scholarly journals The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation

2019 ◽  
Vol 5 (11) ◽  
pp. eaaw9807 ◽  
Author(s):  
Qian Zhang ◽  
Fengcai Wen ◽  
Siqi Zhang ◽  
Jiachuan Jin ◽  
Lulu Bi ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Single Molecule ◽  
Dna Interactions ◽  
Base Pairs ◽  
Complementary Dna ◽  
Interaction Site ◽  
Protospacer Adjacent Motif ◽  
Dna Loss ◽  
Target Binding

Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.

scholarly journals Implementation of single molecule FRET for visualizing intramolecular movement in CRISPR-Cas9

2020 ◽  
Author(s):  
Haruka Narita ◽  
Hiroshi Ebata ◽  
Karibu Sakai ◽  
Katsuhiko Minami ◽  
Sotaro Uemura ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Dna Sequence ◽  
Single Molecule ◽  
Fluorescence Anisotropy ◽  
Molecular Basis ◽  
Catalytic Process ◽  
Single Molecule Fret ◽  
Protospacer Adjacent Motif ◽  
A Genome ◽  
Cas9 Protein

SHORT ABSTRACTThis paper summarizes how to visualize the flexible inter-domain movements of CRISPR-associated protein Cas9 using single molecule FRETLONG ABSTRACTThe CRISPR-associated protein Cas9 is widely used as a genome editing tool because of its ability to be programmed to cleave any DNA sequence that is followed by a protospacer adjacent motif. The continuing expansion of Cas9 technologies has stimulated studies regarding the molecular basis of the Cas9 catalytic process. Here we summarize methods for single molecule FRET (smFRET) to visualize the inter-domain movements of Cas9 protein. Our measurements and analysis demonstrate flexible and reversible movements of the Cas9 domains. Such flexible movements allow Cas9 to adopt transient conformations beyond those solved by crystal structures and play important roles in the Cas9 catalytic process. In addition to the smFRET measurement itself, to obtain precise results, it is necessary to validate Cas9 catalytic activity. Also, fluorescence anisotropy data are required to interpret smFRET data properly. Thus, in this paper, we describe the details of these important additional experiments for smFRET measurements.

scholarly journals CRISPR Cas9 searches for a protospacer adjacent motif by one-dimensional diffusion

2018 ◽  
Author(s):  
Viktorija Globyte ◽  
Seung Hwan Lee ◽  
Taegun Bae ◽  
Jin-Soo Kim ◽  
Chirlmin Joo
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Lateral Diffusion ◽  
Resonance Energy ◽  
Dynamic Mode ◽  
Target Sequence ◽  
Dimensional Diffusion ◽  
Search Mechanism ◽  
Protospacer Adjacent Motif ◽  
Target Binding

AbstractSince its discovery, the CRISPR/Cas9 system has been at the focus of fundamental researchers, genome engineers, and the general public alike. Despite being in the spotlight for several years, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. We use single-molecule Foerster resonance energy transfer (smFRET) to reveal Cas9 target search mechanism with nanometer sensitivity. We have developed single-molecule assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the protospacer adjacent motif (PAM) sequence weakly, yet probing neighboring sequences via lateral diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with 3-dimensional diffusion and thus might aid PAM finding and consequently on-target binding.

scholarly journals A machine learning approach for accurate and real-time DNA sequence identification

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yiren Wang ◽  
Mashari Alangari ◽  
Joshua Hihath ◽  
Arindam K. Das ◽  
M. P. Anantram
Keyword(s):  
Real Time ◽  
Dna Sequence ◽  
Single Molecule ◽  
Identification Accuracy ◽  
Measuring System ◽  
Identification System ◽  
Genetic Sequencing ◽  
Sequence Identification ◽  
Tree Classifier ◽  
Single Mismatch

