New advances in CRISPR/Cas-mediated precise gene-editing techniques

Disease Models & Mechanisms, Год журнала: 2023, Номер 16(2)

Опубликована: Фев. 1, 2023

ABSTRACT Over the past decade, CRISPR/Cas-based gene editing has become a powerful tool for generating mutations in variety of model organisms, from Escherichia coli to zebrafish, rodents and large mammals. effectively generates insertions or deletions (indels), which allow rapid disruption. However, proportion human genetic diseases are caused by single-base-pair substitutions, result more subtle alterations protein function, require complex precise recreate systems. Precise genome (PGE) methods, however, typically have efficiencies less than tenth those that generate less-specific indels, so there been great deal effort improve PGE efficiency. Such optimisations include optimal guide RNA mutation-bearing donor DNA template design, modulation repair pathways underpin how edits Cas-induced cuts, development Cas9 fusion proteins introduce via alternative mechanisms. In this Review, we provide an overview recent progress optimising methods their potential models disease.

Язык: Английский

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Zebrafish: unraveling genetic complexity through duplicated genes

Development Genes and Evolution, Год журнала: 2024, Номер unknown

Опубликована: Июль 30, 2024

Abstract The zebrafish is an invaluable model organism for genetic, developmental, and disease research. Although its high conservation with humans often cited as justification use, the harbors oft-ignored genetic characteristics that may provide unique insights into gene structure function. Zebrafish, along other teleost fish, underwent additional round of whole genome duplication after their split from tetrapods—resulting in abundance duplicated genes when compared to vertebrates. These have evolved distinct ways over ensuing 350 million years. Thus, each within a pair has nuanced differences create identity. By investigating both members together, we can elucidate mechanisms underly protein function drive complex interplay biological systems, such signal transduction cascades, regulatory networks, evolution tissue organ It crucial leverage studies explore these molecular dynamics, which could far-reaching implications basic science therapeutic development. Here, will review role duplications existing models divergence retention following events. We also highlight examples where comparing yielded key structure, function, regulation.

Язык: Английский

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Knock-in tagging in zebrafish facilitated by insertion into non-coding regions

Development, Год журнала: 2021, Номер 148(19)

Опубликована: Сен. 8, 2021

Zebrafish provide an excellent model for in vivo cell biology studies because of their amenability to live imaging. Protein visualization zebrafish has traditionally relied on overexpression fluorescently tagged proteins from heterologous promoters, making it difficult recapitulate endogenous expression patterns and protein function. One way circumvent this problem is tag the by modifying genomic loci. Such approach not widely available researchers inefficient homologous recombination error-prone nature targeted integration zebrafish. Here, we report a simple tagging N or C termini with fluorescent inserting PCR-generated donor amplicons into non-coding regions corresponding genes. Using approach, generated endogenously alleles several genes that are crucial epithelial organ development, including tight junction components ZO-1 Cldn15la, trafficking effector Rab11a, apical polarity aPKC ECM receptor Integrin β1b. Our facilitates generation knock-in lines zebrafish, opening accurate quantitative imaging studies.

Язык: Английский

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In search of an ideal template for therapeutic genome editing: A review of current developments for structure optimization

Frontiers in Genome Editing, Год журнала: 2023, Номер 5

Опубликована: Фев. 22, 2023

Gene therapy is a fast developing field of medicine with hundreds ongoing early-stage clinical trials and numerous preclinical studies. Genome editing (GE) now an increasingly important technology for achieving stable therapeutic effect in gene correction, hematopoietic cells representing key target cell population novel treatments number hereditary diseases, infections cancer. By introducing double strand break (DSB) the defined locus genomic DNA, GE tools allow to knockout desired or knock-in if provided appropriate repair template. Currently, efficiency methods GE-mediated limited. Significant efforts were focused on improving parameters interaction nuclease proteins. However, emerging data suggests that optimal characteristics templates may play role mechanisms. While viral vectors notable example AAVs as donor template carrier remain mainstay many trials, non-viral templates, including plasmid linear dsDNA, long ssDNA single double-stranded ODNs, represent promising alternative. Furthermore, tuning conditions chosen well its structure, length, sequence optimization, homology arm (HA) modifications have paramount importance highly efficient favorable safety profile. This review outlines current developments optimization mediated correction.

