scholarly journals Mechanistic Insights into theCis-andTrans-acting Deoxyribonuclease Activities of Cas12a

2018 ◽  
Author(s):  
Daan C. Swarts ◽  
Martin Jinek
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Cleavage Product ◽  
List Type ◽  
Francisella Novicida ◽  
Ssdna Binding ◽  
Double Stranded Dna ◽  
Target Dna ◽  
Dna Strands ◽  
Dna Strand

HIGHLIGHTSTarget ssDNA binding allosterically induces unblocking of the RuvC active sitePAM binding facilitates unwinding of dsDNA targetsNon-target DNA strand cleavage is prerequisite for target DNA strand cleavageAfter DNA cleavage, Cas12a releases the PAM-distal DNA productSUMMARYCRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA incisand non-target single stranded DNAs (ssDNA) intrans.To elucidate the molecular basis for both deoxyribonuclease cleavage modes, we performed structural and biochemical studies onFrancisella novicidaCas12a. We show how crRNA-target DNA strand hybridization conformationally activates Cas12a, triggering itstrans-acting, non-specific, single-stranded deoxyribonuclease activity. In turn,cis-cleavage of double-stranded DNA targets is a result of PAM-dependent DNA duplex unwinding and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent,trans-active state. Together, these results provide a revised model for the molecular mechanism of Cas12a enzymes that explains theircis- andtrans-acting deoxyribonuclease activities, and additionally contribute to improving Cas12a-based genome editing.

scholarly journals Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bo Zhang ◽  
Diyin Luo ◽  
Yu Li ◽  
Vanja Perčulija ◽  
Jing Chen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Binary Complex ◽  
Loop Formation ◽  
Dna Duplex ◽  
Target Dna ◽  
Before And After ◽  
Dna Strand ◽  
Type V ◽  
Functionally Diverse

AbstractCas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.

scholarly journals Structure of the Cpf1 endonuclease R-Loop complex after target DNA cleavage

2017 ◽  
Author(s):  
Stefano Stella ◽  
Pablo Alcón ◽  
Guillermo Montoya
Keyword(s):  
Dna Cleavage ◽  
Longitudinal Axis ◽  
Modification System ◽  
Francisella Novicida ◽  
Helix Loop Helix ◽  
Phosphate Backbone ◽  
Target Dna ◽  
Catalytically Active ◽  
Dna Strand ◽  
Type V

AbstractCpf1 is a single RNA-guided endonuclease of class 2 type V CRISPR-Cas system, emerging as a powerful genome editing tool 1,2. To provide insight into its DNA targeting mechanism, we have determined the crystal structure of Francisella novicida Cpf1 (FnCpf1) in complex with the triple strand R-loop formed after target DNA cleavage. The structure reveals a unique machinery for target DNA unwinding to form a crRNA-DNA hybrid and a displaced DNA strand inside FnCpf1. The protospacer adjacent motif (PAM) is recognised by the PAM interacting (PI) domain. In this domain, the conserved K667, K671 and K677 are arranged in a dentate manner in a loop-lysine helix-loop motif (LKL). The helix is inserted at a 45° angle to the dsDNA longitudinal axis. Unzipping of the dsDNA in a cleft arranged by acidic and hydrophobic residues facilitates the hybridization of the target DNA strand with crRNA. K667 initiates unwinding by pushing away the guanine after the PAM sequence of the dsDNA. The PAM ssDNA is funnelled towards the nuclease site, which is located 70 Å away, through a hydrophobic protein cavity with basic patches that interact with the phosphate backbone. In this catalytically active conformation the PI and the helix-loop-helix (HLH) motif in the REC1 domain adopt a “rail shape” and “flap-on” conformations, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms a “septum” that separates the displaced PAM strand and the crRNA-DNA hybrid, avoiding re-annealing of the DNA. Mutations in key residues reveal a novel mechanism to determine the DNA product length, thereby linking the PAM and DNAase sites. Our study reveals a singular working model of RNA-guided DNA cleavage by Cpf1, opening up new avenues for engineering this genome modification system2-4.

