CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.
CRISPR/Cas9 editing efficacies in tetraploid potato were highly improved through the use of endogenous potato
- NSF-PAR ID:
- 10153863
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 9
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Intracellular immune receptor nucleotide‐binding leucine‐rich repeats (NLRs) are highly regulated transcriptionally and post‐transcriptionally for balanced plant defence and growth. NLR genes often exist in gene clusters and are usually co‐expressed under various conditions. Despite of intensive studies of regulation of NLR proteins, cis‐acting elements for NLR gene induction, repression or co‐expression are largely unknown due to a larger than usual cis‐region for their expression regulation. Here we used the CRISPR/Cas9 genome editing technology to generate a series of in situ deletions at the endogenous location of a NLR gene
SNC1 residing in theRPP5 gene cluster. These deletions that made in the wild type and theSNC1 constitutive expressing autoimmune mutantbon1 revealed both positive and negative cis‐acting elements forSNC1 expression. Two transcription factors that could bind to these elements were found to have an impact on the expression ofSNC1 . In addition, co‐expression of two genes withSNC1 in the same cluster is found to be mostly dependent on theSNC1 function. Therefore,SNC1 expression is under complex local regulation involving multiple cis elements andSNC1 itself is a critical regulator of gene expression of other NLR genes in the same gene cluster. -
Abstract Use of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated 9)-mediated genome editing has proliferated for use in numerous plant species to modify gene function and expression, usually in the context of either transient or stably inherited genetic alternations. While extremely useful in many applications, modification of some loci yields outcomes detrimental to further experimental evaluation or viability of the target organism. Expression of Cas9 under a promoter conferring gene knockouts in a tissue-specific subset of genomes has been demonstrated in insect and animal models, and recently in
Arabidopsis . We developed an in planta GFP (green fluorescent protein) assay system to demonstrate fruit-specific gene editing in tomato using aphosphoenolpyruvate carboxylase 2 gene promoter. We then targeted a SET-domain containing polycomb protein, SlEZ2, previously shown to yield pleiotropic phenotypes when targeted via35S-driven RNA interference and we were able to characterize fruit phenotypes absent additional developmental perturbations. Tissue-specific gene editing will have applications in assessing function of essential genes otherwise difficult to study via germline modifications and will provide routes to edited genomes in tissues that could not otherwise be recovered when their germline modification perturbs their normal development. -
null (Ed.)Cell cultures are effective supplemental models to study specific biochemical pathways used for environmental adaption in animals. They enable isolation from system influence and facilitate control the extracellular environment. For work focusing on fish species many representative cell lines now exist, including a tilapia brain cell line (OmB) developed in our lab. CRISPR/Cas9 gene editing is an additional tool aiding these studies by allowing manipulation of specific genetic loci and evaluating their causal relationship between phenotypes of interest. However, established CRISPR/Cas9 gene targeting tools and methods often have not functioned as efficiently in fish cells as seen in other animal cell models such as mammalian cell lines, consistent with our initial attempts to apply CRISPR/Cas9 in OmB cells that failed to indicate genomic alteration at the targeted sites. Poor expression of heterologous promoters in OmB cells was hypothesized to be a primary cause for this occurrence so we constructed a custom plasmid vector based system utilizing tilapia endogenous promoters (EF1 alpha to express Cas9 and a U6 to express gRNAs). This system demonstrated substantial editing of most target sites attempted with mutational efficiency as high 80%. This work specifically highlighted the importance of phylogenetic proximity in selection of a polymerase III promoter for gRNA expression as commonly used interspecies U6 promoters (human and zebrafish) yielded no detectable gene editing when applied in this system with a common gRNA target sequence. These new tools will allow generation of knockout cell lines for gene targeting studies in tilapia and other phylogenetically close fish species.more » « less
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Summary The ability to edit plant genomes through gene targeting (
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