MedComm – Biomaterials and Applications, Год журнала: 2024, Номер 3(4)
Опубликована: Дек. 1, 2024
Язык: Английский
Опубликована: Март 27, 2025
The intuitive manipulation of specific amino acids to alter the activity or specificity CRISPR-Cas9 has been a topic great interest. As large multi-domain RNA-guided endonuclease, intricate molecular crosstalk within Cas9 protein hinges on its conformational dynamics, but comprehensive understanding extent and timescale motions that drive allosteric function association with nucleic remains elusive. Here, we investigated structure multi-timescale recognition (Rec) lobe Geo Cas9, thermophilic from Geobacillus stearothermophilus. Our results provide new atomic details about Rec subdomains ( Rec1, Rec2) full-length domain in solution. Two rationally designed mutants, K267E R332A, enhanced redistributed micro-millisecond flexibility throughout Rec, NMR studies interaction between guide RNA showed mutations reduced this affinity stability ribonucleoprotein complex.Despite measured biophysical differences due mutations, DNA cleavage assays reveal no functional on-target activity, similar specificity. These data suggest interactions can be tuned at level absence major losses, also raise questions underlying mechanism since analogous single-point have significantly impacted on- off-target editing mesophilic S. pyogenes Cas9. A K267E/R332A double mutant did not enhance specificity, highlighting robust tolerance species-dependent complexity across paralogs. Ultimately, work provides an avenue by which modulate structure, motion, setting stage for future variants their effect mechanism.
Язык: Английский
Процитировано
0
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown
Опубликована: Март 26, 2025
CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer-adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability only ∼1% of typical genome. To mitigate this constraint, we used bacterial-based directed evolution assay combined rational engineering identify variants Lachnospiraceae bacterium Cas12a (LbCas12a) expanded PAM recognition. The resulting use range non-canonical PAMs while retaining recognition the canonical PAM. In particular, biochemical cell-based assays show that variant Flex-Cas12a utilizes 5'-NYHV-3' expand sites ∼25% With enhanced versatility, unlocks access previously inaccessible genomic loci, providing new opportunities for both therapeutic agricultural genome engineering.
Язык: Английский
Процитировано
0
The CRISPR Journal, Год журнала: 2025, Номер unknown
Опубликована: Март 27, 2025
Design of guide RNA (gRNA) with high efficiency and specificity is vital for successful application the CRISPR gene editing technology. Although many machine learning (ML) deep (DL)-based tools have been developed to predict gRNA activities, a systematic unbiased evaluation their predictive performance still needed. Here, we provide brief overview in silico design assess datasets statistical metrics used evaluating model performance. We benchmark seven ML DL-based CRISPR-Cas9 prediction across nine covering six cell types three species. The DL models CRISPRon DeepHF outperform other exhibiting greater accuracy higher Spearman correlation coefficient multiple datasets. compile all into GuideNet resource web portal, aiming facilitate streamline sharing Furthermore, summarize features affecting activity, providing important insights further development more accurate models.