scholarly journals Programmable Cleavage of Double-stranded DNA by Combined Action of Argonaute CbAgo from Clostridium butyricum and Nuclease Deficient RecBC Helicase from E.coli

2021 ◽  
Author(s):  
Rita Vaiskunaite ◽  
Jogirdas Vainauskas ◽  
Janna Morris ◽  
Vladimir Potapov ◽  
Jurate Bitinaite
Keyword(s):  
Dna Cleavage ◽  
Dna Helicase ◽  
Clostridium Butyricum ◽  
Dna Targeting ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
Linear Dsdna ◽  
Combined Action ◽  
Dna Strand

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Discovery of mesophilic Argonautes active at physiological temperature places pAgos closer to their possible application for genome editing as a simpler alternative to CRISPR/Cas nucleases. Currently, the use of mesophilic pAgos as programmable DNA endonucleases is hampered by their poor action on double-stranded DNA (dsDNA), mainly due to their inability to invade the DNA duplex. The present study demonstrates that efficient in vitro cleavage of double-stranded DNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecBexo-C). Properties of CbAgo and characteristics of simultaneous cleavage of complementary DNA strands in concurrence with DNA strand unwinding by RecBexo-C were thoroughly explored using 0.3-25 kb DNA substrates. When combined with RecBexo-C helicase, CbAgo was capable of cleaving target sequences located 11-12.5 kb from the ends of linear dsDNA at 37 C. Our study demonstrates that CbAgo with RecBexo-C can be programmed to generate dsDNA fragments flanked with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. At present, the combination of CbAgo and RecBexo-C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of dsDNA at any desired location.

scholarly journals Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase

2017 ◽  
Vol 114 (22) ◽  
pp. E4492-E4500 ◽  
Author(s):  
Pan F. Chan ◽  
Thomas Germe ◽  
Benjamin D. Bax ◽  
Jianzhong Huang ◽  
Reema K. Thalji ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Dna Gyrase ◽  
Dna Breaks ◽  
Antibiotic Development ◽  
X Ray Crystallography ◽  
Dna Mutations ◽  
Target Dna ◽  
Antibiotic Drug ◽  
Dna Strands ◽  
Dna Strand

A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase–DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.

scholarly journals Staring at the Naked Goddess: Unraveling the Structure and Reactivity of Artemis Endonuclease Interacting with a DNA Double Strand

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3986
Author(s):  
Cécilia Hognon ◽  
Antonio Monari
Keyword(s):  
Dna Cleavage ◽  
Catalytic Mechanism ◽  
Dna Lesions ◽  
Cleavage Reaction ◽  
Dynamic Simulations ◽  
Important Target ◽  
System Adaptation ◽  
Dna Strands ◽  
Dna Strand ◽  
Dna Substrate

Artemis is an endonuclease responsible for breaking hairpin DNA strands during immune system adaptation and maturation as well as the processing of potentially toxic DNA lesions. Thus, Artemis may be an important target in the development of anticancer therapy, both for the sensitization of radiotherapy and for immunotherapy. Despite its importance, its structure has been resolved only recently, and important questions concerning the arrangement of its active center, the interaction with the DNA substrate, and the catalytic mechanism remain unanswered. In this contribution, by performing extensive molecular dynamic simulations, both classically and at the hybrid quantum mechanics/molecular mechanics level, we evidenced the stable interaction modes of Artemis with a model DNA strand. We also analyzed the catalytic cycle providing the free energy profile and key transition states for the DNA cleavage reaction.

scholarly journals Efficient target cleavage by Type V Cas12a effector programmed with split CRISPR RNA

2020 ◽  
Author(s):  
Regina Tkach ◽  
Natalia Nikitchina ◽  
Nikita Shebanov ◽  
Vladimir Mekler ◽  
Egor Ulashchik ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Target Recognition ◽  
Human Cells ◽  
Stable Complex ◽  
Slight Effect ◽  
Target Dna ◽  
Type V

