Health research in the era of artificial intelligence: Advances in gene-editing study DOI Creative Commons
Mengyao Dai, Xiangyang Li, Qí Zhāng

et al.

Medicine Plus, Journal Year: 2024, Volume and Issue: 1(2), P. 100027 - 100027

Published: May 2, 2024

In recent years, gene editing technology, represented by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has made a revolutionary breakthrough. Base techniques developed from CRISPR/Cas9 technologies have demonstrated the ability to efficiently achieve single base substitutions at specific genomic sites without double-strand breaks. systems evolved rapidly over past few efficient single-base high-throughput screening, and in vitro vivo applications. particular, screening facilitated functional studies single-nucleotide resolution, offering unprecedented insights into genetic function. Meanwhile, rapid advancement of artificial intelligence (AI) is playing pivotal role assisting genome streamlining data analysis within systems. This review encompasses progress research, focusing on development technologies, exploring editing-based level, underscoring potential synergies between AI studies.

Language: Английский

Phage-assisted evolution and protein engineering yield compact, efficient prime editors DOI Creative Commons
Jordan L. Doman, Smriti Pandey, Monica E. Neugebauer

et al.

Cell, Journal Year: 2023, Volume and Issue: 186(18), P. 3983 - 4002.e26

Published: Aug. 1, 2023

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size improved efficiency. Using phage-assisted evolution, efficiencies compact reverse transcriptases by up 22-fold generated that are 516–810 base pairs smaller than the current-generation editor PEmax. We discovered different specialize types used this insight outperform PEmax PEmaxΔRNaseH, truncated dual-AAV delivery systems. Finally, Cas9 domains improve editing. These resulting (PE6a-g) enhance therapeutically relevant patient-derived fibroblasts primary human T-cells. PE6 variants also enable longer insertions be installed vivo following delivery, achieving 40% loxP insertion cortex murine brain, 24-fold improvement compared previous state-of-the-art editors.

Language: Английский

Citations

136
CRISPR technologies for genome, epigenome and transcriptome editing DOI
Lukas Villiger,

Julia Joung,

Luke W. Koblan

et al.

Nature Reviews Molecular Cell Biology, Journal Year: 2024, Volume and Issue: 25(6), P. 464 - 487

Published: Feb. 2, 2024

Language: Английский

Citations

75
Targeted genome-modification tools and their advanced applications in crop breeding DOI
Boshu Li, Chao Sun, Jiayang Li

et al.

Nature Reviews Genetics, Journal Year: 2024, Volume and Issue: 25(9), P. 603 - 622

Published: April 24, 2024

Language: Английский

Citations

58
Chromatin context-dependent regulation and epigenetic manipulation of prime editing DOI Creative Commons
Xiaoyi Li, Wei Chen, Beth Martin

et al.

Cell, Journal Year: 2024, Volume and Issue: 187(10), P. 2411 - 2427.e25

Published: April 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.

Language: Английский

Citations

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

et al.

Nature Biotechnology, Journal Year: 2024, Volume and Issue: unknown

Published: March 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.

Language: Английский

Citations

27
Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells DOI Creative Commons

Alexander A. Sousa,

Colin Hemez, Lei Lei

et al.

Nature Biomedical Engineering, Journal Year: 2024, Volume and Issue: unknown

Published: July 10, 2024

Abstract Prime editing (PE) enables precise and versatile genome without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) F508del, a three-nucleotide deletion that is predominant cause CF. By combining six efficiency optimizations for PE—engineered guide RNAs, PEmax architecture, transient expression dominant-negative mismatch repair protein, strategic silent edits, PE6 variants proximal ‘dead’ single-guide RNAs—we increased correction efficiencies F508del from less than 0.5% in HEK293T cells 58% immortalized bronchial epithelial (a 140-fold improvement) 25% patient-derived airway cells. The also resulted minimal off-target editing, edit-to-indel ratios 3.5-fold greater those achieved by nuclease-mediated homology-directed repair, functional restoration ion channels over 50% wild-type levels (similar via combination treatment with elexacaftor, tezacaftor ivacaftor) primary Our findings support feasibility durable one-time

Language: Английский

Citations

23
Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells DOI Creative Commons
Mattijs Bulcaen,

Phéline Kortleven,

Ronald B. Liu

et al.

Cell Reports Medicine, Journal Year: 2024, Volume and Issue: 5(5), P. 101544 - 101544

Published: May 1, 2024

Prime editing is a recent, CRISPR-derived genome technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime approaches to correct L227R- N1303K-CFTR, two mutations that cause cystic fibrosis are not eligible for current market-approved modulator therapies. We show that, upon DNA correction the CFTR gene, complex glycosylation, localization, and, most importantly, function protein restored in HEK293T 16HBE cell lines. These findings were subsequently validated patient-derived rectal organoids human nasal epithelial cells. Through analysis predicted experimentally identified candidate off-target sites primary stem cells, confirm previous reports on high editor (PE) specificity its potential curative CF gene therapy. To facilitate future screening genetic strategies translational model, machine learning algorithm was developed dynamic quantification (DETECTOR: "detection targeted organoids").

Language: Английский

Citations

21
CRISPR–Cas applications in agriculture and plant research DOI
Aytug Tuncel, Changtian Pan,

Joshua S. Clem

et al.

Nature Reviews Molecular Cell Biology, Journal Year: 2025, Volume and Issue: unknown

Published: March 7, 2025

Language: Английский

Citations

3
Randomizing the human genome by engineering recombination between repeat elements DOI
Jonas Koeppel, Raphaël Ferreira,

Thomas Vanderstichele

et al.

Science, Journal Year: 2025, Volume and Issue: 387(6733)

Published: Jan. 30, 2025

We lack tools to edit DNA sequences at scales necessary study 99% of the human genome that is noncoding. To address this gap, we applied CRISPR prime editing insert recombination handles into repetitive sequences, up 1697 per cell line, which enables generating large-scale deletions, inversions, translocations, and circular DNA. Recombinase induction produced more than 100 stochastic megabase-sized rearrangements in each cell. tracked these over time measure selection pressures, finding a preference for shorter variants avoided essential genes. characterized 29 clones with multiple rearrangements, an impact deletions on expression genes variant but not nearby This genome-scrambling strategy large sequence relocations, insertion regulatory elements explore dispensability organization.

Language: Английский

Citations

2
High-throughput PRIME-editing screens identify functional DNA variants in the human genome DOI Creative Commons

Xingjie Ren,

Han Yang, Jovia L. Nierenberg

et al.

Molecular Cell, Journal Year: 2023, Volume and Issue: 83(24), P. 4633 - 4645.e9

Published: Dec. 1, 2023

Despite tremendous progress in detecting DNA variants associated with human disease, interpreting their functional impact a high-throughput and single-base resolution manner remains challenging. Here, we develop pooled prime-editing screen method, PRIME, that can be applied to characterize thousands of coding non-coding single experiment high reproducibility. To showcase its applications, first identified essential nucleotides for 716 bp MYC enhancer via PRIME-mediated analysis. Next, PRIME functionally 1,304 genome-wide association study (GWAS)-identified breast cancer 3,699 from ClinVar. We discovered 103 156 uncertain significance are affecting cell fitness. Collectively, demonstrate is capable characterizing genetic at scale, advancing accurate genome annotation disease risk prediction, diagnosis, therapeutic target identification.

Language: Английский

Citations

32