Язык: Английский

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From DNA break repair pathways to CRISPR/Cas-mediated gene knock-in methods

Life Sciences, Год журнала: 2022, Номер 295, С. 120409 - 120409

Опубликована: Фев. 16, 2022

Язык: Английский

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Targeted large fragment deletion in plants using paired crRNAs with type I CRISPR system

Plant Biotechnology Journal, Год журнала: 2023, Номер 21(11), С. 2196 - 2208

Опубликована: Авг. 29, 2023

The CRISPR-Cas systems have been widely used as genome editing tools, with type II and V typically introducing small indels, I system mediating long-range deletions. However, the precision of for large fragment deletion is still remained to be optimized. Here, we developed a compact Cascade-Cas3 Dvu I-C Cas11c plant editing. was efficient introduce controllable up at least 20 kb using paired crRNAs. paired-crRNAs design also improved controllability deletions I-E system. sensitive spacer length mismatch, which benefit target specificity. In addition, showed that generating stable transgenic lines in maize rice efficiency 86.67%. Overall, here powerful achieving

Язык: Английский

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ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells

BMC Genomics, Год журнала: 2023, Номер 24(1)

Опубликована: Май 29, 2023

Recent advances in CRISPR technology have enabled us to perform gene knock-in various species and cell lines. CRISPR-mediated requires donor DNA which serves as a template for homology-directed repair (HDR). For of short sequences or base substitutions, ssDNA donors are frequently used among other forms HDR donors, such linear dsDNA. However, partly due the complexity long preparation, it remains unclear whether is optimal type insertion transgenes fluorescent reporters human cells.In this study, we established nuclease-based simple method preparation with high yield purity, comprehensively compared performance dsDNA 90 bases homology arms endogenous tagging diploid RPE1 HCT116 cells. Quantification using flow cytometry revealed lower efficiency than By analyzing outcomes long-read amplicon sequencing classification framework, variety mis-integration events were detected regardless type. Importantly, ratio precise was Moreover, off-target integration analyses without arms, comparably prone non-homologous integration.These results indicate that not superior relatively

Язык: Английский

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RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

International Journal of Molecular Sciences, Год журнала: 2022, Номер 23(2), С. 631 - 631

Опубликована: Янв. 6, 2022

The efficiency of the RNA-guided AsCas12a nuclease Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions ratio guide RNAs to nuclease, donor templates, and electroporation parameters, target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a Cas9 resulted staggered- blunt-ended strand breaks, respectively. more efficient than gene knockout, as determined TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout both nucleases markedly increased presence single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative non-CRISPR (NT) (T) strands tested, resulting KO efficiencies 15.67, 28.71, 21.43% plus ssODN, NT-ssODN, T-ssODN groups, Trans-cleavage against activated not apparent vitro. precise transgene insertion, knock-in 17.07% KI_Cas9 group, 14.58% KI_Cas12a-NT-ssODN, 12.37% KI_Cas12a-T-ssODN. Although induced fewer per genome SpCas9, phenotypic impact on transcription expression omega-1 similar nucleases.

Язык: Английский

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Generation of Rhesus Macaque Embryos with Expanded CAG Trinucleotide Repeats in the Huntingtin Gene

Cells, Год журнала: 2024, Номер 13(10), С. 829 - 829

Опубликована: Май 13, 2024

Huntington's disease (HD) arises from expanded CAG repeats in exon 1 of the

Язык: Английский

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CRISPR-Cas9-Mediated Genome Modifications in Zebrafish

Methods in molecular biology, Год журнала: 2023, Номер unknown, С. 313 - 324

Опубликована: Янв. 1, 2023

Язык: Английский

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