Optimized CRISPR–Cas9 System for Genome Editing in Zebrafish

Vejnar, Charles E.; Moreno-Mateos, Miguel A.; Cifuentes, Daniel; Bazzini, Ariel

doi:10.1101/pdb.prot086850

Cited by 70 publications

(65 citation statements)

References 5 publications

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“…A ~300 bp fragment surrounding this site was PCR amplified with flanking BamHI restriction sites and cloned into the eGFP bait-E2A-KalTA4-pA plasmid 49 by T4 ligation according to the manufacturer’s protocols (NEB), to produce the miR-223-E2A-KalTA4 bait plasmid. CRISPRScan 50,51 was then used to design a guide RNA (gRNA) that targets both the miR-223 genomic locus and miR-223-E2A-KalTA4 bait plasmid. The gRNA template for in vitro transcription was PCR amplified and purified with Qiaquick PCR purification Kit (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

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The N-Glycome regulates the endothelial-to-hematopoietic transition

Kasper

Hintzen

et al. 2019

Preprint

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20Hematopoietic stem and progenitor cells (HSPCs) are required to establish and maintain the 21 adult blood system in vertebrates. During development, hemogenic endothelial cells undergo an 22 endothelial-to-hematopoietic transition (EHT) to generate HSPCs 1-4 . Growth factors and 23 epigenetic changes can promote EHT 1,3,5 , but these mechanisms do not explain its tight 24 spatiotemporal regulation during development. Here, we show that microRNA (miR) miR-223-25 mediated regulation of N-glycan biosynthesis intrinsically restrains EHT, representing the first 26 pathway that prevents excessive HSPC production. We find that miR-223 is uniquely expressed 27 in hemogenic endothelial cells undergoing EHT and in nascent HSPCs. Loss of miR-223 28 promotes the expansion of these cells in the zebrafish and mouse aorta-gonad-mesonephros 29 (AGM), where EHT occurs 6-8 . miR-223 targets alg2 (α1,3/ α1,6 mannosyltransferase) and 30 st3gal2 (α2,3 sialyltransferase) for repression in the AGM endothelium. These two enzymes are 31 involved in the biosynthesis of N-glycans, a common co-translational modification 9,10 that 32 influences several pathophysiological processes 11,12 , but has not yet been implicated in EHT. 33 Using an N-glycosensor, we demonstrate that vascular N-glycosylation increases during EHT, 34 and this process is disrupted upon loss of miR-223. Specifically, high-throughput glycome 35 analysis revealed terminal α1,3 mannose and α2,3 sialic acid modifications of membrane 36 proteins are altered upon loss of miR-223. Importantly, pharmacological manipulation targeting 37 these N-glycan types in wild-type embryos phenocopies the loss of miR-223 and enhances EHT 38 as well as HSPC production. Thus, the N-glycome plays a previously unappreciated role as an 39 intrinsic negative regulator of EHT, with specific mannose and sialic acid modifications serving 40as key endothelial determinants of the hematopoietic fate. 41 43Zebrafish endothelial-to-hematopoietic transition (EHT) has emerged as an instrumental 44 model to understand the formation and regulation of definitive hematopoietic stem and 45 progenitor cells (HSPCs) with long-term engraftment capability. As in their mammalian 46 counterparts, EHT occurs in endothelial cells that co-express vascular and hematopoietic genes 47

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Section: Methodsmentioning

confidence: 99%

“…CRISPRScan 50,51 was used to design two gRNAs to mutate the miR-223 responsive element region in the alg2 3’UTR with CRISPR/Cas9 genome editing. gRNA preparation was performed as above for miR-223:Gal4 zebrafish generation.…”

Section: Methodsmentioning

confidence: 99%

The N-Glycome regulates the endothelial-to-hematopoietic transition

Kasper

Hintzen

et al. 2019

Preprint

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“…crRNAs were visualized in a 2% agarose stained by ethidium bromide to check for RNA integrity. sgRNAs were generated as previously described 18,21 . sgRNAs and crRNAs targeting slc45a2 in zebrafish were individually in vitro transcribed.…”

Section: Methods Crrna and Sgrna Target Sites Designmentioning

confidence: 99%

CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

Moreno-Mateos

Fernández

Rouet

et al. 2017

Preprint

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Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows posttranslational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.

