Posts Tagged ‘CRISPR/Cas3’

Innovations on the CRISPR System for Gene Editing: (1) Cryo-electron microscopy-based visualization of Cas3 Enzyme Cleavage (2) New tool testing an entire genome against a CRISPR molecule to predict potential errors and interactions, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

Innovations on the CRISPR System for Gene Editing: (1) Cryo-electron microscopy-based visualization of Cas3 Enzyme Cleavage (2) New tool testing an entire genome against a CRISPR molecule to predict potential errors and interactions

Curator and Reporter: Aviva Lev-Ari, PhD, RN


Boom in human gene editing as 20 CRISPR trials gear up

A pioneering CRISPR trial in China will be the first to try editing the genomes of cells inside the body, in an effort to eliminate cancer-causing HPV virus



(1) Cryo-electron microscopy-based visualization of Cas3 Enzyme Cleavage

Harvard Medical School and Cornell University scientists have now generated near-atomic resolution snapshots of CRISPR that reveal key steps in its mechanism of action. The findings, published in Cell on June 29, provide the structural data necessary for efforts to improve the efficiency and accuracy of CRISPR for biomedical applications.

Through cryo-electron microscopy, the researchers describe for the first time the exact chain of events as the CRISPR complex loads target DNA and prepares it for cutting by the Cas3 enzyme. These structures reveal a process with multiple layers of error detection—a molecular redundancy that prevents unintended genomic damage, the researchers say.


Image Source: CRISPR forms a “seed bubble” state, which acts as an initial fail-safe mechanism to ensure that CRISPR RNA matches its target DNA. Image: Liao Lab/HMS


In contrast to the scalpel-like Cas9, CRISPR-Cas3 acts like a shredder that chews DNA up beyond repair. While CRISPR-Cas3 has, thus far, limited utility for precision gene editing, it is being developed as a tool to combat antibiotic-resistant strains of bacteria. A better understanding of its mechanisms may broaden the range of potential applications for CRISPR-Cas3.

In addition, all CRISPR-Cas subtypes utilize some version of an R-loop formation to detect and prepare target DNA for cleavage. The improved structural understanding of this process can now enable researchers to work toward modifying multiple types of CRISPR-Cas systems to improve their accuracy and reduce the chance of off-target effects in biomedical applications.


Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System

Yibei Xiao3


Min Luo3


Robert P. Hayes4


Jonathan Kim


Sherwin Ng


Fang Ding


Maofu Liao'Correspondence information about the author Maofu Liao


Ailong Ke5,'Correspondence information about the author Ailong Ke
3These authors contributed equally
4Present address: Merck & Co., 770 Sumneytown Pike, West Point, PA 19486, USA
5Lead contact
Bringing CRISPR into Focus – New study reveals key steps in CRISPR-Cas3 function at near-atomic resolution
June 29, 2017
Scientists from The University of Texas at Austin may have come up with a possible solution. They’ve developed something that works like a predictive editor for CRISPR: a method for anticipating and catching the tool’s mistakes as it works, thereby allowing for the editing of disease-causing errors out of genomes.
Many forms of cancer, Huntington’s disease, and even HIV can be targeted using CRISPR. CRISPR can “correct” something that was actually right — the consequences of which can make it a dangerous mistake. One that actually causes a disease. CRISPR molecules—proteins that find and edit genes—sometimes target the wrong genes, acting more like an auto-correct feature that turns correctly spelled words into typos. Editing the wrong gene could create new problems, such as causing healthy cells to become cancerous.

“You and I differ in about 1 million spots in our genetic code,” says Ilya Finkelstein, an assistant professor in the Department of Molecular Biosciences at UT Austin and the project’s principal investigator. “Because of this genetic diversity, human gene editing will always be a custom-tailored therapy.”

Image Source: The heart of the new technique developed by Finkelstein, et al. for detecting interactions between CRISPR and off-target DNA segments is a standard next generation gene sequencing slide (a.k.a. flowcell), produced by Illumina. Image by Wikimedia user Bainscou, via Creative Commons Attribution 3.0 license
CHAMP, or Chip Hybridized Affinity Mapping Platform. The heart of the test is a standard next generation genome sequencing chip already widely used in research and medicine. Two other key elements—designs for a 3-D printed mount that holds the chip under a microscope and software the team developed for analyzing the results—are open source. As a result, other researchers can easily replicate the technique in experiments involving CRISPR.

Andy Ellington, a professor in the Department of Molecular Biosciences and vice president for research of the Applied Research Laboratories at UT Austin, is a co-author of the paper. He says this method also illustrates the unpredictable side benefits of new technologies.

“Next generation genome sequencing was invented to read genomes, but here we’ve turned the technology on its head to allow us to characterize how CRISPR interacts with genomes,” says Ellington. “Inventive folks like Ilya take new technologies and extend them into new realms.”

they found that the CRISPR molecule they tested, called Cascade, pays less attention to every third letter in a DNA sequence than to the others.


CHAMP repurposes sequenced and discarded chips from modern next-generation Illumina sequencers for high-throughput association profiling of proteins to nucleic acids. A key difference between CHAMP and prior NGS-based approaches is that it does not require any hardware or software modifications to discontinued Illumina sequencers (Nutiu et al., 2011Tome et al., 2014Buenrostro et al., 2014). In CHAMP, all association-profiling experiments are carried out on sequenced MiSeq chips and imaged in a conventional TIRF microscope. CHAMP’s computational strategy uses phiX clusters as alignment markers to align the spatial information obtained via Illumina sequencing with the fluorescent association profiling experiments. This strategy offers three key advantages over previous approaches. First, using a conventional fluorescence microscope opens new experimental configurations, including multi-color co-localization and time-dependent kinetic experiments. The excitation and emission optics can also be readily adapted for FRET (Figure S6) and other advanced imaging modalities. Second, complete fluidic access to the chip allows addition of other protein components during a biochemical reaction. Third, the computational strategy for aligning sequencer outputs to fluorescent datasets is applicable to all modern Illumina sequencers, including the MiSeq, NextSeq, and HiSeq platforms. Indeed, we also used the CHAMP imaging and bioinformatics pipeline to regenerate, image, and spatially align the DNA clusters in a HiSeq flowcell (Figure S6), providing an avenue for massively parallel profiling of protein-nucleic acid interactions on both synthetic libraries and entire genomes. Future extensions will leverage on-chip transcription and translation (e.g., ribosome display) to facilitate high-throughput studies of RNA or peptide association landscapes. These studies will permit quantitative biophysical studies of diverse protein-nucleic acid interactions.



Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips

Cheulhee Jung8


John A. Hawkins8


Stephen K. Jones Jr.8


Yibei Xiao


James R. Rybarski


Kaylee E. Dillard


Jeffrey Hussmann


Fatema A. Saifuddin


Cagri A. Savran


Andrew D. Ellington


Ailong Ke


William H. Press


Ilya J. Finkelstein9,'Correspondence information about the author Ilya J. Finkelstein
8These authors contributed equally
9Lead Contact

This New Gene-Editing Technique Can Spot CRISPR’s Mistakes

New Technique Enables Safer Gene-Editing Therapy Using CRISPR

Other related articles on CRISPR published on this Open Access Online Scientific Journal include the following:

255 Articles on CRISPR

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