Язык: Английский
Процитировано
0
Advanced Healthcare Materials, Год журнала: 2025, Номер unknown
Опубликована: Апрель 10, 2025
Abstract DNA hydrogels have emerged as promising materials in tissue engineering due to their biocompatibility, programmability, and responsiveness stimuli. Synthesized through physical chemical crosslinking, these can be categorized into functionalized types, such those based on aptamers, stimuli‐responsive types that react pH, temperature, light. This review highlights applications engineering, including drug delivery, cell culture, biosensing, gene editing. encapsulate therapeutic agents, support growth, respond dynamically environmental changes, making them ideal for engineering. A comprehensive bibliometric analysis is included, identifying key research trends emerging areas of interest hydrogel design, synthesis, biomedical applications. The provides a deeper understanding the field's development future directions. Challenges mechanical strength, stability, biosafety persist, but integration AI design shows promise advancing functionality clinical
Язык: Английский
Процитировано
0
MedComm, Год журнала: 2024, Номер 5(7)
Опубликована: Июль 1, 2024
The development of gene editing tools has been a significant area research in the life sciences for nearly 30 years. These have widely utilized disease detection and mechanism research. In new century, they shown potential addressing various scientific challenges saving lives through therapies, particularly combating cardiovascular (CVD). rapid advancement therapies provided optimism CVD patients. progress therapy CVDs is comprehensive reflection practical implementation technology both clinical basic settings, as well steady treatment CVDs. This article provides an overview commonly DNA-targeted developed thus far, with specific focus on application these tools, clustered regularly interspaced short palindromic repeat/CRISPR-associated genes (Cas) (CRISPR/Cas) system, therapy. It also delves into limitations current while summarizing ongoing trials related to CVD. aim facilitate further exploration by relevant researchers successful applications field
Язык: Английский
Процитировано
2
ACS Catalysis, Год журнала: 2024, Номер 14(17), С. 13227 - 13235
Опубликована: Авг. 20, 2024
CRISPR-Cas9 has been developed as a powerful gene editing tool, but the mechanism that governs its intricate catalytic process remains incompletely resolved. Here, cryo-electron microscopy structures of thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) in complex with sgRNA and target DNA are reported. The structure GeoCas9 reveals slit termed an L1 crevice comprising HNH, RuvC, helix transient storage site 5′ spacer fragment sgRNA. When loosely bound segment is extracted to pair DNA, collapses trigger subsequent HNH domain translocation. In addition, structural biochemical analyses suggest resilience at elevated temperatures related unique PI conformation higher proline content WED domain. These results advance our understanding unveil molecular accounts for superior thermal profile GeoCas9.
Язык: Английский
Процитировано
1
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown
Опубликована: Сен. 5, 2024
Cas endonucleases, like Cas9 and Cas12a, are RNA-guided immune effectors that provide bacterial defense against bacteriophages. endonucleases rely on divalent metal ions for their enzymatic activities to facilitate conformational changes required specific recognition cleavage of target DNA. While typically produce double-strand breaks (DSBs) in DNA targets, reduced, physiologically relevant Mg
Язык: Английский
Процитировано
1
International Journal of Molecular Sciences, Год журнала: 2024, Номер 25(23), С. 12686 - 12686
Опубликована: Ноя. 26, 2024
CRISPR-Cas system, a natural acquired immune system in prokaryotes that defends against exogenous DNA invasion because of its simple structure and easy operation, has been widely used many research fields such as synthetic biology, crop genetics breeding, precision medicine, so on. The miniature CRISPR-Cas12 an emerging genome editing tool recent years. Compared to the commonly CRISPR-Cas9 CRISPR-Cas12a, unique advantages, rich PAM sites, higher specificity, smaller volume, cytotoxicity. However, application Cas12 proteins methods improve efficiency have not systematically summarized. In this review, we introduce classification summarize structural characteristics type V cleavage mechanism five proteins. gene animals, plants, microorganisms is summarized, strategies are discussed, aiming provide reference for further understanding functional engineering modification system.
Язык: Английский
Процитировано
1
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown
Опубликована: Ноя. 26, 2024
Abstract While the most widely used CRISPR-Cas enzyme is S. pyogenes Cas9 endonuclease (Cas9), it exhibits single-turnover kinetics which leads to long residence times on product DNA. This blocks access DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Although can eventually be forcibly removed by extrinsic factors (translocating polymerases, helicases, chromatin modifying complexes, etc), mechanisms contributing dissociation following cleavage remain poorly understood. Interestingly, it’s been shown that more easily dislodged when complexes collide with PAM-distal region of complex or strength interactions in this are weakened. Here, we employ truncated guide RNAs strategy weaken nucleic acid still support activity. We find truncation promotes much faster turnover capture previously uncharacterized reaction states. Kinetics-guided cryo-EM enabled us enrich for rare, transient states often difficult standard workflows. From single dataset, examine entire conformational landscape multi-turnover Cas9, including first detailed snapshots dissociating from discovered while dissociates cleavage, tight binding PAM-proximal directly inhibits re-binding new targets. Our work provides direct evidence why will future engineering efforts.
Язык: Английский
Процитировано
1
Molecular Cell, Год журнала: 2024, Номер 84(12), С. 2221 - 2222
Опубликована: Июнь 1, 2024
Язык: Английский
Процитировано
0