scholarly journals Type II and type V CRISPR effector nucleases from a structural biologist’s perspective

2016 ◽  
Vol 62 (3) ◽  
pp. 315-326
Author(s):  
Humberto Fernandes ◽  
Michal Pastor ◽  
Matthias Bochtler
Keyword(s):  
Target Sequence ◽  
Type Ii ◽  
Molecular Architecture ◽  
Genomic Engineering ◽  
Double Stranded Dna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strands ◽  
Catalytic Role ◽  
Type V

The type II and type V CRISPR effector nucleases Cas9 and Cpf1 are “universal” DNA endonucleases, which can be programmed by an appropriate crRNA or sgRNA strand to cleave almost any DNA duplex at a preselected position (constrained only by short, so-called PAMs). In this review, we briefly introduce CRISPR bacterial adaptive immunity as the biological context in which Cas9 and Cpf1 proteins operate, and then present the structural insights that have been obtained in the last two or three years that illustrate the mode of operation of these proteins. We describe the R-loop structures at the core of the Cas9 and Cpf1 complexes, and the structure of the 5’- or 3’-handles that help anchor the nucleic acid complexes to the proteins in a manner that is independent of the target sequence. Next, we describe the molecular architecture of the Cas9 and Cpf1 proteins. We illustrate how Cas9 and Cpf1 proteins scan double stranded DNA for so-called protospacer associated motifs (PAMs), we explain how the phosphate loop (PLL) and basic helix (BH) promote the separation of target and non-target DNA strands and the formation of hybrids between crRNA or sgRNA and the target strand of DNA. We also describe the current understanding of the catalytic mechanisms of RuvC and HNH domains, and a possible, but still very uncertain catalytic role of the Nuc domain. At the end of the review, we briefly summarize key developments that have initiated the field of genomic engineering using Cas9 or Cpf1 nucleases..

scholarly journals CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

2020 ◽  
Author(s):  
Joshua C. Cofsky ◽  
Deepti Karandur ◽  
Carolyn J. Huang ◽  
Isaac P. Witte ◽  
John Kuriyan ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Strand Break ◽  
Loop Structure ◽  
Loop Formation ◽  
Strand Breaks ◽  
Guide Rna ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
Type V

ABSTRACTMost type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing applications. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one of the DNA strands of a double-helical substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we show that Cas12a-mediated R-loop formation destabilizes DNA at the second-strand cleavage site, which is located outside of the R-loop structure and beyond the 3′ end of the guide RNA. Chemical and fluorescent DNA probes reveal that this destabilization is an intrinsic feature of DNA flanking the RNA-3′ side of R-loops and does not require direct protein interactions. Interestingly, DNA flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

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 Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage

2021 ◽  
Vol 118 (49) ◽  
pp. e2113747118
Author(s):  
Heyjin Son ◽  
Jaeil Park ◽  
Injoo Hwang ◽  
Youngri Jung ◽  
Sangsu Bae ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Resonance Energy ◽  
Distal Region ◽  
Catalytic Site ◽  
Nucleic Acid Detection ◽  
Double Stranded Dna ◽  
Target Strand ◽  
Target Dna ◽  
Conformational Rearrangements

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)–distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

scholarly journals The cell cycle-regulated DNA adenine methyltransferase CcrM opens a bubble at its DNA recognition site

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
John R. Horton ◽  
Clayton B. Woodcock ◽  
Sifa B. Opot ◽  
Norbert O. Reich ◽  
Xing Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Methyltransferase ◽  
Caulobacter Crescentus ◽  
Recognition Site ◽  
Van Der Waals Interactions ◽  
Target Sequence ◽  
Recognition Sequence ◽  
Methyltransferase Domain ◽  
Target Strand ◽  
Dna Strands

Abstract The Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM) methylates the adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity.

scholarly journals Detection of Target ssDNA Using a Microfabricated Hall Magnetometer with Correlated Optical Readout

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Steven M. Hira ◽  
Khaled Aledealat ◽  
Kan-Sheng Chen ◽  
Mark Field ◽  
Gerard J. Sullivan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Signal Amplification ◽  
Point Of Care ◽  
Target Sequence ◽  
Large Signal ◽  
Optical Readout ◽  
Hall Magnetometer ◽  
Target Dna ◽  
Dna Strands ◽  
Target Binding

Sensing biological agents at the genomic level, while enhancing the response time for biodetection over commonly used, optics-based techniques such as nucleic acid microarrays or enzyme-linked immunosorbent assays (ELISAs), is an important criterion for new biosensors. Here, we describe the successful detection of a 35-base, single-strand nucleic acid target by Hall-based magnetic transduction as a mimic for pathogenic DNA target detection. The detection platform has low background, large signal amplification following target binding and can discriminate a single, 350 nm superparamagnetic bead labeled with DNA. Detection of the target sequence was demonstrated at 364 pM (<2 target DNA strands per bead) target DNA in the presence of 36 μM nontarget (noncomplementary) DNA (<10 ppm target DNA) using optical microscopy detection on a GaAs Hall mimic. The use of Hall magnetometers as magnetic transduction biosensors holds promise for multiplexing applications that can greatly improve point-of-care (POC) diagnostics and subsequent medical care.

