Engineering TadA ortholog-derived cytosine base editor without motif preference and adenosine activity limitation

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: Sept. 16, 2024

The engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to that rely on natural deaminases. However, the current demonstrate preference for editing DNA with specific motif sequences possess dual deaminase activity, acting both adenosine adjacent positions, limiting their application scope. To address these issues, we employ orthologs screening multi sequence alignment (MSA)-guided protein engineering techniques create highly effective editor (aTdCBE) without activity limitations. Notably, delivery of aTdCBE humanized mouse model Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping restoration dystrophin expression. Our advancement ortholog enriches toolkits gene-editing therapy other potential applications.

Language: Английский

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Research Progress and Application of Miniature CRISPR-Cas12 System in Gene Editing

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(23), P. 12686 - 12686

Published: Nov. 26, 2024

CRISPR-Cas system, a natural acquired immune system in prokaryotes that defends against exogenous DNA invasion because of its simple structure and easy operation, has been widely used many research fields such as synthetic biology, crop genetics breeding, precision medicine, so on. The miniature CRISPR-Cas12 an emerging genome editing tool recent years. Compared to the commonly CRISPR-Cas9 CRISPR-Cas12a, unique advantages, rich PAM sites, higher specificity, smaller volume, cytotoxicity. However, application Cas12 proteins methods improve efficiency have not systematically summarized. In this review, we introduce classification summarize structural characteristics type V cleavage mechanism five proteins. gene animals, plants, microorganisms is summarized, strategies are discussed, aiming provide reference for further understanding functional engineering modification system.

Language: Английский

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Current Non-Viral-Based Strategies to Manufacture CAR-T Cells

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(24), P. 13685 - 13685

Published: Dec. 21, 2024

The successful application of CAR-T cells in the treatment hematologic malignancies has fundamentally changed cancer therapy. With increasing numbers registered cell clinical trials, efforts are being made to streamline and reduce costs manufacturing while improving their safety. To date, all approved products have relied on viral-based gene delivery genomic integration methods. While viral vectors offer high transfection efficiencies, concerns regarding potential malignant transformation coupled with costly time-consuming vector constant drivers search for cheaper, easier-to-use, safer, more efficient alternatives. In this review, we examine different non-viral transfer methods as alternatives production, advantages disadvantages, examples applications. Transposon-based lead stable but non-targeted integration, easy handle, achieve rates. Programmable endonucleases allow targeted reducing risk integration-mediated cells. Non-integrating CAR-encoding avoid completely only transient CAR expression. these promising alternative techniques transfer, avenues open fully exploiting next-generation therapy applying it a wide range

Language: Английский

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Improvements in the genetic editing technologies: CRISPR-Cas and beyond

Gene, Journal Year: 2022, Volume and Issue: 852, P. 147064 - 147064

Published: Nov. 24, 2022

Language: Английский

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Generation of eco-friendly channel catfish,Ictalurus punctatus, harboring alligator cathelicidin gene with robust disease resistance by harnessing different CRISPR/Cas9-mediated systems

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Jan. 5, 2023

Abstract The CRISPR/Cas9 platform holds promise for modifying fish traits of interest as a precise and versatile tool genome manipulation. To reduce introgression transgene control reproduction, catfish species have been studied upscaled disease resistance intervening reproduction to lower the potential environmental risks escapees’ transgenic animals. Taking advantage CRISPR/Cas9-mediated system, we succeeded in integrating cathelicidin gene from an alligator ( Alligator sinensis ; As-Cath ) into target luteinizing hormone LH locus channel Ictalurus punctatus using two delivery systems assisted by double-stranded DNA (dsDNA) single-stranded oligodeoxynucleotides (ssODNs), respectively. In this study, high knock-in (KI) efficiency (22.38%, 64/286) but low on-target was achieved ssODN strategy, whereas adopting dsDNA donor template led efficient KI (10.80%, 23/213). On-target instrumental establishing knockout (LH − _As-Cath + line, which displayed heightened reduced fecundity compared wild-type sibling fish. Furthermore, implanting with HCG LHRHa can restore fecundity, spawnability hatchability new line. Overall, replaced then administered therapy gain complete reproductive disease-resistant environmentally sound manner. This strategy not only effectively improves consumer-valued traits, also guards against genetic contamination. is breakthrough aquaculture genetics confine prevent establishment or domestic genotypes natural environment.

Language: Английский

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