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1.
BMC Genomics ; 23(1): 12, 2022 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-34986794

RESUMO

BACKGROUND: Zebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create 'knockout' models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies. METHODS: To systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression. RESULTS: We compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (< 1%). To characterize if commonly used 'mock' CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5 days post fertilization. While we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundred differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the 'mock' injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potentially lasting effect from the microinjection process that requires further investigation. CONCLUSION: Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.


Assuntos
Edição de Genes , Peixe-Zebra , Animais , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , RNA Guia de Cinetoplastídeos/genética , Peixe-Zebra/genética
2.
bioRxiv ; 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39314374

RESUMO

Recent expansion of duplicated genes unique in the Homo lineage likely contributed to brain evolution and other human-specific traits. One hallmark example is the expansion of the human SRGAP2 family, resulting in a human-specific paralog SRGAP2C . Introduction of SRGAP2C in mouse models is associated with altering cortical neuronal migration, axon guidance, synaptogenesis, and sensory-task performance. Truncated, human-specific SRGAP2C heterodimerizes with the full-length ancestral gene product SRGAP2A and antagonizes its functions. However, the significance of SRGAP2 duplication beyond neocortex development has not been elucidated due to the embryonic lethality of complete Srgap2 knockout in mice. Using zebrafish, we showed that srgap2 knockout results in viable offspring that phenocopy "humanized" SRGAP2C larvae. Specifically, human SRGAP2C protein interacts with zebrafish Srgap2, demonstrating similar Srgap2 functional antagonism observed in mice. Shared traits between knockout and humanized zebrafish larvae include altered morphometric features (i.e., reduced body length and inter-eye distance) and differential expression of synapse-, axogenesis-, vision-related genes. Through single-cell transcriptome analysis, we further observed a skewed balance of excitatory and inhibitory neurons that likely contributes to increased susceptibility to seizures displayed by Srgap2 mutant larvae, a phenotype resembling SRGAP2 loss-of-function in a child with early infantile epileptic encephalopathy. Single-cell data also pointed to strong microglia expression of srgap2 with mutants exhibiting altered membrane dynamics and likely delayed maturation of microglial cells. srgap2 -expressing microglia cells were also detected in the developing eye together with altered expression of genes related to axogenesis and synaptogenesis in mutant retinal cells. Consistent with the perturbed gene expression in the retina, we found that SRGAP2 mutant larvae exhibited increased sensitivity to broad and fine visual cues. Finally, comparing the transcriptomes of relevant cell types between human (+ SRGAP2C ) and non-human primates (- SRGAP2C ) revealed significant overlaps of gene alterations with mutant cells in our zebrafish models; this suggests that SRGAP2C plays similar roles altering microglia and the visual system in modern humans. Together, our functional characterization of zebrafish Srgap2 and human SRGAP2C in zebrafish uncovered novel gene functions and highlights the strength of cross-species analysis in understanding the development of human-specific features.

3.
Front Cell Dev Biol ; 8: 586296, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33330465

RESUMO

In recent years, zebrafish have become commonly used as a model for studying human traits and disorders. Their small size, high fecundity, and rapid development allow for more high-throughput experiments compared to other vertebrate models. Given that zebrafish share >70% gene homologs with humans and their genomes can be readily edited using highly efficient CRISPR methods, we are now able to rapidly generate mutations impacting practically any gene of interest. Unfortunately, our ability to phenotype mutant larvae has not kept pace. To address this challenge, we have developed a protocol that obtains multiple phenotypic measurements from individual zebrafish larvae in an automated and parallel fashion, including morphological features (i.e., body length, eye area, and head size) and movement/behavior. By assaying wild-type zebrafish in a variety of conditions, we determined optimal parameters that avoid significant developmental defects or physical damage; these include morphological imaging of larvae at two time points [3 days post fertilization (dpf) and 5 dpf] coupled with motion tracking of behavior at 5 dpf. As a proof-of-principle, we tested our approach on two novel CRISPR-generated mutant zebrafish lines carrying predicted null-alleles of syngap1b and slc7a5, orthologs to two human genes implicated in autism-spectrum disorder, intellectual disability, and epilepsy. Using our optimized high-throughput phenotyping protocol, we recapitulated previously published results from mouse and zebrafish models of these candidate genes. In summary, we describe a rapid parallel pipeline to characterize morphological and behavioral features of individual larvae in a robust and consistent fashion, thereby improving our ability to better identify genes important in human traits and disorders.

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