scholarly journals Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA

2019 ◽  
Author(s):  
Ryan T. Fuchs ◽  
Jennifer Curcuru ◽  
Megumu Mabuchi ◽  
Paul Yourik ◽  
G. Brett Robb
Keyword(s):  
Dna Sequences ◽  
Nuclease Activity ◽  
Target Sequence ◽  
Sequence Composition ◽  
Guide Rna ◽  
Dna Targeting ◽  
Single Turnover ◽  
Target Dna ◽  
Buffer Composition

ABSTRACTCRISPR-Cas12a (Cpf1) are RNA-guided nuclease effectors of acquired immune response that act in their native organisms by cleaving targeted DNA sequences. Like CRISPR-Cas9 RNA-guided DNA targeting enzymes, Cas12a orthologs have been repurposed for genome editing in non-native organisms and for DNA manipulationin vitro. Recent studies have shown that activation of Cas12a via guide RNA-target DNA pairing causes multiple turnover, non-specific ssDNA degradation intrans, after single turnover on-target cleavage incis. We find that the non-specifictransnuclease activity affects RNA and dsDNA in addition to ssDNA, an activity made more evident by adjustment of reaction buffer composition. The magnitude of thetransnuclease activity varies depending on features of the guide RNA being used, specifically target sequence composition and length. We also find that the magnitude oftransnuclease activity varies between the three most well-studied Cas12a orthologs and that the Cas12a fromLachnospiraceaebacterium ND2006 appears to be the most active.

In vitro Assay Revealed Mismatches between Guide RNA and Target DNA can Enhance Cas9 Nuclease Activity

2020 ◽  
Vol 1 (1) ◽  
pp. 69-72
Author(s):  
Ji Luan ◽  
Zhen Li ◽  
Hailong Wang ◽  
Jun Fu ◽  
Youming Zhang
Keyword(s):  
Dna Sequences ◽  
High Efficiency ◽  
Nuclease Activity ◽  
Ease Of Use ◽  
Unexpected Finding ◽  
Vitro Assay ◽  
Guide Rna ◽  
Cleavage Activity ◽  
Target Dna

Background: CRISPR-Cas9 is a powerful technology that allows us to modify DNA sequences in a specific manner across a variety of organisms. Due to its high efficiency and specificity, and ease of use, it becomes a commonly used method for gene editing. Although many structural and biochemical studies have been carried out to understand the fundamental mechanism of CRISPR/Cas9, our understanding of CRISPR/Cas9 caused off-target effects is still lacking. Methods: The enhanced in vitro cleavage activity of Cas9 protein from Streptococcus pyogenes (SpCas9) was evaluated by both synthetic crRNA-tracrRNA duplexes and in vitro transcribed single guide RNAs. Results: Here, we report an unexpected finding that mismatches between the guide RNA and target DNA significantly enhanced the in vitro cleavage activity of SpCas9 by more than 2 folds. Conclusion: Our observation that mismatches between the guide RNA and target DNA can dramatically increase the in vitro cleavage of Cas9 suggests the potential sequence preference for the CRSIPR/Cas9 system.

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 Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

2020 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Seung-Hun Kang ◽  
Junho K. Hur ◽  
Hyomin Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Genetic Mutation ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Effective Manner ◽  
Target Dna

AbstractThe CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 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 Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 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-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less off-target mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable diseases caused by genetic mutation.

scholarly journals Purification of specific DNA species using the CRISPR system

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Toshitsugu Fujita ◽  
Hodaka Fujii
Keyword(s):  
Dna Sequences ◽  
Chromatin Immunoprecipitation ◽  
In Vitro Method ◽  
Guide Rna ◽  
Crispr System ◽  
Target Dna ◽  
Genomic Regions ◽  
Generation Sequencing ◽  
Subsequent Identification

AbstractIn 2013, we developed a new method of engineered DNA-binding molecule-mediated chromatin immunoprecipitation that incorporates the clustered regularly interspaced short palindromic repeats (CRISPR) system to purify specific DNA species. This CRISPR-mediated purification can be performed in-cell or in vitro; CRISPR complexes can be expressed to tag target DNA sequences in the cells to be analyzed, or a CRISPR ribonucleoprotein complex consisting of recombinant nuclease-dead Cas9 (dCas9) and synthetic guide RNA can be used to tag target DNA sequences in vitro. Both methods enable purification of specific DNA sequences in chromatin structures for subsequent identification of molecules (proteins, RNAs, and other genomic regions) associated with the target sequences. The in vitro method also enables enrichment of purified DNA sequences from a pool of heterogeneous sequences for next-generation sequencing or other applications. In this review, we outline the principle of CRISPR-mediated purification of specific DNA species and discuss recent advances in the technology.

scholarly journals In vitro analysis of site specific nuclease selectivity by NGS

2021 ◽  
Vol 8 (4) ◽  
pp. 235-242
Author(s):  
Vincent Brondani ◽  
Keyword(s):  
Restriction Endonuclease ◽  
Genome Engineering ◽  
Direct Interaction ◽  
Target Sequence ◽  
Vitro Assay ◽  
Guide Rna ◽  
Phosphate Backbone ◽  
Target Dna ◽  
Vitro Analysis

