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

Past, present, and future of CRISPR genome editing technologies DOI Creative Commons

Martin Pacesa,

Oana Pelea, Martin Jínek

и другие.

Cell, Год журнала: 2024, Номер 187(5), С. 1076 - 1100

Опубликована: Фев. 1, 2024

Genome editing has been a transformative force in the life sciences and human medicine, offering unprecedented opportunities to dissect complex biological processes treat underlying causes of many genetic diseases. CRISPR-based technologies, with their remarkable efficiency easy programmability, stand at forefront this revolution. In Review, we discuss current state CRISPR gene technologies both research therapy, highlighting limitations that constrain them technological innovations have developed recent years address them. Additionally, examine summarize landscape applications context health therapeutics. Finally, outline potential future developments could shape coming years.

Язык: Английский

Процитировано

146
Plant organellar genomes: much done, much more to do DOI
Jie Wang, Shenglong Kan, Xuezhu Liao

и другие.

Trends in Plant Science, Год журнала: 2024, Номер 29(7), С. 754 - 769

Опубликована: Янв. 19, 2024

Язык: Английский

Процитировано

109
CRISPR technologies for genome, epigenome and transcriptome editing DOI
Lukas Villiger,

Julia Joung,

Luke W. Koblan

и другие.

Nature Reviews Molecular Cell Biology, Год журнала: 2024, Номер 25(6), С. 464 - 487

Опубликована: Фев. 2, 2024

Язык: Английский

Процитировано

75
Improving prime editing with an endogenous small RNA-binding protein DOI Creative Commons
Jun Yan, Paul Oyler, Purnima Ravisankar

и другие.

Nature, Год журнала: 2024, Номер 628(8008), С. 639 - 647

Опубликована: Апрель 3, 2024

Abstract Prime editing enables the precise modification of genomes through reverse transcription template sequences appended to 3′ ends CRISPR–Cas guide RNAs 1 . To identify cellular determinants prime editing, we developed scalable reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as strongest mediator editing: small RNA-binding exonuclease protection La. Further investigation revealed that La promotes across approaches (PE2, PE3, PE4 PE5), edit types (substitutions, insertions deletions), endogenous loci cell but has no consistent effect on genome-editing rely standard, unextended RNAs. Previous work shown binds polyuridine tracts at RNA polymerase III transcripts 2 We found functionally interacts with polyuridylated (pegRNAs). Guided by results, editor protein (PE7) fused RNA-binding, N-terminal domain This improved expressed pegRNAs engineered (epegRNAs), well synthetic optimized for binding. Together, our results provide key insights into how components interact environment suggest general strategies stabilizing exogenous therein.

Язык: Английский

Процитировано

66
Targeted genome-modification tools and their advanced applications in crop breeding DOI
Boshu Li, Chao Sun, Jiayang Li

и другие.

Nature Reviews Genetics, Год журнала: 2024, Номер 25(9), С. 603 - 622

Опубликована: Апрель 24, 2024

Язык: Английский

Процитировано

58
Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing DOI Creative Commons
Smriti Pandey, Xin D. Gao, N Krasnow

и другие.

Nature Biomedical Engineering, Год журнала: 2024, Номер unknown

Опубликована: Июнь 10, 2024

Abstract Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, efficiencies or specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples programmability prime with ability recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 eeBxb1) mediated up 60% donor (3.2-fold wild-type Bxb1) human cell lines pre-installed landing sites. In single-transfection experiments at safe-harbour therapeutically relevant sites, PASSIGE eeBxb1 led an average targeted-gene-integration 23% (4.2-fold Bxb1). Notably, exceeded 30% multiple sites primary fibroblasts. evoBxb1 outperformed PASTE (for ‘programmable addition via targeting elements’, a method uses editors fused recombinases) on by 9.1-fold 16-fold, respectively. continuously evolved is unusually efficient cells.

Язык: Английский

Процитировано

44
Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases DOI
Joana Ferreira da Silva, Connor J. Tou, Emily M. King

и другие.

Nature Biotechnology, Год журнала: 2024, Номер unknown

Опубликована: Июль 22, 2024

Язык: Английский

Процитировано

30
Chromatin context-dependent regulation and epigenetic manipulation of prime editing DOI Creative Commons
Xiaoyi Li, Wei Chen, Beth Martin

и другие.

Cell, Год журнала: 2024, Номер 187(10), С. 2411 - 2427.e25

Опубликована: Апрель 11, 2024

We set out to exhaustively characterize the impact of cis-chromatin environment on prime editing, a precise genome engineering tool. Using highly sensitive method for mapping genomic locations randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% 94% an identical target site and edit. Position effects efficiency are well predicted chromatin marks, e.g., positively H3K79me2 negatively H3K9me3. Next, developed multiplex perturbational framework assess interaction trans-acting factors with outcomes. Applying this DNA repair factors, identify HLTF as context-dependent repressor editing. Finally, several lines evidence suggest that active transcriptional elongation enhances Consistent this, show can robustly decrease or increase preceding it CRISPR-mediated silencing activation, respectively.

Язык: Английский

Процитировано

29
High-throughput evaluation of genetic variants with prime editing sensor libraries DOI Creative Commons
Samuel I. Gould, Alexandra Wuest, Kexin Dong

и другие.

Nature Biotechnology, Год журнала: 2024, Номер unknown

Опубликована: Март 12, 2024

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install evaluate genetic variants, but previous approaches have limited by the variable efficiency prime guide RNAs. Here we present high-throughput sensor strategy couples RNAs with synthetic versions their cognate target sites quantitatively assess functional impact endogenous variants. We screen over 1,000 cancer-associated variants TP53-the most frequently mutated gene in cancer-to identify alleles p53 function mechanistically diverse ways. find certain TP53 particularly those oligomerization domain, display opposite phenotypes exogenous overexpression systems. Our results emphasize physiological importance dosage shaping native stoichiometry protein-protein interactions, establish framework for studying sequence context at scale.

Язык: Английский

Процитировано

27
Continuous evolution of compact protein degradation tags regulated by selective molecular glues DOI
Jaron A. M. Mercer, Stephan J. DeCarlo, Shourya S. Roy Burman

и другие.

Science, Год журнала: 2024, Номер 383(6688)

Опубликована: Март 14, 2024

Conditional protein degradation tags (degrons) are usually >100 amino acids long or triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame SD40 using prime editing degraded otherwise inert PT-179. Cryo-electron microscopy structures ligand-bound revealed mechanistic insights into basis SD40's activity specificity. Our efforts establish system provide ZF overcome shortcomings associated existing degrons.

Язык: Английский

Процитировано

26