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“…1 nl of morpholino solution (0.6mM) was injected into wild-type dechorionated embryos at the one-cell stage. CRISPR/Cas9-mediated gene editing was performed as described previously (Vejnar et al 2016). Briefly, 3 different sgRNAs (20 pg each) targeting bud13 gene ( Supplemental Table S4) were co-injected together with 100 pg of mRNA coding for zebrafish codon optimized Cas9-nanos in one-cell stage embryos (Supplemental Fig.…”

Section: Morpholino Injections and Gene Editing Using Crispr-cas9mentioning

confidence: 99%

RES complex is associated with intron definition and required for zebrafish early embryogenesis

Fernández

Moreno-Mateos

Gohr

et al. 2017

Preprint

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Pre-mRNA splicing is a critical step of gene expression in eukaryotes. Transcriptome-wide splicing patterns are complex and primarily regulated by a diverse set of recognition elements and associated RNA-binding proteins. The retention and splicing (RES) complex is formed by three different proteins (Bud13p, Pml1p and Snu17p) and is involved in splicing in yeast. However, the importance of the RES complex for vertebrate splicing, the intronic features associated with its activity, and its role in development are unknown. In this study, we have generated loss-of-function mutants for the three components of the RES complex in zebrafish and showed that they are required during early development. The mutants showed a marked neural phenotype with increased cell death in the brain and a decrease in differentiated neurons. Transcriptomic analysis of bud13, snip1 (pml1) and rbmx2 (snu17) mutants revealed a global defect in intron splicing, with strong mis-splicing of a subset of introns. We found these RES-dependent introns were short, rich in GC and flanked by GC depleted exons, all of which are features associated with intron definition. Using these features we developed a predictive model that classifies RES dependent introns. Altogether, our study uncovers the essential role of the RES complex during vertebrate development and provides new insights into its function during splicing.Splicing is critical step in eukaryotic gene expression and is an important source of transcriptomic complexity (Licatalosi and Darnell 2010). Splicing is carried out by the spliceosome, a large macromolecular complex that includes five small nuclear ribonucleoproteins (snRNPs; U1, U2, U4, U5 and U6) and hundreds of core and accessory proteins that ensure the accurate removal of introns from pre-mRNAs (Wahl et al. 2009). Canonically s p l i c e d i n t r o n s a r e r e m o v e d t h r o u g h t w o transesterification reactions during a complex process involving the recruitment and release of multiple core splicing factors (Will and Luhrmann 2011). However, many introns are recognized by different mechanisms depending on their specific features. Short introns with high GC content are believed to be spliced through an "intron definition" mechanism, in which initial U1-U2 pairing occurs across the intron. On the other hand, long introns surrounding short exons are recognized and spliced through "exon definition" mechanisms, in which the initial pairing bridges across the exon (De Conti et al. 2013). While these mechanisms are widely accepted, little is known about the specific factors associated with each process. The pre-mRNA REtention and Splicing (RES) complex is a spliceosomal complex conserved from yeast to human. It is organized around the U2 snRNP-associated protein Snu17p/Ist3p (RBMX2 in human), which binds to both the pre-mRNA-leakage protein 1 (Pml1p; SNIP1 in human) and bud site-selection protein 13 (Bud13p; BUD13 in human) (Gottschalk et al. 2001;Dziembowski et al. 2004;Bessonov et al. 2010). Snu17p interacts directly with the ...

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Optimized CRISPR–Cas9 System for Genome Editing in Zebrafish

Cited by 70 publications

References 5 publications

The N-Glycome regulates the endothelial-to-hematopoietic transition

The N-Glycome regulates the endothelial-to-hematopoietic transition

CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing

RES complex is associated with intron definition and required for zebrafish early embryogenesis

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