scholarly journals Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

Cell Research ◽  
2016 ◽  
Vol 26 (8) ◽  
pp. 901-913 ◽  
Author(s):  
Pu Gao ◽  
Hui Yang ◽  
Kanagalaghatta R Rajashankar ◽  
Zhiwei Huang ◽  
Dinshaw J Patel
Keyword(s):  
Dna Recognition ◽  
Target Dna ◽  
Type V ◽  
Unique Mechanism

scholarly journals Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition

2021 ◽  
Author(s):  
Renjian Xiao ◽  
Shukun Wang ◽  
Ruijie Han ◽  
Zhuang Li ◽  
Clinton Gabel ◽  
...  
Keyword(s):  
Dna Recognition ◽  
Structural Basis ◽  
Structural Elements ◽  
Dna Transposition ◽  
Guide Rna ◽  
Promising Tool ◽  
Target Dna ◽  
Dna Insertion ◽  
Type V

The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryo-EM structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT PAM sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for 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 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.

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.

scholarly journals Structural basis for DNA recognition by the transcription regulator MetR

2016 ◽  
Vol 72 (6) ◽  
pp. 417-426 ◽  
Author(s):  
Avinash S. Punekar ◽  
Jonathan Porter ◽  
Stephen B. Carr ◽  
Simon E. V. Phillips
Keyword(s):  
Molecular Level ◽  
Dna Recognition ◽  
Data Driven ◽  
Structural Basis ◽  
Methionine Biosynthesis ◽  
Dna Complexes ◽  
Winged Helix ◽  
Target Dna ◽  
Operator Sequence ◽  
Dna Complex

MetR, a LysR-type transcriptional regulator (LTTR), has been extensively studied owing to its role in the control of methionine biosynthesis in proteobacteria. A MetR homodimer binds to a 24-base-pair operator region of themetgenes and specifically recognizes the interrupted palindromic sequence 5′-TGAA-N5-TTCA-3′. Mechanistic details underlying the interaction of MetR with its target DNA at the molecular level remain unknown. In this work, the crystal structure of the DNA-binding domain (DBD) of MetR was determined at 2.16 Å resolution. MetR-DBD adopts a winged-helix–turn–helix (wHTH) motif and shares significant fold similarity with the DBD of the LTTR protein BenM. Furthermore, a data-driven macromolecular-docking strategy was used to model the structure of MetR-DBD bound to DNA, which revealed that a bent conformation of DNA is required for the recognition helix α3 and the wing loop of the wHTH motif to interact with the major and minor grooves, respectively. Comparison of the MetR-DBD–DNA complex with the crystal structures of other LTTR-DBD–DNA complexes revealed residues that may confer operator-sequence binding specificity for MetR. Taken together, the results show that MetR-DBD uses a combination of direct base-specific interactions and indirect shape recognition of the promoter to regulate the transcription ofmetgenes.

scholarly journals Key determinants of target DNA recognition by retroviral intasomes

Retrovirology ◽  
2015 ◽  
Vol 12 (1) ◽  
Author(s):  
Erik Serrao ◽  
Allison Ballandras-Colas ◽  
Peter Cherepanov ◽  
Goedele N Maertens ◽  
Alan N Engelman
Keyword(s):  
Dna Recognition ◽  
Target Dna

scholarly journals PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease

Cell ◽  
2016 ◽  
Vol 167 (7) ◽  
pp. 1814-1828.e12 ◽  
Author(s):  
Hui Yang ◽  
Pu Gao ◽  
Kanagalaghatta R. Rajashankar ◽  
Dinshaw J. Patel
Keyword(s):  
Dna Recognition ◽  
Target Dna

scholarly journals Dynamic Conformational Change Regulates the Protein-DNA Recognition: An Investigation on Binding of a Y-Family Polymerase to Its Target DNA

2014 ◽  
Vol 10 (9) ◽  
pp. e1003804 ◽  
Author(s):  
Xiakun Chu ◽  
Fei Liu ◽  
Brian A. Maxwell ◽  
Yong Wang ◽  
Zucai Suo ◽  
...  
Keyword(s):  
Conformational Change ◽  
Dna Recognition ◽  
Target Dna ◽  
Y Family

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 Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xue Huang ◽  
Wei Sun ◽  
Zhi Cheng ◽  
Minxuan Chen ◽  
Xueyan Li ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Metal Ion ◽  
Catalytic Domain ◽  
Dna Recognition ◽  
Structural Basis ◽  
Seed Region ◽  
Dna Complexes ◽  
Metal Ion Catalysis ◽  
Type V ◽  
Cas Proteins

Abstract To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2–crRNA–DNA complexes and a Cas12i2–crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1–8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9–15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.

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