Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Abstract
We seek to manipulate gene function here through CRISPR-Cas9 editing of cis-regulatory sequences, rather than the more typical mutation of coding regions. This approach would minimize secondary effects of cellular responses to nonsense mediated decay pathways or to mutant protein products by premature stops. This strategy also allows for reducing gene activity in cases where a complete gene knockout would result in lethality, and it can be applied to the rapid identification of key regulatory sites essential for gene expression. We tested this strategy here with genes of known function as a proof of concept, and then applied it to examine the upstream genomic region of the germline gene Nanos2 in the sea urchin, Strongylocentrotus purpuratus. We first used CRISPR-Cas9 to target established genomic cis-regulatory regions of the skeletogenic cell transcription factor, Alx1, and the TGF-β signaling ligand, Nodal, which produce obvious developmental defects when altered in sea urchin embryos. Importantly, mutation of cis-activator sites (Alx1) and cis-repressor sites (Nodal) result in the predicted decreased and increased transcriptional output, respectively. Upon identification of efficient gRNAs by genomic mutations, we then used the same validated gRNAs to target a deadCas9-VP64 transcriptional activator to increase Nodal transcription directly. Finally, we paired these new methodologies with a more traditional, GFP reporter construct approach to further our understanding of the transcriptional regulation of Nanos2, a key gene required for germ cell identity in S. purpuratus. With a series of reporter assays, upstream Cas9-promoter targeted mutagenesis, coupled with qPCR and in situ RNA hybridization, we concluded that the promoter of Nanos2 drives strong mRNA expression in the sea urchin embryo, indicating that its primordial germ cell (PGC)-specific restriction may rely instead on post-transcriptional regulation. Overall, we present a proof-of-principle tool-kit of Cas9-mediated manipulations of promoter regions that should be applicable in most cells and embryos for which CRISPR-Cas9 is employed.
Aihara,
Inversion of left-right asymmetry in the formation of the adult rudiment in sea urchin larvae: removal of a part of embryos at the gastrula stage.
2000, Pubmed,
Echinobase
Aihara,
Inversion of left-right asymmetry in the formation of the adult rudiment in sea urchin larvae: removal of a part of embryos at the gastrula stage.
2000,
Pubmed
,
Echinobase
Aihara,
Left-right positioning of the adult rudiment in sea urchin larvae is directed by the right side.
2001,
Pubmed
,
Echinobase
Anderson,
mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.
2017,
Pubmed
Angerer,
Sea urchin goosecoid function links fate specification along the animal-vegetal and oral-aboral embryonic axes.
2001,
Pubmed
,
Echinobase
Anzalone,
Search-and-replace genome editing without double-strand breaks or donor DNA.
2019,
Pubmed
Arenas-Mena,
Expression of the Hox gene complex in the indirect development of a sea urchin.
1998,
Pubmed
,
Echinobase
Brinkman,
Easy quantitative assessment of genome editing by sequence trace decomposition.
2014,
Pubmed
Brinkman,
Rapid Quantitative Evaluation of CRISPR Genome Editing by TIDE and TIDER.
2019,
Pubmed
Cameron,
SpBase: the sea urchin genome database and web site.
2009,
Pubmed
,
Echinobase
Campanale,
Methods to label, isolate, and image sea urchin small micromeres, the primordial germ cells (PGCs).
2019,
Pubmed
,
Echinobase
Cheers,
P16 is an essential regulator of skeletogenesis in the sea urchin embryo.
2005,
Pubmed
,
Echinobase
Coffman,
A hyaline layer protein that becomes localized to the oral ectoderm and foregut of sea urchin embryos.
1990,
Pubmed
,
Echinobase
Damle,
Precise cis-regulatory control of spatial and temporal expression of the alx-1 gene in the skeletogenic lineage of s. purpuratus.
2011,
Pubmed
,
Echinobase
Davidson,
A genomic regulatory network for development.
2002,
Pubmed
,
Echinobase
Duboc,
Lefty acts as an essential modulator of Nodal activity during sea urchin oral-aboral axis formation.
2008,
Pubmed
,
Echinobase
Duboc,
A conserved role for the nodal signaling pathway in the establishment of dorso-ventral and left-right axes in deuterostomes.
2008,
Pubmed
,
Echinobase
Duboc,
Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo.
2004,
Pubmed
,
Echinobase
Duboc,
Left-right asymmetry in the sea urchin embryo is regulated by nodal signaling on the right side.
2005,
Pubmed
,
Echinobase
Ettensohn,
Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-class homeodomain proteins, is an essential component of the gene network controlling skeletogenic fate specification in the sea urchin embryo.
2003,
Pubmed
,
Echinobase
Ewen-Campen,
The molecular machinery of germ line specification.
2010,
Pubmed
Extavour,
Mechanisms of germ cell specification across the metazoans: epigenesis and preformation.
2003,
Pubmed
Flowers,
Nodal/activin signaling establishes oral-aboral polarity in the early sea urchin embryo.
2004,
Pubmed
,
Echinobase
Fresques,
Nodal induces sequential restriction of germ cell factors during primordial germ cell specification.
2018,
Pubmed
,
Echinobase
Gandhi,
CRISPR Knockouts in Ciona Embryos.
2018,
Pubmed
Haeussler,
When needles look like hay: how to find tissue-specific enhancers in model organism genomes.
2011,
Pubmed
Hardin,
Commitment along the dorsoventral axis of the sea urchin embryo is altered in response to NiCl2.
