MedComm – Biomaterials and Applications, Journal Year: 2024, Volume and Issue: 3(4)
Published: Dec. 1, 2024
Language: Английский
Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)
Published: Jan. 7, 2025
Abstract Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a nuclease (747 amino acids in length) multiple functional states, revealing stepwise process DNA targeting involving conformational switch REC2 domain insertion. Our provide insights into intricately folded guide RNA which acts as structural scaffold to anchor small, flexible protein domains for recognition. The sgRNA can be truncated by up ~25% yet still retain activity vivo. Using ancestral sequence reconstruction, generated capable efficient mammalian cells. Collectively, our results mechanistic evolution and diverse systems, providing blueprint future re-engineering minimal RNA-guided endonucleases.
Language: Английский
Citations
2
Nature Biotechnology, Journal Year: 2024, Volume and Issue: unknown
Published: Oct. 16, 2024
Language: Английский
Citations
11
Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 52(16), P. 9343 - 9359
Published: July 18, 2024
Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing other biotechnology tools. The specificity subject extensive investigation both in vitro experiments. However, studies have often performed at high magnesium ion concentrations that are inconsistent with free Mg2+ would be present cells. By profiling orthologs a range concentrations, we find switches its depending on metal concentration. Lowering concentration decreases cleavage defects caused by seed mismatches, while increasing PAM-distal mismatches. We show can bind mutant targets more rapidly low resulting faster cleavage. In contrast, mismatches cause substantial following formation Cas12a-target complex concentrations. observe differences switching between three results variations routes phage escape from Cas12a-mediated immunity. Overall, our reveal importance physiological conditions Cas effectors used different cellular environments.
Language: Английский
Citations
8
Biotechnology Advances, Journal Year: 2025, Volume and Issue: 81, P. 108561 - 108561
Published: March 12, 2025
Language: Английский
Citations
1
Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 52(15), P. 9014 - 9027
Published: July 12, 2024
Abstract CasX (also known as Cas12e), a Class 2 CRISPR-Cas system, shows promise in genome editing due to its smaller size compared the widely used Cas9 and Cas12a. Although structures of CasX–sgRNA–DNA ternary complexes have been resolved uncover distinctive NTSB domain, dynamic behaviors are not well characterized. In this study, we employed single-molecule biochemical assays investigate conformational dynamics two homologs, DpbCasX PlmCasX, from DNA binding target cleavage fragment release. Our results indicate that cleaves non-target strand sequentially with relative irreversible dynamics. The homologs exhibited different patterns specificities. characterization also reveals PAM-proximal seed region, providing guidance for CasX-based effector design. Further studies elucidate mechanistic basis why modification sgRNA domain can affect activity. Interestingly, has less effective search efficiency than Cas12a, potentially accounting lower efficiency. This observation opens new avenue future protein engineering.
Language: Английский
Citations
5
Transgenic Research, Journal Year: 2024, Volume and Issue: 33(5), P. 323 - 357
Published: Aug. 19, 2024
Language: Английский
Citations
5
Scientia Horticulturae, Journal Year: 2025, Volume and Issue: 341, P. 113957 - 113957
Published: Jan. 16, 2025
Language: Английский
Citations
0
Methods in enzymology on CD-ROM/Methods in enzymology, Journal Year: 2025, Volume and Issue: unknown, P. 117 - 142
Published: Jan. 1, 2025
Language: Английский
Citations
0
Scientific Reports, Journal Year: 2025, Volume and Issue: 15(1)
Published: Feb. 10, 2025
Language: Английский
Citations
0
Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown
Published: March 15, 2025
Clustered regularly interspaced short palindromic repeats-associated (CRISPR/Cas) proteins have been used for a growing class of in vitro molecular diagnostics due to their modularity and high specificity targeting nucleic acid. However, the requirement protospacer adjacent motif (PAM) Cas protein-catalyzed trans-cleavage poses challenge random acid detection. Here, we demonstrate that dithiothreitol (DTT) enables LbCas12a adopt relaxed preference PAM base pairing, thereby expanding target sequence space. Accordingly, propose DTT-mediated CRISPR/Cas12a toolbox (DTT-deCRISPR) exhibits is readily compatible with amplification techniques including recombinase polymerase (RPA) chain reaction (PCR). As proof concept, integrate DTT-deCRISPR frequently PCR sensitively selectively detecting high-risk human papillomavirus (HPV) 16 18. The platform demonstrates ability detect synthesized HPV 18 plasmids down 1 aM within 60 min. Based on receiver operating characteristic curve analysis, clinical sensitivities developed method are 93.75% 80.00%, respectively. We further incorporate it into lateral flow assay (LFA) point-of-care detection, abundances determined by LFA samples consistent fluorescence analysis results. Together, this work uncovers an unexpected connection between DTT preferences LbCas12a, promoting universality flexibility CRISPR technology diagnostics.
Language: Английский
Citations
0