CRISPR-Cas9 editing of non-coding genomic loci as a means of controlling gene expression in the sea urchin

• Published in: Developmental biology Vol. 472; pp. 85 - 97
• Main Authors: Pieplow, Alice, Dastaw, Meseret, Sakuma, Tetsushi, Sakamoto, Naoaki, Yamamoto, Takashi, Yajima, Mamiko, Oulhen, Nathalie, Wessel, Gary M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2021
• Subjects: Alx1; Animals; Animals, Genetically Modified; Cis-regulatory elements; CRISPR-Associated Protein 9 - genetics; CRISPR-Cas Systems; CRISPR/Cas9; Embryo, Nonmammalian - metabolism; Embryonic Development - genetics; Gene Editing - methods; Gene Expression; Gene Expression Regulation, Developmental; Gene Knockout Techniques; Genetic Loci; Germ Cells - metabolism; Germ line; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Nanos; Nodal; Nodal Protein - genetics; Nodal Protein - metabolism; Primordial germ cells; Promoter Regions, Genetic - genetics; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Messenger - genetics; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Sea urchin; Strongylocentrotus purpuratus - embryology; Strongylocentrotus purpuratus - genetics; Transcription, Genetic - genetics; Transcriptional regulation; Nodal; CRISPR/Cas9; Cis-regulatory elements; Germ line; Nanos; Primordial germ cells; Alx1; Sea urchin; Transcriptional regulation
• ISSN: 0012-1606; 1095-564X
• DOI: 10.1016/j.ydbio.2021.01.003

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. •We seek to test gene function by CRISPR-Cas9 editing of cis-regulatory sequences.•Targeting cis-regulatory sites enables unique control over endogenous transcriptional levels.•We optimize this approach using the well annotated test genes, Alx1 and Nodal.•Specific accumulation of Nanos2 in the germ line appears largely post-transcriptional.