International Journal of Biological Macromolecules, Год журнала: 2025, Номер unknown, С. 142992 - 142992
Опубликована: Апрель 1, 2025
Язык: Английский
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
126
Nature Communications, Год журнала: 2023, Номер 14(1)
Опубликована: Сен. 5, 2023
Abstract Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools identify molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage the presence split activators. Specifically, PAM-distal region crRNA recognizes RNA targets provided PAM-proximal seed target. Our method, Split Activator for Highly Accessible Analysis (SAHARA), detects picomolar concentrations without sample amplification, reverse-transcription, or strand-displacement by simply supplying short sequence complementary region. Beyond detection, SAHARA outperforms wild-type CRISPR-Cas12a specificity towards point-mutations can detect pooled crRNA/Cas12a arrays via distinct DNAs. In conclusion, simple, yet powerful nucleic acid detection platform based on be applied multiplexed fashion potentially expanded other CRISPR-Cas enzymes.
Язык: Английский
Процитировано
85
Nature, Год журнала: 2023, Номер 613(7944), С. 588 - 594
Опубликована: Янв. 4, 2023
Abstract Bacterial abortive-infection systems limit the spread of foreign invaders by shutting down or killing infected cells before can replicate 1,2 . Several RNA-targeting CRISPR–Cas (that is, types III and VI) cause phenotypes activating indiscriminate nucleases 3–5 However, a CRISPR-mediated abortive mechanism that leverages DNase activity an RNA-guided single-effector nuclease has yet to be observed. Here we report RNA targeting type V Cas12a2 drives infection through non-specific cleavage double-stranded DNA (dsDNA). After recognizing target with protospacer-flanking sequence, efficiently degrades single-stranded (ssRNA), (ssDNA) dsDNA. Within cells, activation induces SOS DNA-damage response impairs growth, preventing dissemination invader. Finally, harnessed collateral for direct detection, demonstrating repurposed as tool. These findings expand known defensive abilities create additional opportunities CRISPR technologies.
Язык: Английский
Процитировано
45
Nature Communications, Год журнала: 2024, Номер 15(1)
Опубликована: Фев. 17, 2024
Abstract CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by sequential mechanism in which initial cleavage of the non-target strand followed target cleavage. How spatially distant traverses toward catalytic core presently not understood. Here, continuous tens microsecond-long molecular dynamics and free-energy simulations reveal an α-helical lid, located within domain, plays pivotal role traversal anchoring crRNA:target duplex guiding core, as also corroborated experiments. In this mechanism, REC2 pushes enzyme, while Nuc aids bending accommodation inward. Understanding critical process underlying Cas12a activity will enrich fundamental knowledge facilitate further engineering strategies for genome editing.
Язык: Английский
Процитировано
30
Angewandte Chemie International Edition, Год журнала: 2024, Номер 63(20)
Опубликована: Март 22, 2024
Abstract The CRISPR‐Cas12a system has emerged as a powerful tool for next‐generation nucleic acid‐based molecular diagnostics. However, it long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA‐enabled trans‐ cleavage activity of AsCas12a and LbCas12a discover that they can directly activated by full‐size RNA targets, although exhibits weaker than both single‐stranded substrates. Remarkably, find RNA‐activated Cas12a possesses higher specificity in recognizing mutated target sequences compared activation. Based these findings, develop “ U niversal N uclease I dentification V irus E mpowered R NA‐ Se nsing” (UNIVERSE) assay acid testing. We incorporate T7 transcription step into this assay, thereby eliminating requirement protospacer adjacent motif (PAM) sequence target. Additionally, successfully detect multiple PAM‐less targets HIV clinical samples are undetectable conventional based double‐stranded activation, demonstrating unrestricted selection with UNIVERSE assay. further validate utility testing HPV 16 samples. envision targeting capability may bring paradigm shift Cas12a‐based detection enhance understanding CRISPR‐Cas biochemistry.
Язык: Английский
Процитировано
26
Journal of the American Chemical Society, Год журнала: 2024, Номер 146(39), С. 26657 - 26666
Опубликована: Авг. 26, 2024
Active clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) systems possess both
Язык: Английский
Процитировано
18
Nature, Год журнала: 2024, Номер 630(8018), С. 961 - 967
Опубликована: Май 13, 2024
Язык: Английский
Процитировано
14
Biotechnology Advances, Год журнала: 2024, Номер 74, С. 108382 - 108382
Опубликована: Май 25, 2024
Язык: Английский
Процитировано
14
Molecular Cell, Год журнала: 2024, Номер unknown
Опубликована: Июль 1, 2024
The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. intermediates DNA delivery into RuvC active site. Stages formation-starting from 5-bp seed-are marked by distinct REC domain arrangements. Dramatic flexibility limits contacts until nearly complete formation, when non-target strand is pulled across coordinated docking promotes efficient cleavage. Next, substantial movements enable repositioning Between cleavage events, lid conformationally resets occlude site, requiring re-activation. These snapshots build structural model depicting targeting that rationalizes observed specificity highlights mechanistic comparisons other class 2 effectors.
Язык: Английский
Процитировано
11
ACS Sensors, Год журнала: 2024, Номер 9(4), С. 2194 - 2202
Опубликована: Апрель 15, 2024
Breast cancer is one of the most diagnosed cancers worldwide. Precise diagnosis and subtyping have important significance for targeted therapy prognosis prediction breast cancer. Herein, we design a proximity-guaranteed DNA machine accurate identification extracellular vesicles (EVs), which beneficial to explore subtype features In our design, two proximity probes are located close on same EV through specific recognition coexisting surface biomarkers, thus being ligated with help click chemistry. Then, product initiates operation involving catalytic hairpin assembly clusters regularly interspaced short palindromic repeats (CRISPR)-Cas12a-mediated trans-cleavage, finally generates significant response that enables EVs expressing both biomarkers. Principle-of-proof studies performed using derived from cell line BT474 as models, confirming high sensitivity specificity machine. When further applied clinical samples, shown be capable not only distinguishing patients special subtypes but also realizing tumor staging regarding disease progression. Therefore, work may provide new insights into subtype-based well more potential therapeutic targets in future.
Язык: Английский
Процитировано
10