1992,
Pubmed
,
Echinobase
Irish,
The Drosophila posterior-group gene nanos functions by repressing hunchback activity.
1989,
Pubmed
Juliano,
Germ line determinants are not localized early in sea urchin development, but do accumulate in the small micromere lineage.
2006,
Pubmed
,
Echinobase
Juliano,
Nanos functions to maintain the fate of the small micromere lineage in the sea urchin embryo.
2010,
Pubmed
,
Echinobase
Khor,
Functional divergence of paralogous transcription factors supported the evolution of biomineralization in echinoderms.
2017,
Pubmed
,
Echinobase
Kobayashi,
Essential role of the posterior morphogen nanos for germline development in Drosophila.
1996,
Pubmed
Konermann,
Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.
2015,
Pubmed
Lai,
Xenopus Nanos1 is required to prevent endoderm gene expression and apoptosis in primordial germ cells.
2012,
Pubmed
Lapraz,
Patterning of the dorsal-ventral axis in echinoderms: insights into the evolution of the BMP-chordin signaling network.
2009,
Pubmed
,
Echinobase
Lasko,
The product of the Drosophila gene vasa is very similar to eukaryotic initiation factor-4A.
1988,
Pubmed
Lasko,
Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development.
1990,
Pubmed
Lin,
Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.
2016,
Pubmed
,
Echinobase
Lin,
A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary.
1997,
Pubmed
Longabaugh,
Computational representation of developmental genetic regulatory networks.
2005,
Pubmed
Luo,
Opposing nodal and BMP signals regulate left-right asymmetry in the sea urchin larva.
2012,
Pubmed
,
Echinobase
Magnúsdóttir,
How to make a primordial germ cell.
2014,
Pubmed
Makabe,
Cis-regulatory control of the SM50 gene, an early marker of skeletogenic lineage specification in the sea urchin embryo.
1995,
Pubmed
,
Echinobase
McMahon,
Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis.
1985,
Pubmed
,
Echinobase
Moreno-Mateos,
CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.
2015,
Pubmed
Nam,
Cis-regulatory control of the nodal gene, initiator of the sea urchin oral ectoderm gene network.
2007,
Pubmed
,
Echinobase
Oulhen,
Transient translational quiescence in primordial germ cells.
2017,
Pubmed
,
Echinobase
Oulhen,
Distinct transcriptional regulation of Nanos2 in the germ line and soma by the Wnt and delta/notch pathways.
2019,
Pubmed
,
Echinobase
Oulhen,
Albinism as a visual, in vivo guide for CRISPR/Cas9 functionality in the sea urchin embryo.
2016,
Pubmed
,
Echinobase
Oulhen,
Every which way--nanos gene regulation in echinoderms.
2014,
Pubmed
,
Echinobase
Oulhen,
Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.
2016,
Pubmed
,
Echinobase
Oulhen,
The 3'UTR of nanos2 directs enrichment in the germ cell lineage of the sea urchin.
2013,
Pubmed
,
Echinobase
Parisi,
Translational repression: a duet of Nanos and Pumilio.
2000,
Pubmed
Range,
Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-beta related to Vg1.
2007,
Pubmed
,
Echinobase
Revilla-i-Domingo,
R11: a cis-regulatory node of the sea urchin embryo gene network that controls early expression of SpDelta in micromeres.
2004,
Pubmed
,
Echinobase
Safari,
CRISPR Cpf1 proteins: structure, function and implications for genome editing.
2019,
Pubmed
Saudemont,
Ancestral regulatory circuits governing ectoderm patterning downstream of Nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm.
2010,
Pubmed
,
Echinobase
Su,
Telling left from right: left-right asymmetric controls in sea urchins.
2014,
Pubmed
,
Echinobase
Suh,
Small RNAs in early mammalian development: from gametes to gastrulation.
2011,
Pubmed
Triglia,
A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences.
1988,
Pubmed
Tuladhar,
CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation.
2019,
Pubmed
Voronina,
Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development.
2008,
Pubmed
,
Echinobase
Weidmann,
Drosophila Pumilio protein contains multiple autonomous repression domains that regulate mRNAs independently of Nanos and brain tumor.
2012,
Pubmed
Weidmann,
Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio.
2016,
Pubmed
Wessel,
The biology of the germ line in echinoderms.
2014,
Pubmed
,
Echinobase
Wessel,
Somatic cell conversion to a germ cell lineage: A violation or a revelation?
2021,
Pubmed
,
Echinobase
Williams,
Genome and epigenome engineering CRISPR toolkit for in vivo modulation of cis-regulatory interactions and gene expression in the chicken embryo.
2018,
Pubmed
Wylie,
Germ cells.
1999,
Pubmed
Yaguchi,
Microinjection methods for sea urchin eggs and blastomeres.
2019,
Pubmed
,
Echinobase
Yaguchi,
TGFβ signaling positions the ciliary band and patterns neurons in the sea urchin embryo.
2010,
Pubmed
,
Echinobase
Yajima,
Ars insulator protects transgenes from long-term silencing in sea urchin larva.
2007,
Pubmed
,
Echinobase
Yajima,
Implication of HpEts in gene regulatory networks responsible for specification of sea urchin skeletogenic primary mesenchyme cells.
2010,
Pubmed
,
Echinobase
Yajima,
Small micromeres contribute to the germline in the sea urchin.
2011,
Pubmed
,
Echinobase