Generation of eco-friendly channel catfish,Ictalurus punctatus, harboring alligator cathelicidin gene with robust disease resistance by harnessing different CRISPR/Cas9-mediated systems DOI Open Access
Jinhai Wang, Baofeng Su, De Xing

et al.

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

Fanzor is a eukaryotic programmable RNA-guided endonuclease DOI Creative Commons
Makoto Saito, Peiyu Xu, Guilhem Faure

et al.

Nature, Journal Year: 2023, Volume and Issue: 620(7974), P. 660 - 668

Published: June 28, 2023

Abstract RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have central role in biological processes both prokaryotes eukaryotes. For example, the prokaryotic CRISPR–Cas systems provide adaptive immunity bacteria archaea against foreign elements. Cas effectors such as Cas9 Cas12 perform guide-RNA-dependent DNA cleavage 1 . Although few eukaryotic been studied, including interference 2 ribosomal modification 3 , it remains unclear whether eukaryotes endonucleases. Recently, new class (termed OMEGA) was reported 4,5 The OMEGA effector TnpB is putative ancestor has endonuclease activity 4,6 may also be transposon-encoded Fanzor (Fz) proteins 4,7 raising possibility that are equipped with or OMEGA-like programmable Here we report biochemical characterization Fz, showing an endonuclease. We show Fz can reprogrammed human genome engineering applications. Finally, resolve structure Spizellomyces punctatus at 2.7 Å using cryogenic electron microscopy, conservation core regions among Cas12, despite diverse cognate structures. Our results system, demonstrating endonucleases present all three domains life.

Language: Английский

Citations

125
Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox DOI Creative Commons
Eugene V. Koonin, Jonathan S. Gootenberg, Omar O. Abudayyeh

et al.

Biochemistry, Journal Year: 2023, Volume and Issue: 62(24), P. 3465 - 3487

Published: May 16, 2023

CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms have been repurposed for versatile applications therapeutics diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting interference are mediated by effectors that either components of multisubunit complexes class 1 or multidomain single-effector proteins 2. The compact 2 broadly adopted multiple applications, especially genome editing, leading a transformation the molecular biology biotechnology toolkit. diversity enzymes, initially limited Cas9 nuclease, was substantially expanded via computational metagenome mining include numerous variants Cas12 Cas13, providing substrates development versatile, orthogonal tools. Characterization these uncovered many new features, including distinct protospacer adjacent motifs (PAMs) expand space, improved editing specificity, rather than DNA targeting, smaller crRNAs, staggered blunt end cuts, miniature promiscuous cleavage, etc. These unique properties enabled such as harnessing RNase activity type VI effector, supersensitive nucleic acid detection. well, despite challenge expressing delivering multiprotein effectors. rich enzymes led rapid maturation toolbox, capabilities gene knockout, base prime insertion, imaging, epigenetic modulation, transcriptional editing. Combined rational design engineering associated RNAs, natural related bacterial provides vast resource expanding repertoire tools biotechnology.

Language: Английский

Citations

51
Development of miniature base editors using engineered IscB nickase DOI
Dingyi Han, Qingquan Xiao, Yifan Wang

et al.

Nature Methods, Journal Year: 2023, Volume and Issue: 20(7), P. 1029 - 1036

Published: May 25, 2023

Language: Английский

Citations

40
Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants DOI Creative Commons
Liyang Zhang, Gen Li, Yingxiao Zhang

et al.

Genome biology, Journal Year: 2023, Volume and Issue: 24(1)

Published: April 30, 2023

Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of most robust nucleases, LbCas12a general is less efficient than SpCas9 human cells, animals, plants.

Language: Английский

Citations

27
Genome editing in rice using CRISPR/Cas12i3 DOI Creative Commons

Ping Lv,

Fei Su, Fangyuan Chen

et al.

Plant Biotechnology Journal, Journal Year: 2023, Volume and Issue: 22(2), P. 379 - 385

Published: Oct. 11, 2023

The CRISPR/Cas type V-I is a family of programmable nuclease systems that prefers T-rich protospacer adjacent motif (PAM) and guided by short crRNA. In this study, the genome-editing application Cas12i3, endonuclease, was characterized in rice. We developed CRIPSR/Cas12i3-based Multiplex direct repeats (DR)-spacer Array Genome Editing (iMAGE) system efficient editing various genes Interestingly, iMAGE produced chromosomal structural variations with higher frequency than CRISPR/Cas9. addition, we base editors using deactivated Cas12i3 generated herbicide-resistant rice plants editors. These genome will facilitate precision molecular breeding plants.

