scholarly journals Structural basis for target site selection in RNA-guided DNA transposition systems

Science ◽  
2021 ◽  
Vol 373 (6556) ◽  
pp. 768-774
Author(s):  
Jung-Un Park ◽  
Amy Wei-Lun Tsai ◽  
Eshan Mehrotra ◽  
Michael T. Petassi ◽  
Shan-Chi Hsieh ◽  
...  
Keyword(s):  
Site Selection ◽  
Information Transfer ◽  
Structural Basis ◽  
Target Sequence ◽  
Guide Rna ◽  
Target Site Selection ◽  
Cryo Electron Microscopy ◽  
Fixed Distance ◽  
Type V ◽  
Universal Mechanism

CRISPR-associated transposition systems allow guide RNA–directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo–electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate–bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.

scholarly journals Structural basis for target-site selection in RNA-guided DNA transposition systems

2021 ◽  
Author(s):  
Jung-Un Park ◽  
Amy Tsai ◽  
Eshan Mehrotra ◽  
Michael T Petassi ◽  
Shan-Chi Hsieh ◽  
...  
Keyword(s):  
Site Selection ◽  
Information Transfer ◽  
Structural Basis ◽  
Target Sequence ◽  
Guide Rna ◽  
Target Site Selection ◽  
Fixed Distance ◽  
Dna Insertion ◽  
Type V ◽  
Universal Mechanism

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA insertion in one orientation at a fixed distance from a programmable target sequence. We define the mechanism explaining this process by characterizing the transposition regulator, TnsC, from a Type V-K CRISPR-transposase system using cryo-EM. Polymerization of ATP-bound TnsC helical filaments explains how polarity information is passed to the transposase. Our Cryo-EM structure of TniQ-TnsC reveals that TniQ caps the TnsC filament, establishing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, structures with the transition state mimic, ADPᐧAlF3, reveals how TnsC transitions to define the fixed point of insertion. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.

scholarly journals Structural basis for DNA targeting by the Tn7 transposon

2021 ◽  
Author(s):  
Yao Shen ◽  
Josue Gomez-Blanco ◽  
Michael Thomas Petassi ◽  
Joseph E Peters ◽  
Joaquin Ortega ◽  
...  
Keyword(s):  
Site Selection ◽  
Atp Hydrolysis ◽  
Target Site ◽  
Structural Basis ◽  
Target Site Selection ◽  
Dna Targeting ◽  
Target Dna ◽  
Integration Sites ◽  
Selection Mechanisms ◽  
Aaa Atpases

Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7-family utilize a AAA+ adaptor (TnsC) to coordinates target-site selection with transposase activation and prevent insertions at sites already containing a Tn7 element. Due to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. We find that TnsC forms an asymmetric ring on target DNA that segregates target-site selection and transposase recruitment to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP-hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.

scholarly journals CRISPR-Cas12a target binding unleashes single-stranded DNase activity

2017 ◽  
Author(s):  
Janice S. Chen ◽  
Enbo Ma ◽  
Lucas B. Harrington ◽  
Xinran Tian ◽  
Jennifer A. Doudna
Keyword(s):  
Dna Binding ◽  
Fundamental Property ◽  
Dnase Activity ◽  
Target Sequence ◽  
Sequence Matching ◽  
Genetic Changes ◽  
Guide Rna ◽  
Circular Ssdna ◽  
Target Binding ◽  
Type V

AbstractCRISPR-Cas12a (Cpf1) proteins are RNA-guided DNA targeting enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a can be used as a powerful genome editing tool based on its ability to induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. Here we show that RNA-guided DNA binding unleashes robust, non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to completely degrade both linear and circular ssDNA molecules within minutes. This activity, catalyzed by the same active site responsible for site-specific dsDNA cutting, indiscriminately shreds ssDNA with rapid multiple-turnover cleavage kinetics. Activation of ssDNA cutting requires faithful recognition of a DNA target sequence matching the 20-nucleotide guide RNA sequence with specificity sufficient to distinguish between closely related viral serotypes. We find that target-dependent ssDNA degradation, not observed for CRISPR-Cas9 enzymes, is a fundamental property of type V CRISPR-Cas12 proteins, revealing a fascinating parallel with the RNA-triggered general RNase activity of the type VI CRISPR-Cas13 enzymes.One Sentence SummaryCas12a (Cpf1) and related type V CRISPR interference proteins possess non-specific, single-stranded DNase activity upon activation by guide RNA-dependent DNA binding.

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.