scholarly journals SPECIFICITY OF TRANSPOSON Tn5 INSERTION

Genetics ◽  
1983 ◽  
Vol 105 (4) ◽  
pp. 813-828
Author(s):  
Douglas E Berg ◽  
Margaret A Schmandt ◽  
John B Lowe
Keyword(s):  
Molecular Level ◽  
Target Sequence ◽  
Restriction Endonuclease Digestion ◽  
Plasmid Pbr322 ◽  
Base Pairs ◽  
Transposon Tn5 ◽  
Target Dna ◽  
Dna Strands ◽  
Endonuclease Digestion ◽  
Bacterial Transposon

ABSTRACT Genetic mapping studies had shown that the bacterial transposon Tn5 can insert into many sites in a gene, but that some sites are preferred. To begin understanding Tn5's insertion specificity at the molecular level, we selected transpositions of Tn5 from the Escherichia coli chromosome to the plasmid pBR322 and analyzed the resultant pBR322::Tn5 plasmids by restriction endonuclease digestion and DNA sequencing. Seventy-five insertions in the tet gene were found at 28 sites including one major hotspot (with 21 insertions) and four lesser hotspots (with four to ten insertions each). All five hotspots are within the first 300 of the 1250-base pair (bp) tet gene. In contrast, 31 independent insertions in the amp gene were found in at least 27 distinct sites.—Tn5 generates 9 bp target sequence duplications when it transposes. Such transposon-induced duplications are generally taken to indicate that cleavages of complementary target DNA strands are made 9 bp apart during transposition. DNA sequence analysis indicated that GC base pairs occupy positions 1 and 9 in the duplications at each of the five hotspots examined, suggesting a GC-cutting preference during Tn5 transposition.

The distribution and density of ciliary tufts on the siphons of Anodonta cygnea (Mollusca: Bivalvia)

1990 ◽  
Vol 48 (3) ◽  
pp. 518-519
Author(s):  
Wen-lung Wu
Keyword(s):  
Osmium Tetroxide ◽  
Internal Surface ◽  
Mechanical Stimuli ◽  
Type Ii ◽  
Sensory Receptors ◽  
Anodonta Cygnea ◽  
Type Iv ◽  
Sem Study ◽  
Inhalant Siphon ◽  
Type V

The mantle of bivalves has come entirely to enclose the laterally compressed body and the mantle margin has assumed a variety of functions, one of the pricipal ones being sensory. Ciliary tufts, which are probably sensory, have been reported from the mantle and siphons of several bivalves1∽4. Certain regions of the mantle margin are likely to be more or less, sensitive to certain stimuli than others. The inhalant siphon is likely to be particularly sensitive to both chemical and mechanical stimuli, whereas the exhalant siphon will be less sensitive to both. The distribution and density of putative sensory receptors on the in-and ex-halant siphon is compared in this paper.The excised siphons were fixed in glutaraldehyde and osmium tetroxide, the whole procedure of SEM study is recorded in Wu's thesis.Type II cilia cover the tips of tentacles, 6.13um. Type IV and type V cilia are found on the surface of tentacles. Type IV cilia are occasionally present at the tips of tentacles, 8 um long. They are the commonest type on the surface of tentacles. Type VI cilia occor in the internal surface of the inhalant siphon, but are not found on the surface of tentacles, 6.7-10um long.

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 Promiscuous molecules for smarter file operations in DNA-based data storage

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kyle J. Tomek ◽  
Kevin Volkel ◽  
Elaine W. Indermaur ◽  
James M. Tuck ◽  
Albert J. Keung
Keyword(s):  
Data Storage ◽  
Data Access ◽  
Molecular Architecture ◽  
Biomolecular Structure ◽  
Jpeg Images ◽  
Storage Media ◽  
Dna Storage ◽  
Dna Strands ◽  
Background Database ◽  
Organizational Features

AbstractDNA holds significant promise as a data storage medium due to its density, longevity, and resource and energy conservation. These advantages arise from the inherent biomolecular structure of DNA which differentiates it from conventional storage media. The unique molecular architecture of DNA storage also prompts important discussions on how data should be organized, accessed, and manipulated and what practical functionalities may be possible. Here we leverage thermodynamic tuning of biomolecular interactions to implement useful data access and organizational features. Specific sets of environmental conditions including distinct DNA concentrations and temperatures were screened for their ability to switchably access either all DNA strands encoding full image files from a GB-sized background database or subsets of those strands encoding low resolution, File Preview, versions. We demonstrate File Preview with four JPEG images and provide an argument for the substantial and practical economic benefit of this generalizable strategy to organize data.

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