Language: Английский

Citations

16
Structure and engineering of miniature Acidibacillus sulfuroxidans Cas12f1 DOI
Zhaowei Wu, Dongliang Liu, Deng Pan

et al.

Nature Catalysis, Journal Year: 2023, Volume and Issue: 6(8), P. 695 - 709

Published: July 31, 2023

Language: Английский

Citations

15
Assessing and engineering the IscB–ωRNA system for programmed genome editing DOI

Hao Yan,

Xiaoqing Tan, Siyuan Zou

et al.

Nature Chemical Biology, Journal Year: 2024, Volume and Issue: 20(12), P. 1617 - 1628

Published: July 8, 2024

Language: Английский

Citations

5
Generation of Eco-Friendly and Disease-Resistant Channel Catfish (Ictalurus punctatus) Harboring the Alligator Cathelicidin Gene via CRISPR/Cas9 Engineering DOI Creative Commons
Jinhai Wang, Baofeng Su, De Xing

et al.

Engineering, Journal Year: 2024, Volume and Issue: 39, P. 273 - 286

Published: Feb. 1, 2024

As a precise and versatile tool for genome manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) platform holds promise modifying fish traits of interest. With aim reducing transgene introgression controlling reproduction, upscaled disease resistance reproductive intervention in catfish species have been studied to lower potential environmental risks escapees as transgenic animals. Taking advantage CRISPR/Cas9-mediated system, we succeeded integrating cathelicidin gene (As-Cath) from an alligator (Alligator sinensis) 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 (KI) efficiency (22.38%, 64/286) but low on-target events was achieved ssODN strategy, whereas adopting dsDNA donor template led efficient KI (10.80%, 23/213). The As-Cath instrumental establishing lh knockout (LH–_As-Cath+) line, which displayed heightened reduced fecundity compared with wild-type (WT) sibling fish. Furthermore, administration human chorionic gonadotropin (HCG) hormone-releasing analogue (LHRHa) can restore reproduction line. Overall, replaced then administered therapy gain complete control disease-resistant environmentally sound manner. This strategy not only effectively improves consumer-valued also guards against unwanted introgression, providing breakthrough aquaculture genetics confine prevent establishment or domestic genotypes natural environment.

Language: Английский

Citations

4
Allogeneic CD19 CAR T cells armed with an anti-rejection CD70 CAR overcome antigen escape and evade alloimmune responses DOI Creative Commons
Elvin J. Lauron, Kristen Zhang, Zhe Li

et al.

Research Square (Research Square), Journal Year: 2025, Volume and Issue: unknown

Published: March 20, 2025

Abstract Allogeneic chimeric antigen receptor (CAR) T cells can achieve sustained clinical benefit in B cell malignancies and autoimmune diseases. Despite the many potential advantages over autologous products, allogeneic CAR carry a higher risk of rejection, which may limit persistence therapeutic efficacy. We report design evaluation an optimized CD70 that prevents rejection by targeting activated alloreactive lymphocytes. Co-expression this with CD19 resulted presence lymphocytes prolonged antitumor activity escape model. In vivo, CD19/CD70 dual resisted eliminated CD70+ from patients systemic lupus erythematosus, lowering immunoglobulin production. An therapy therefore reduce need for lymphodepleting conditioning regimens required prior to infusion.

Language: Английский

Citations

0
Genome Editing in Mouse Embryo Using the CRISPR/Cas12i3 System DOI Open Access
Jiale He,

Juan Liu,

Yuan Yue

et al.

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(7), P. 3036 - 3036

Published: March 26, 2025

The CRISPR/Cas system is a sizable family that currently popular and efficient gene editing tool. Cas12i3, as member of the Type V-I family, has characteristics recognizing T-rich PAM sequences being guided by shorter crRNA higher efficiency than Cas9 in rice. However, potential tool accelerating breeding process, application Cas12i3 mammalian embryos not yet been reported. Our study systematically evaluated feasibility applying CRISPR/Cas12i3 to mouse embryos, with core pluripotency regulator Nanog target. We successfully constructed loss-of-function embryo model using CRISPR/Cas12i3. At targeted locus, its exceeded under matched experimental conditions; no off-target phenomenon was detected. Moreover, exhibited side effect on development proliferation blastocyst cells. Finally, we obtained healthy chimeric gene-edited offspring optimizing concentration mixture. These results confirm safety for mammals, which provides reliable one-step generation animals applications biology, medical research, large livestock breeding.

Language: Английский

Citations

0