Target site selection and remodelling by type V CRISPR-transposon systems

Nature ◽  
2021 ◽  
Author(s):  
Irma Querques ◽  
Michael Schmitz ◽  
Seraina Oberli ◽  
Christelle Chanez ◽  
Martin Jinek
Keyword(s):  
Site Selection ◽  
Target Site ◽  
Target Site Selection ◽  
Type V

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

Development of a mito-CRISPR system for generating mitochondrial DNA-deleted strain in Saccharomyces cerevisiae

2020 ◽  
Vol 85 (4) ◽  
pp. 895-901
Author(s):  
Takamitsu Amai ◽  
Tomoka Tsuji ◽  
Mitsuyoshi Ueda ◽  
Kouichi Kuroda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Nuclear Genome ◽  
Target Sequence ◽  
Guide Rna ◽  
Crispr System ◽  
Mitochondrial Target ◽  
Gene Treatment ◽  
Mtdna Deletion

ABSTRACT Mitochondrial dysfunction can occur in a variety of ways, most often due to the deletion or mutation of mitochondrial DNA (mtDNA). The easy generation of yeasts with mtDNA deletion is attractive for analyzing the functions of the mtDNA gene. Treatment of yeasts with ethidium bromide is a well-known method for generating ρ° cells with complete deletion of mtDNA from Saccharomyces cerevisiae. However, the mutagenic effects of ethidium bromide on the nuclear genome cannot be excluded. In this study, we developed a “mito-CRISPR system” that specifically generates ρ° cells of yeasts. This system enabled the specific cleavage of mtDNA by introducing Cas9 fused with the mitochondrial target sequence at the N-terminus and guide RNA into mitochondria, resulting in the specific generation of ρ° cells in yeasts. The mito-CRISPR system provides a concise technology for deleting mtDNA in yeasts.

scholarly journals Structural basis for the regulation of nucleosome recognition and HDAC activity by histone deacetylase assemblies

2021 ◽  
Vol 7 (2) ◽  
pp. eabd4413
Author(s):  
Jung-Hoon Lee ◽  
Daniel Bollschweiler ◽  
Tillman Schäfer ◽  
Robert Huber
Keyword(s):  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Active Sites ◽  
Chromatin Modification ◽  
Structural Basis ◽  
Amino Terminal ◽  
Cryo Electron Microscopy ◽  
Multiple Contacts ◽  
Lysine Residues ◽  
Nucleosome Binding

The chromatin-modifying histone deacetylases (HDACs) remove acetyl groups from acetyl-lysine residues in histone amino-terminal tails, thereby mediating transcriptional repression. Structural makeup and mechanisms by which multisubunit HDAC complexes recognize nucleosomes remain elusive. Our cryo–electron microscopy structures of the yeast class II HDAC ensembles show that the HDAC protomer comprises a triangle-shaped assembly of stoichiometry Hda12-Hda2-Hda3, in which the active sites of the Hda1 dimer are freely accessible. We also observe a tetramer of protomers, where the nucleosome binding modules are inaccessible. Structural analysis of the nucleosome-bound complexes indicates how positioning of Hda1 adjacent to histone H2B affords HDAC catalysis. Moreover, it reveals how an intricate network of multiple contacts between a dimer of protomers and the nucleosome creates a platform for expansion of the HDAC activities. Our study provides comprehensive insight into the structural plasticity of the HDAC complex and its functional mechanism of chromatin modification.

scholarly journals The integration preference of Sleeping Beauty at non-TA site is related to the transposon end sequences

2019 ◽  
Author(s):  
Yiting Zhou ◽  
Guangwei Ma ◽  
Jiawen Yang ◽  
Yabin Guo
Keyword(s):  
Site Selection ◽  
Genomic Dna ◽  
Consensus Sequence ◽  
Mouse Cell ◽  
Sleeping Beauty ◽  
Consensus Sequences ◽  
Target Site Selection ◽  
Target Sites ◽  
End Sequences ◽  
Selection Of

Abstract Background: Sleeping Beauty (SB) transposon had been thought to strictly integrate into TA dinucleotides. Recently, we found that SB also integrates into non-TA sites at a lower frequency. Here we performed further study on the non-TA integration of SB. Results: 1) SB can integrate into non-TA sites in HEK293T cells as well as in mouse cell lines. 2) Both the hyperactive transposase SB100X and the traditional SB11 catalyze integrations at non-TA sites. 3) The consensus sequence of the non-TA target sites only occur at the opposite side of the sequenced junction between transposon end and the genomic sequences, indicating that the integrations at non-TA sites are mainly aberrant integrations. 4) The consensus sequence of the non-TA target sites is corresponding to the transposon end sequence. When the transposon end sequence is mutated, the consensus sequences changed too. Conclusion: The interaction between the SB transposon end and genomic DNA may be involved in the target site selection of the SB integrations at non-TA sites.

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