ABSTRACTCRISPR RNAs (crRNAs) directing target DNA cleavage by type V-A Cas12a nucleases consist of repeat-derived 5’-scaffold moiety and 3’-spacer moiety. We demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by Cas12a ortholog from Acidaminococcus sp (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer part only, while crRNAs split into two individual moieties (scaffold and spacer RNAs) catalyzed highly specific and efficient cleavage of target DNA. Our data also indicate that AsCas12a combined with split crRNA forms a stable complex with the target. These observations were also confirmed in lysates of human cells expressing AsCas12a. The ability of the AsCas12a nuclease to be programmed with split crRNAs opens new lines of inquiry into the mechanisms of target recognition and cleavage and will further facilitate genome editing techniques based on Cas12a nucleases.

scholarly journals Mechanism of R-loop formation and conformational activation of Cas9

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Martin Jinek
Keyword(s):  
Dna Cleavage ◽  
Structural Basis ◽  
Seed Region ◽  
Loop Formation ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strand ◽  
Dna Substrate

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease

2020 ◽  
Author(s):  
Renjian Xiao ◽  
Zhuang Li ◽  
Shukun Wang ◽  
Ruijie Han ◽  
Leifu Chang
Keyword(s):  
Genome Editing ◽  
Conformational Changes ◽  
Dna Cleavage ◽  
Substrate Recognition ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Target Dna ◽  
Type V ◽  
Underlying Substrate

ABSTRACTCas12f, also known as Cas14, is an exceptionally small type V-F CRISPR-Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 Å and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR-Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.

Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination

2003 ◽  
Vol 36 (4) ◽  
pp. 429-453 ◽  
Author(s):  
Chantal Prévost ◽  
Masayuki Takahashi
Keyword(s):  
Homologous Recombination ◽  
Structural Information ◽  
Double Helix ◽  
Genetic Material ◽  
Strand Exchange ◽  
Double Stranded Dna ◽  
Dna Deformation ◽  
Architectural Proteins ◽  
Dna Strands ◽  
Dna Strand

1. Introduction 4302. Transformations of the RecA filament 4312.1 The different forms of the RecA filament 4312.2 Orientation and position of the RecA monomers in the active filament 4332.3 Transmission of structural information along the filament 4333. RecA-induced DNA deformations 4353.1 Characteristics of RecA-bound DNA 4353.2 Stretching properties of double-stranded DNA 4363.3 DNA bound to architectural proteins 4373.4 Implications for RecA-induced DNA deformations 4383.5 Axial distribution of the DNA stretching deformation 4384. Contacts between RecA and the DNA strands 4404.1 The DNA-binding sites 4404.2 Possible arrangement of loops L1 and L2 and the three bound strands of DNA 4425. Strand arrangement during pairing reorganization 4445.1 Hypotheses for DNA strand association 4445.2 Association via major or minor grooves 4465.3 Post-strand exchange geometries 4466. Conclusion 4477. Acknowledgments 4488. References 448Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.

scholarly journals Phosphorothioate-modified DNA oligonucleotides inactivate CRISPR-Cpf1 mediated genome editing

2018 ◽  
Author(s):  
Bin Li ◽  
Chunxi Zeng ◽  
Wenqing Li ◽  
Xinfu Zhang ◽  
Xiao Luo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Adaptive Immune System ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna ◽  
Modified Dna ◽  
Target Dna ◽  
Editing Activity ◽  
Dose Dependent

CRISPR-Cpf1, a microbial adaptive immune system discovered from Prevotella and Francisella 1, employs a single-stranded CRISPR RNA (crRNA) to induce double stranded DNA breaks1. To modulate genome editing activity of Cpf1 in human cells, we designed a series of crRNA variants including DNA-crRNA and RNA-crRNA duplexes, and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1 mediated gene editing. More importantly, without prehybridization, this PS-modified DNA was able to regulate Cpf1 activity in a time-and dose-dependent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder the binding between Cpf1-crRNA complex and target DNA substrate. Consequently, phosphorothioate-modified DNA oligonucleotides provide a tunable platform to inactivate Cpf1 mediated genome editing.

Sign in / Sign up

Export Citation Format

Share Document