<abstract> <p>Nucleases currently used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. The RNA guided nucleases describe today are recognizing a sequence with two distinct molecular interactions: first, like a restriction endonuclease, by direct interaction between the protein and the DNA; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report an in vitro assay to assess the cleavage specificity and the selectivity of the nucleases. The assay is designed using a plasmid encompassing the DNA target site degenerated at positions determined on structural feature. The results demonstrate that the Cpf1 RNA guided nuclease is highly specific for the target sequence, nevertheless its substrate selectivity is low compare to a restriction endonuclease.</p> </abstract>

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 CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet

2021 ◽  
Vol 12 ◽  
Author(s):  
Vanitharani Ramachandran ◽  
John J. Weiland ◽  
Melvin D. Bolton
Keyword(s):  
Virus Detection ◽  
Detection Methods ◽  
Target Sequence ◽  
Field Soil ◽  
Human Virus ◽  
Guide Rna ◽  
Highly Sensitive ◽  
Infested Field ◽  
Virus Diagnostics

Rhizomania is a disease of sugarbeet caused by beet necrotic yellow vein virus (BNYVV) that significantly affects sugarbeet yield globally. Accurate and sensitive detection methods for BNYVV in plants and field soil are necessary for growers to make informed decisions on variety selection to manage this disease. A recently developed CRISPR-Cas-based detection method has proven highly sensitive and accurate in human virus diagnostics. Here, we report the development of a CRISPR-Cas12a-based method for detecting BNYVV in the roots of sugarbeet. A critical aspect of this technique is the identification of conditions for isothermal amplification of viral fragments. Toward this end, we have developed a reverse transcription (RT) recombinase polymerase amplification (RPA) for detecting BNYVV in sugarbeet roots. The RT-RPA product was visualized, and its sequence was confirmed. Subsequently, we designed and validated the cutting efficiency of guide RNA targeting BNYVV via in vitro activity assay in the presence of Cas12a. The sensitivity of CRISPR-Cas12a trans reporter-based detection for BNYVV was determined using a serially diluted synthetic BNYVV target sequence. Further, we have validated the developed CRISPR-Cas12a assay for detecting BNYVV in the root-tissue of sugarbeet bait plants reared in BNYVV-infested field soil. The results revealed that BNYVV detection is highly sensitive and specific to the infected roots relative to healthy control roots as measured quantitatively through the reporter signal. To our knowledge, this is the first report establishing isothermal RT-RPA- and CRISPR-based methods for virus diagnostic approaches for detecting BNYVV from rhizomania diseased sugarbeet roots.

scholarly journals A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

2016 ◽  
Vol 2 (8) ◽  
pp. e1600699 ◽  
Author(s):  
Hiroshi Arakawa
Keyword(s):  
Dna Sequences ◽  
Genetic Screening ◽  
Restriction Enzymes ◽  
Mrna Sequence ◽  
Complementary Sequence ◽  
Complementary Dna ◽  
Random Primer ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Target Dna

The clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously impedes experimentally making gRNA. I describe a method to construct a gRNA library via molecular biology techniques without relying on bioinformatics. Briefly, one synthesizes complementary DNA from the mRNA sequence using a semi-random primer containing a PAM complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it applicable to any species.

scholarly journals Universal microbial diagnostics using random DNA probes

2016 ◽  
Vol 2 (9) ◽  
pp. e1600025 ◽  
Author(s):  
Amirali Aghazadeh ◽  
Adam Y. Lin ◽  
Mona A. Sheikh ◽  
Allen L. Chen ◽  
Lisa M. Atkins ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pathogenic Bacteria ◽  
Disease Outbreaks ◽  
Dna Probes ◽  
Signal Recovery ◽  
Human Pathogens ◽  
Infectious Disease Outbreaks ◽  
Target Dna ◽  
Detection Schemes

Early identification of pathogens is essential for limiting development of therapy-resistant pathogens and mitigating infectious disease outbreaks. Most bacterial detection schemes use target-specific probes to differentiate pathogen species, creating time and cost inefficiencies in identifying newly discovered organisms. We present a novel universal microbial diagnostics (UMD) platform to screen for microbial organisms in an infectious sample, using a small number of random DNA probes that are agnostic to the target DNA sequences. Our platform leverages the theory of sparse signal recovery (compressive sensing) to identify the composition of a microbial sample that potentially contains novel or mutant species. We validated the UMD platform in vitro using five random probes to recover 11 pathogenic bacteria. We further demonstrated in silico that UMD can be generalized to screen for common human pathogens in different taxonomy levels. UMD’s unorthodox sensing approach opens the door to more efficient and universal molecular diagnostics.

scholarly journals Engineered dual selection for directed evolution of SpCas9 PAM specificity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gregory W. Goldberg ◽  
Jeffrey M. Spencer ◽  
David O. Giganti ◽  
Brendan R. Camellato ◽  
Neta Agmon ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Negative Selection ◽  
Selection Procedures ◽  
Guide Rna ◽  
Dna Targeting ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Selection For ◽  
Reduced Activity

AbstractThe widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.

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