A Cas9-guide RNA complex preorganized for target DNA recognition

Science ◽  
2015 ◽  
Vol 348 (6242) ◽  
pp. 1477-1481 ◽  
Author(s):  
F. Jiang ◽  
K. Zhou ◽  
L. Ma ◽  
S. Gressel ◽  
J. A. Doudna
Keyword(s):  
Dna Recognition ◽  
Guide Rna ◽  
Target Dna ◽  
Rna Complex

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 CRISPR as a Diagnostic Tool

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1162
Author(s):  
Seohyun Kim ◽  
Sangmin Ji ◽  
Hye Ran Koh
Keyword(s):  
Diagnostic Tool ◽  
Specific Binding ◽  
Diagnostic Methods ◽  
Specific Gene ◽  
Guide Rna ◽  
Engineering Technique ◽  
Target Dna ◽  
Target Binding ◽  
Genetic Engineering Technique ◽  
Disease Related Gene

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.

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.

scholarly journals Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

2020 ◽  
Vol 48 (15) ◽  
pp. 8601-8616 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Yeounsun Oh ◽  
Seung-Hun Kang ◽  
Junho K Hur ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Protospacer Adjacent Motif ◽  
Effective Manner ◽  
Target Dna

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

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

Determination of Protein−RNA Interaction Sites in the Cbf5-H/ACA Guide RNA Complex by Mass Spectrometric Protein Footprinting†

Biochemistry ◽  
2008 ◽  
Vol 47 (6) ◽  
pp. 1500-1510 ◽  
Author(s):  
Daniel L. Baker ◽  
Nicholas T. Seyfried ◽  
Hong Li ◽  
Ron Orlando ◽  
Rebecca M. Terns ◽  
...  
Keyword(s):  
Mass Spectrometric ◽  
Protein Footprinting ◽  
Guide Rna ◽  
Interaction Sites ◽  
Rna Interaction ◽  
Rna Complex

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 Spelling changes and fluorescent tagging with prime editing vectors for plants

2020 ◽  
Author(s):  
Li Wang ◽  
Hilal Betul Kaya ◽  
Ning Zhang ◽  
Rhitu Rai ◽  
Matthew R. Willmann ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Binding Site ◽  
Nicotiana Benthamiana ◽  
Primer Binding Site ◽  
First Report ◽  
Guide Rna ◽  
Insertions And Deletions ◽  
Target Dna ◽  
Fluorescent Tagging

AbstractPrime editing (PE) is a recent adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase (RT) fusion and a guide RNA (pegRNA) amended with template and primer binding site (PBS) sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of PE in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for PE in dicots and monocots, their validation in Nicotiana benthamiana, rice and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

Sign in / Sign up

Export Citation Format

Share Document