Sensors and Actuators B Chemical, Journal Year: 2024, Volume and Issue: unknown, P. 137001 - 137001
Published: Nov. 1, 2024
Language: Английский
Cell, Journal Year: 2024, Volume and Issue: 187(13), P. 3249 - 3261.e14
Published: May 22, 2024
Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations the wedge (WED) domain that produce >100-fold-higher levels. Cryoelectron microscopy (cryo-EM) structures wild-type improved (iGeoCas9) reveal contacts between WED iGeoCas9 DNA substrates. Biochemical analysis shows accelerates unwinding capture substrates under magnesium-restricted conditions typical mammalian but not bacterial These findings enabled rational engineering other orthologs enhance levels, pointing a general strategy for editing enzyme improvement. Together, these results uncover new role demonstrate how accelerated target dramatically improves Cas9-induced activity.
Language: Английский
Citations
23
Molecular Cell, Journal Year: 2024, Volume and Issue: unknown
Published: July 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.
Language: Английский
Citations
11
bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown
Published: Jan. 10, 2025
ABSTRACT CRISPR-Cas12a is widely used for genome editing and biomarker detection since it can create targeted double-stranded DNA breaks promote non-specific cleavage after identifying specific DNA. To mitigate the off-target of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12a KD2P ) by introducing double proline substitutions (K969P/D970P) in conserved helix called bridge (BH). In this work, cryogenic electron microscopy (cryoEM) to understand molecular mechanisms BH- mediated activation Cas12a. We captured five structures FnoCas12a at different states conformational activation. Comparison with wild-type WT unravels mechanism where BH acts as trigger that allosterically activates REC lobe movements tracking number base pairs growing RNA-DNA hybrid undergo loop-to-helical transition bending latch onto hybrid. The coupled reported loop-to-helix “lid”, essential opening RuvC endonuclease, through direct interactions residues lid. also observe structural details cooperativity “helix-1” activation, proposed interaction. Overall, our study enables development high-fidelity Cas9 variants BH-modifications.
Language: Английский
Citations
0
Nucleic Acids Research, Journal Year: 2025, Volume and Issue: 53(3)
Published: Jan. 24, 2025
Abstract CRISPR-Cas12a technology has transformative potential, but as its applications grow, enhancing inherent functionalities is essential to meet diverse demands. Here, we reveal a regulatory mechanism for LbCas12a through direct repeat (DR) region 3′ end modifications and de-modifications, which can regulate LbCas12a’s cis- trans-cleavage activities. We extensively explored the effects of introducing phosphorylation, DNA, photo-cleavable linker, DNA at DR on functionality. find that temporary inhibitory function Cas12a be reactivated by modification corresponding substances, such alkaline phosphatase (ALP), immunoglobulin G (IgG), alpha-fetoprotein (AFP), exonucleases, ultraviolet radiation, glycosylases, greatly expand scope application Cas12a. Clinical demonstrated promising results in ALP, AFP, trace Epstein–Barr virus detection compared gold standard methods. Our research provides valuable insights into regulating activity significantly expands potential clinical targets, paving way future universal clustered regularly interspaced short palindromic repeats (CRISPR) diagnostic strategies.
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
Journal of Molecular Biology, Journal Year: 2024, Volume and Issue: 436(10), P. 168550 - 168550
Published: April 3, 2024
Language: Английский
Citations
1
bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown
Published: Sept. 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
Language: Английский
Citations
1
Sensors and Actuators B Chemical, Journal Year: 2024, Volume and Issue: unknown, P. 137001 - 137001
Published: Nov. 1, 2024
Language: Английский
Citations
0