Structural basis for pegRNA-guided reverse transcription by a prime editor DOI Creative Commons
Yutaro Shuto, Ryoya Nakagawa, Shiyou Zhu

и другие.

Nature, Год журнала: 2024, Номер 631(8019), С. 224 - 231

Опубликована: Май 29, 2024

Abstract The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a editing guide RNA (pegRNA) to facilitate wide variety precise genome edits in living cells 1 . However, owing lack structural information, the molecular mechanism pegRNA-guided transcription by remains poorly understood. Here we present cryo-electron microscopy structures SpCas9–M-MLV RTΔRNaseH–pegRNA–target DNA complex multiple states. termination structure, along our functional analysis, reveals that M-MLV RT extends beyond expected site, resulting scaffold-derived incorporations cause undesired at target loci. Furthermore, comparisons among pre-initiation, initiation elongation states show consistent position relative SpCas9 during transcription, whereas pegRNA–synthesized heteroduplex builds up surface SpCas9. On basis insights, rationally pegRNA variants prime-editor which is fused within Collectively, findings provide insights into stepwise editing, will pave way for development versatile toolbox.

Язык: Английский

Recent advances in prime editing technologies and their promises for therapeutic applications DOI Creative Commons
Hongzhi Zeng, T. Daniel,

Ananya Lingineni

и другие.

Current Opinion in Biotechnology, Год журнала: 2024, Номер 86, С. 103071 - 103071

Опубликована: Фев. 7, 2024

Язык: Английский

Процитировано

10
Enhancing prime editor activity by directed protein evolution in yeast DOI Creative Commons
Yanik Weber,

Desirée Böck,

A. Ivascu

и другие.

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Март 7, 2024

Prime editing is a highly versatile genome technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, yeast-based platform for directed protein evolution, enhance efficiency PEs. After several rounds evolution with increased selection pressure, identify multiple mutations have positive effect on PE activity in yeast cells biochemical assays. Combining two most effective - A259D amino acid substitution nCas9 K445T M-MLV RT results variant PE_Y18. Delivery PE_Y18, encoded DNA, mRNA or as ribonucleoprotein complex into mammalian cell lines increases rates up 3.5-fold PEmax. addition, PE_Y18 supports higher when delivered vivo liver brain. Our demonstrates proof-of-concept application OrthoRep optimize tools eukaryotic cells.

Язык: Английский

Процитировано

10
Mutagenesis techniques for evolutionary engineering of microbes – exploiting CRISPR-Cas, oligonucleotides, recombinases, and polymerases DOI Creative Commons
Anna Zimmermann, Julian E. Prieto-Vivas, Karin Voordeckers

и другие.

Trends in Microbiology, Год журнала: 2024, Номер 32(9), С. 884 - 901

Опубликована: Март 15, 2024

The natural process of evolutionary adaptation is often exploited as a powerful tool to obtain microbes with desirable traits. For industrial microbes, engineering used generate variants that show increased yields or resistance stressful environments, thus obtaining superior microbial cell factories. However, even in large populations, the supply beneficial mutations typically low, which implies improved time-consuming and inefficient. To overcome this limitation, different techniques have been developed boost mutation rates. While some these methods simply increase overall rate across genome, others use recent developments DNA synthesis, synthetic biology, CRISPR-Cas control type location mutations. This review summarizes most important field model microorganisms. It discusses how both vitro vivo approaches can genetic diversity host, special emphasis on for optimization metabolic pathways precision fermentation.

Язык: Английский

Процитировано

10
Harnessing the evolving CRISPR/Cas9 for precision oncology DOI Creative Commons
Tianye Li,

Shuiquan Li,

Yue Kang

и другие.

Journal of Translational Medicine, Год журнала: 2024, Номер 22(1)

Опубликована: Авг. 8, 2024

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system, a groundbreaking innovation in genetic engineering, has revolutionized our approach to surmounting complex diseases, culminating CASGEVY™ approved for sickle cell anemia. Derived from microbial immune defense mechanism, CRISPR/Cas9, characterized as precision, maneuverability and universality gene editing, been harnessed versatile tool precisely manipulating DNA mammals. In the process of applying it practice, consecutive exploitation novel orthologs variants never ceases. It's conducive understanding essentialities particularly cancer, which is crucial diagnosis, prevention, treatment. CRISPR/Cas9 used not only investigate tumorous genes functioning but also model disparate cancers, providing valuable insights into tumor biology, resistance, evasion. Upon cancer therapy, instrumental developing individual precise therapies that can selectively activate or deactivate within cells, aiming cripple growth invasion sensitize cells treatments. Furthermore, facilitates development innovative treatments, enhancing targeting efficiency reprogrammed exemplified by advancements CAR-T regimen. Beyond potent screening susceptible genes, offering possibility intervening before initiative progresses. However, despite its vast potential, application research therapy accompanied significant efficacy, efficiency, technical, safety considerations. Escalating technology innovations are warranted address these issues. system revolutionizing treatment, opening up new avenues management cancers. integration this evolving clinical practice promises era precision oncology, with targeted, personalized, potentially curative patients.

Язык: Английский

Процитировано

10
Structural basis for pegRNA-guided reverse transcription by a prime editor DOI Creative Commons
Yutaro Shuto, Ryoya Nakagawa, Shiyou Zhu

и другие.

Nature, Год журнала: 2024, Номер 631(8019), С. 224 - 231

Опубликована: Май 29, 2024

Abstract The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a editing guide RNA (pegRNA) to facilitate wide variety precise genome edits in living cells 1 . However, owing lack structural information, the molecular mechanism pegRNA-guided transcription by remains poorly understood. Here we present cryo-electron microscopy structures SpCas9–M-MLV RTΔRNaseH–pegRNA–target DNA complex multiple states. termination structure, along our functional analysis, reveals that M-MLV RT extends beyond expected site, resulting scaffold-derived incorporations cause undesired at target loci. Furthermore, comparisons among pre-initiation, initiation elongation states show consistent position relative SpCas9 during transcription, whereas pegRNA–synthesized heteroduplex builds up surface SpCas9. On basis insights, rationally pegRNA variants prime-editor which is fused within Collectively, findings provide insights into stepwise editing, will pave way for development versatile toolbox.

Язык: Английский

Процитировано

9