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 Sequence specificity in DNA binding is determined by association rather than dissociation

2021 ◽  
Author(s):  
Emil Marklund ◽  
Guanzhong Mao ◽  
Sebastian Deindl ◽  
Johan Elf
Keyword(s):  
Dna Binding ◽  
Genetic Information ◽  
Specific Binding ◽  
Theoretical Framework ◽  
Simple Equation ◽  
Target Sequence ◽  
Sequence Specificity ◽  
Target Search ◽  
Target Binding ◽  
Site Recognition

AbstractSequence-specific binding of proteins to DNA is essential for accessing genetic information. Here, we derive a simple equation for target-site recognition, which uncovers a previously unrecognized coupling between the macroscopic association and dissociation rates of the searching protein. Importantly, this relationship makes it possible to recover the relevant microscopic rates from experimentally determined macroscopic ones. We directly test the equation by observing the binding and unbinding of individual lac repressor (LacI) molecules during target search. We find that LacI dissociates from different target sequences with essentially identical microscopic dissociation rates. Instead, sequence specificity is determined by the efficiency with which the protein recognizes different targets, effectively reducing its risk of being retained on a non-target sequence. Our theoretical framework also accounts for the coupling between off-target binding and unbinding of the catalytically inactive Cas9 (dCas9), showing that the binding pathway can be obtained from macroscopic data.One Sentence SummaryAssociation and dissociation rates are anti-correlated for reactions that include a nonspecific probing step.

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 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.

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 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 CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

2020 ◽  
Author(s):  
Anindya Bandyopadhyay ◽  
Nagesh Kancharla ◽  
vivek javalkote ◽  
santanu dasgupta ◽  
Thomas Brutnell
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Temperature Stability ◽  
Double Strand Breaks ◽  
Plant Genome ◽  
Strand Breaks ◽  
Guide Rna ◽  
Versatile Tool ◽  
Type V ◽  
Global Population

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

scholarly journals CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1260 ◽  
Author(s):  
Arturo Temblador ◽  
Dimitrios Topalis ◽  
Graciela Andrei ◽  
Robert Snoeck
Keyword(s):  
Cell Cycle ◽  
Cell Carcinoma ◽  
Tumor Cells ◽  
Merkel Cell Carcinoma ◽  
Tumor Antigens ◽  
Therapeutic Option ◽  
Target Sequence ◽  
Merkel Cell ◽  
Guide Rna

Merkel cell carcinoma (MCC) is an aggressive type of skin cancer whose main causative agent is Merkel cell polyomavirus (MCPyV). MCPyV is integrated into the genome of the tumor cells in most MCCs. Virus-positive tumor cells constitutively express two viral oncoproteins that promote cell growth: the small (sT) and the large (LT) tumor antigens (TAs). Despite the success of immunotherapies in patients with MCC, not all individuals respond to these treatments. Therefore, new therapeutic options continue to be investigated. Herein, we used CRISPR/Cas9 to target the viral oncogenes in two virus-positive MCC cell lines: MS-1 and WAGA. Frameshift mutations introduced in the target sequence upon repair of the Cas9-induced DNA break resulted in decreased LT protein levels, which subsequently impaired cell proliferation, caused cell cycle arrest, and led to increased apoptosis. Importantly, a virus-negative non-MCC cell line (HEK293T) remained unaffected, as well as those cells expressing a non-targeting single-guide RNA (sgRNA). Thus, we presumed that the noted effects were not due to the off-target activity of the TAs-targeting sgRNAs. Additionally, WAGA cells had altered levels of cellular proteins involved in cell cycle regulation, supporting the observed cell cycle. Taken together, our findings provide evidence for the development of a CRISPR/Cas9-based therapeutic option for virus-positive MCC.

scholarly journals A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 458 ◽  
Author(s):  
François M. Seys ◽  
Peter Rowe ◽  
Edward L. Bolt ◽  
Christopher M. Humphreys ◽  
Nigel P. Minton
Keyword(s):  
Gold Standard ◽  
Mutant Allele ◽  
Target Sequence ◽  
Proof Of Concept ◽  
Wild Type ◽  
Guide Rna ◽  
Simple Strategy ◽  
Clostridium Autoethanogenum ◽  
Gene Knockouts

Phenotypic complementation of gene knockouts is an essential step in establishing function. Here, we describe a simple strategy for ‘gold standard’ complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) ‘bookmark’ sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele. The bookmark comprises a 23 nt Cas9 target sequence plus an additional nt to ensure the deletion is in-frame. Here, bookmarks are tailored to Streptococcus pyogenes CRISPR/Cas9 but could be designed for any CRISPR/Cas system. For proof of concept, nine bookmarks were tested in Clostridium autoethanogenum. Complementation efficiencies reached 91%. As complemented strains are indistinguishable from their progenitors, concerns over contamination may be satisfied by the incorporation of ‘watermark’ sequences into the complementing genes.

Sign in / Sign up

Export Citation Format

Share Document