposon mediated transgenesis.

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Section: Introductionsupporting

confidence: 87%

Section: Crispr/cas9 Mediated Knock-in Of 2a-cre Into Ascl1b Olig2 mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

Almeida

Welker

Ekker³

et al. 2020

Preprint

Self Cite

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“…Zebrafish are a powerful animal model to study the nervous system in general and the retina specifically. First, there are multiple available toolboxes for the manipulation of genes of interest (Kawakami, 2007;Ablain et al, 2015;Kawakami et al, 2016;Niklaus & Neuhauss, 2017;Wierson et al, 2020). Second, zebrafish husbandry is relatively easy, mating has high yields and development is fast when compared to mammalian models.…”

Section: Introductionmentioning

confidence: 99%

Expression and distribution of synaptotagmin isoforms in the zebrafish retina

Henry

Joselevitch

Matthews

et al. 2020

Preprint

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Synaptotagmins belong to a large family of proteins. While various synaptotagmins have been implicated as Ca2+ sensors for vesicle replenishment and release at conventional synapses, their roles at retinal ribbon synapses remain incompletely understood. Zebrafish is a widely used experimental model for retinal research. We therefore investigated the homology between human, rat, mouse, and zebrafish synaptotagmins 1 to 10 using a bioinformatics approach. We also characterized the expression and distribution of various synaptotagmin (syt) genes in the zebrafish retina using RT-PCR and in situ hybridization, focusing on the family members whose products likely underlie Ca2+-dependent exocytosis in the central nervous system (synaptotagmins 1, 2, 5 and 7). We find that most zebrafish synaptotagmins are well conserved and can be grouped in the same classes as mammalian synaptotagmins, based on crucial amino acid residues needed for coordinating Ca2+ binding and determining phospholipid binding affinity. The only exception is synaptotagmin 1b, which lacks 34 amino acid residues in the C2B domain and is therefore unlikely to bind Ca2+ there. Additionally, the products of zebrafish syt5a and syt5b genes share identity with mammalian class 1 and 5 synaptotagmins. Zebrafish syt1, syt2, syt5 and syt7 paralogues are found in the zebrafish brain, eye, and retina, excepting syt1b, which is only present in the brain. The complementary expression pattern of the remaining paralogues in the retina suggests that syt1a and syt5a may underlie synchronous release and syt7a and syt7b may mediate asynchronous release or other Ca2+ dependent processes in different types of retinal neurons.

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Expression and distribution of synaptotagmin family members in the zebrafish retina

Henry

Joselevitch

Matthews

et al. 2021

J of Comparative Neurology

View full text Add to dashboard Cite

Synaptotagmins belong to a large family of proteins. Although various synaptotagmins have been implicated as Ca2+ sensors for vesicle replenishment and release at conventional synapses, their roles at retinal ribbon synapses remain incompletely understood. Zebrafish is a widely used experimental model for retinal research. We therefore investigated the homology between human, rat, mouse, and zebrafish synaptotagmins 1–10 using a bioinformatics approach. We also characterized the expression and distribution of various synaptotagmin (syt) genes in the zebrafish retina using RT‐PCR, qPCR, and in situhybridization, focusing on the family members whose products likely underlie Ca2+‐dependent exocytosis in the central nervous system (synaptotagmins 1, 2, 5, and 7). Most zebrafish synaptotagmins are well conserved and can be grouped in the same classes as mammalian synaptotagmins, based on crucial amino acid residues needed for coordinating Ca2+ binding and determining phospholipid binding affinity. The only exception is synaptotagmin 1b, which lacks 34 amino acid residues in the C2B domain and is therefore unlikely to bind Ca2+ there. Additionally, the products of zebrafish syt5a and syt5b genes share identity with mammalian class 1 and 5 synaptotagmins. Zebrafish syt1, syt2, syt5, and syt7 paralogues are found in the zebrafish brain, eye, and retina, excepting syt1b, which is only present in the brain. The complementary expression pattern of the remaining paralogues in the retina suggests that syt1a and syt5a may underlie synchronous release and syt7a and syt7b may mediate asynchronous release or other Ca2+‐dependent processes in different retinal neurons.

show abstract

Efficient targeted integration directed by short homology in zebrafish and mammalian cells

Cited by 122 publications

References 38 publications

Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

Expression and distribution of synaptotagmin isoforms in the zebrafish retina

Expression and distribution of synaptotagmin family members in the zebrafish retina

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