An official website of the United States government
Abstract Despite the revolutionary impacts of CRISPR-Cas gene editing systems, the effective and widespread use of CRISPR technologies in emerging model organisms still faces significant challenges. These include the inefficiency in generating heritable mutations at the organismal level, limited knowledge about the genomic consequences of gene editing, and an inadequate understanding of the inheritance patterns of CRISPR-Cas-induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for CRISPR editing in the microcrustaceanDaphnia pulex; 2) assessing the editing efficiency of Cas9 and Cas12a nucleases, examining mutation inheritance patterns, and analyzing the local and global mutation spectrum in thescarletmutants; and 3) investigating the transcriptomes ofscarletmutants to understand the pleiotropic effects ofscarletunderlying their swimming behavior changes. Our reengineered CRISPR microinjection method results in efficient biallelic editing with both nucleases. While indels are dominant in Cas-induced mutations, a few on-site large deletions (>1kb) are observed, most likely caused by microhomology-mediated end joining repair. Knock-in of a stop codon cassette to thescarletlocus was successful, despite complex induced mutations surrounding the target site. Moreover, extensive germline mosaicism exists in some mutants, which unexpectedly produce different phenotypes/genotypes in their asexual progenies. Lastly, our transcriptomic analyses unveil significant gene expression changes associated with scarlet knock-out and altered swimming behavior in mutants, including several genes (e.g., NMDA1, ABAT, CNTNAP2) involved in human neurodegenerative diseases. This study expands our understanding of the dynamics of gene editing in the tractable model organismDaphniaand highlights its promising potential as a neurological disease model.
more »
« less
Analysis of the genome-editing activity of microinjected CRISPR/Cas9 ribonucleoprotein complexes in Daphnia pulex
Although Daphnia is a widely used model organism with a completely sequenced genome, molecular tools for analyzing specific gene functions are still being developed. Progress has been made in developing CRISPR/Cas9 gene editing in Daphnia. However, the gene-editing activity of injected ribonucleoprotein complexes (RNPs), the success of co-injected RNPs with different gRNAs, and the heritability of mutations in asexual progeny need further investigation. Here, we show prolonged Cas9 RNP activity past the one-cell stage injected individuals, leading to a wide range of somatic mutations, and germline mosaicism of heritable biallelic mutations.
more »
« less
- Award ID(s):
- 1922914
- PAR ID:
- 10556790
- Publisher / Repository:
- microPublication Biology
- Date Published:
- Journal Name:
- microPublication biology
- Volume:
- 2024
- ISSN:
- 2578-9430
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Cas9 is a metal-dependent nuclease that has revolutionized gene editing across diverse cells and organisms exhibiting varying ion uptake, metabolism, and concentrations. However, how divalent metals impact its catalytic function, and consequently its editing efficiency in different cells, remains unclear. Here, extensive molecular simulations, Markov State Models, biochemical and NMR experiments, demonstrate that divalent metals – Mg2+, Ca2+, and Co2+– promote activation of the catalytic HNH domain by binding within a dynamically forming divalent metal binding pocket (DBP) at the HNH-RuvC interface. Mutations in DBP residues disrupt HNH activation and impair the coupled catalytic activity of both nucleases, identifying this cryptic DBP as a key regulator of Cas9’s metal-dependent activity. The ionic strength thereby promotes Cas9’s conformational activation, while its catalytic activity is metal-specific. These findings are critical to improving the metal-dependent function of Cas9 and its use for genome editing in different cells and organisms.more » « less
-
Slotman, Michel (Ed.)Abstract The wide distribution of Culex (Cx.) pipiens complex mosquitoes makes it difficult to prevent the transmission of mosquito-borne diseases in humans. Gene editing using CRISPR/Cas9 is an effective technique with the potential to solve the growing problem of mosquito-borne diseases. This study uses the ReMOT Control technique in Culex pipiens pallens (L.) to produce genetically modified mosquitoes. A microinjection system was established by injecting 60 adult female mosquitoes—14 µl injection mixture was required, and no precipitation occurred with ≤1 µl of endosomal release reagents (chloroquine or saponin). The efficiency of delivery of the P2C-enhanced green fluorescent protein-Cas9 (P2C-EGFP-Cas9) ribonucleoprotein complex into the ovary was 100% when injected at 24 h post-bloodmeal (the peak of vitellogenesis). Using this method for KMO knockout, we found that gene editing in the ovary could also occur when P2C-Cas9 RNP complex was injected into the hemolymph of adult Cx. pipiens pallens by ReMOT Control. In the chloroquine group, of the 2,251 G0 progeny screened, 9 individuals showed with white and mosaic eye phenotypes. In the saponin group, of the 2,462 G0 progeny screened, 8 mutant individuals were observed. Sequencing results showed 13 bp deletions, further confirming the fact that gene editing occurred. In conclusion, the successful application of ReMOT Control in Cx. pipiens pallens not only provides the basic parameters (injection parameters and injection time) for this method but also facilitates the study of mosquito biology and control.more » « less
-
Abstract The European corn borer (Ostrinia nubilalis) is an agricultural pest and burgeoning model for research on speciation, seasonal adaptation and insect resistance management. Although previous work inO. nubilalishas identified genes associated with differences in life cycle, reproduction, and resistance toBttoxins, the general lack of a robust gene‐editing protocol forO. nubilalishas been a barrier to functional validation of candidate genes. Here, we demonstrate an efficient and practical methodology for heritable gene mutagenesis inO. nubilalisusing the CRISPR/Cas9 genome editing system. Precise loss‐of‐function (LOF) mutations were generated at two circadian clock genes,period(per) andpigment‐dispersing factor receptor(pdfr), and a developmental gene,prothoracicotropic hormone(ptth). Precluding the need for a visible genetic marker, gene‐editing efficiency remained high across different single guide RNAs (sgRNA) and germline transmission of mutations to F1offspring approached 100%. When single or dual sgRNAs were injected at a high concentration, gene‐specific phenotypic differences in behaviour and development were identified in F0mutants. Specifically, F0gene mutants demonstrated that PER, but not PDFR, is essential for normal timing of eclosion. PTTH F0mutants were significantly heavier and exhibited a higher incidence of diapause. This work will accelerate future studies of gene function inO. nubilalisand facilitate the development of similar screens in other Lepidopteran and non‐model insects.more » « less
-
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats associated with protein 9) was first identified as a component of the bacterial adaptive immune system and subsequently engineered into a genome-editing tool. The key breakthrough in this field came with the realization that CRISPR/Cas9 could be used in mammalian cells to enable transformative genetic editing. This technology has since become a vital tool for various genetic manipulations, including gene knockouts, knock-in point mutations, and gene regulation at both transcriptional and post-transcriptional levels. CRISPR/Cas9 holds great potential in human medicine, particularly for curing genetic disorders. However, despite significant innovation and advancement in genome editing, the technology still possesses critical limitations, such as off-target effects, immunogenicity issues, ethical considerations, regulatory hurdles, and the need for efficient delivery methods. To overcome these obstacles, efforts have focused on creating more accurate and reliable Cas9 nucleases and exploring innovative delivery methods. Recently, functional biomaterials and synthetic carriers have shown great potential as effective delivery vehicles for CRISPR/Cas9 components. In this review, we attempt to provide a comprehensive survey of the existing CRISPR-Cas9 delivery strategies, including viral delivery, biomaterials-based delivery, synthetic carriers, and physical delivery techniques. We underscore the urgent need for effective delivery systems to fully unlock the power of CRISPR/Cas9 technology and realize a seamless transition from benchtop research to clinical applications.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.17912/micropub.biology.001310