Abstract Background The all-electronic Single Molecule Break Junction (SMBJ) method is an emerging alternative to traditional polymerase chain reaction (PCR) techniques for genetic sequencing and identification. Existing work indicates that the current spectra recorded from SMBJ experimentations contain unique signatures to identify known sequences from a dataset. However, the spectra are typically extremely noisy due to the stochastic and complex interactions between the substrate, sample, environment, and the measuring system, necessitating hundreds or thousands of experimentations to obtain reliable and accurate results. Results This article presents a DNA sequence identification system based on the current spectra of ten short strand sequences, including a pair that differs by a single mismatch. By employing a gradient boosted tree classifier model trained on conductance histograms, we demonstrate that extremely high accuracy, ranging from approximately 96 % for molecules differing by a single mismatch to 99.5 % otherwise, is possible. Further, such accuracy metrics are achievable in near real-time with just twenty or thirty SMBJ measurements instead of hundreds or thousands. We also demonstrate that a tandem classifier architecture, where the first stage is a multiclass classifier and the second stage is a binary classifier, can be employed to boost the single mismatched pair’s identification accuracy to 99.5 %. Conclusions A monolithic classifier, or more generally, a multistage classifier with model specific parameters that depend on experimental current spectra can be used to successfully identify DNA strands.

scholarly journals A DNA Sequence Based Polymer Model for Chromatin Folding

2021 ◽  
Vol 22 (3) ◽  
pp. 1328
Author(s):  
Rui Zhou ◽  
Yi Qin Gao
Keyword(s):  
Dna Sequence ◽  
Cpg Island ◽  
Coarse Grained ◽  
Chromosome Territories ◽  
Base Pairs ◽  
Single Chromosome ◽  
Power Law Decay ◽  
Multiple Length Scales ◽  
Spatial Domains ◽  
Chromatin Folding

The recent development of sequencing technology and imaging methods has provided an unprecedented understanding of the inter-phase chromatin folding in mammalian nuclei. It was found that chromatin folds into topological-associated domains (TADs) of hundreds of kilo base pairs (kbps), and is further divided into spatially segregated compartments (A and B). The compartment B tends to be located near to the periphery or the nuclear center and interacts with other domains of compartments B, while compartment A tends to be located between compartment B and interacts inside the domains. These spatial domains are found to highly correlate with the mosaic CpG island (CGI) density. High CGI density corresponds to compartments A and small TADs, and vice versa. The variation of contact probability as a function of sequential distance roughly follows a power-law decay. Different chromosomes tend to segregate to occupy different chromosome territories. A model that can integrate these properties at multiple length scales and match many aspects is highly desired. Here, we report a DNA-sequence based coarse-grained block copolymer model that considers different interactions between blocks of different CGI density, interactions of TAD formation, as well as interactions between chromatin and the nuclear envelope. This model captures the various single-chromosome properties and partially reproduces the formation of chromosome territories.

scholarly journals NADH:ubiquinone oxidoreductase from bovine heart mitochondria Complementary DNA sequence of the import precursor of the 10 kDa subunit of the flavoprotein fragment

FEBS Letters ◽  
1991 ◽  
Vol 282 (1) ◽  
pp. 135-138 ◽  
Author(s):  
J.Mark Skehel ◽  
Stephanie J. Pilkington ◽  
Michael J. Runswick ◽  
Ian M. Fearnley ◽  
John E. Walker
Keyword(s):  
Dna Sequence ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Complementary Dna ◽  
Bovine Heart

Complementary DNA sequence of rabbit CAP18—A unique lipopolysaccharide binding protein

1991 ◽  
Vol 179 (1) ◽  
pp. 170-175 ◽  
Author(s):  
James W. Larrick ◽  
John G. Morgan ◽  
Ilona Palings ◽  
Michimasa Hirata ◽  
Michael H. Yen
Keyword(s):  
Dna Sequence ◽  
Binding Protein ◽  
Lipopolysaccharide Binding Protein ◽  
Complementary Dna ◽  
Lipopolysaccharide Binding

scholarly journals Phylogenetic analysis of a reported complementary DNA sequence

Science ◽  
1994 ◽  
Vol 265 (5175) ◽  
pp. 1110-1111 ◽  
Author(s):  
E Zietkiewicz ◽  
W Makalowski ◽  
G. Mitchell ◽  
D Labuda
Keyword(s):  
Phylogenetic Analysis ◽  
Dna Sequence ◽  
Complementary Dna

scholarly journals Supercoiling DNA locates mismatches

2017 ◽  
Author(s):  
Andrew Dittmore ◽  
Sumitabha Brahmachari ◽  
Yasuhara Takagi ◽  
John F. Marko ◽  
Keir C. Neuman
Keyword(s):  
Dna Sequence ◽  
Base Pair ◽  
Dna Supercoiling ◽  
Magnetic Tweezers ◽  
Relative Probability ◽  
Base Pairs ◽  
Damage Sensing ◽  
Mismatched Base Pair ◽  
Monovalent Salt

We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.5 pN force and show that, under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.

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