scholarly journals CRISPR–Cas12a system in fission yeast for multiplex genomic editing and CRISPR interference

2020 ◽  
Vol 48 (10) ◽  
pp. 5788-5798 ◽  
Author(s):  
Yu Zhao ◽  
Jef D Boeke
Keyword(s):  
Fission Yeast ◽  
Pol Ii ◽  
Endogenous Gene ◽  
Crispr Interference ◽  
Interference System ◽  
Potential Applications ◽  
Short Palindromic Repeat ◽  
Genomic Gene ◽  
Genomic Editing ◽  
Type V

Abstract The CRISPR–Cas12a is a class II, type V clustered regularly interspaced short palindromic repeat (CRISPR) system with both RNase and DNase activity. Compared to the CRISPR–Cas9 system, it recognizes T-rich PAM sequences and has the advantage of multiplex genomic editing. Here, in fission yeast Schizosaccharomyces pombe, we successfully implemented the CRISPR–Cas12a system for versatile genomic editing and manipulation. In addition to the rrk1 promoter, we used new pol II promoters from endogenous coding genes to express crRNA for Cas12a and obtained a much higher editing efficiency. This new design expands the promoter choices for potential applications in fission yeast and other organisms. In addition, we expressed a gRNA array using a strong constitutive pol II promoter. The array transcript is processed by Cas12a itself to release multiple mature crRNAs. With this construct, multiplex genomic editing of up to three loci was achieved from a single yeast transformation. We also built a CRISPR interference system using a DNase-dead Cas12a to significantly repress endogenous gene expression. Our study provides the first CRISPR-Cas12a toolkit for efficient and rapid genomic gene editing and regulation in fission yeast.

scholarly journals CRISPR base editors: genome editing without double-stranded breaks

2018 ◽  
Vol 475 (11) ◽  
pp. 1955-1964 ◽  
Author(s):  
Ayman Eid ◽  
Sahar Alshareef ◽  
Magdy M. Mahfouz
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genetic Diseases ◽  
Great Promise ◽  
Cytidine Deaminases ◽  
Strand Breaks ◽  
Base Editing ◽  
Potential Applications ◽  
Short Palindromic Repeat ◽  
Basic Biology

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.

Development of a CRISPR interference system for selective gene knockdown in Acidithiobacillus ferrooxidans

2021 ◽  
Author(s):  
Shohei Yamada ◽  
Yusuke Suzuki ◽  
Atsushi Kouzuma ◽  
Kazuya Watanabe
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Gene Knockdown ◽  
Crispr Interference ◽  
Interference System

scholarly journals CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1843-1845 ◽  
Author(s):  
Luciano A. Marraffini ◽  
Erik J. Sontheimer
Keyword(s):  
Antibiotic Resistance ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Staphylococcus Epidermidis ◽  
Pathogenic Bacteria ◽  
Target Sequence ◽  
Crispr Interference ◽  
Multiple Routes ◽  
Short Palindromic Repeat ◽  
Self Splicing

Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate ofStaphylococcus epidermidisharbors a CRISPR spacer that matches thenickasegene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation inS. epidermidis. Insertion of a self-splicing intron intonickaseblocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.

Abstract 3056: Development of an integrated CRISPR interference system targeting ΔNp63 to treat lung and esophageal squamous cell carcinoma

2019 ◽  
Author(s):  
Masakazu Yoshida ◽  
Etsuko Yokota ◽  
Tetsushi Sakuma ◽  
Nagio Takigawa ◽  
Toshikazu Ushijima ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Esophageal Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Crispr Interference ◽  
Interference System

scholarly journals Investigating essential gene function inMycobacterium tuberculosisusing an efficient CRISPR interference system

2016 ◽  
Vol 44 (18) ◽  
pp. e143-e143 ◽  
Author(s):  
Atul K. Singh ◽  
Xavier Carette ◽  
Lakshmi-Prasad Potluri ◽  
Jared D. Sharp ◽  
Ranfei Xu ◽  
...  
Keyword(s):  
Gene Function ◽  
Essential Gene ◽  
Crispr Interference ◽  
Interference System

scholarly journals Targeted delivery of CRISPR interference system against Fabp4 to white adipocytes ameliorates obesity, inflammation, hepatic steatosis, and insulin resistance

2019 ◽  
Vol 29 (9) ◽  
pp. 1442-1452 ◽  
Author(s):  
Jee Young Chung ◽  
Qurrat Ul Ain ◽  
Yoonsung Song ◽  
Seok-Beom Yong ◽  
Yong-Hee Kim
Keyword(s):  
Insulin Resistance ◽  
Hepatic Steatosis ◽  
Targeted Delivery ◽  
Crispr Interference ◽  
White Adipocytes ◽  
Interference System

scholarly journals PRPS-Associated Disorders and the Drosophila Model of Arts Syndrome

2020 ◽  
Vol 21 (14) ◽  
pp. 4824
Author(s):  
Keemo Delos Santos ◽  
Eunjeong Kwon ◽  
Nam-Sung Moon
Keyword(s):  
Molecular Mechanisms ◽  
Metabolic Diseases ◽  
Fruit Fly ◽  
Nucleotide Metabolism ◽  
Human Diseases ◽  
Past Century ◽  
Missense Mutations ◽  
Phosphoribosyl Pyrophosphate ◽  
Potential Applications ◽  
Short Palindromic Repeat

While a plethora of genetic techniques have been developed over the past century, modifying specific sequences of the fruit fly genome has been a difficult, if not impossible task. clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 truly redefined molecular genetics and provided new tools to model human diseases in Drosophila melanogaster. This is particularly true for genes whose protein sequences are highly conserved. Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme in nucleotide metabolism whose missense mutations are found in several neurological disorders, including Arts syndrome. In addition, PRPS is deregulated in cancer, particularly those that become resistant to cancer therapy. Notably, Drosophila PRPS shares about 90% protein sequence identity with its human orthologs, making it an ideal gene to study via CRISPR/Cas9. In this review, we will summarize recent findings on PRPS mutations in human diseases including cancer and on the molecular mechanisms by which PRPS activity is regulated. We will also discuss potential applications of Drosophila CRISPR/Cas9 to model PRPS-dependent disorders and other metabolic diseases that are associated with nucleotide metabolism.

scholarly journals Reversible regulation of Cas12a activities by AcrVA5-mediated acetylation and CobB-mediated deacetylation

2021 ◽  
Author(s):  
Xiaoman Kang ◽  
Lei Yin ◽  
Songkuan Zhuang ◽  
Tianshuai Hu ◽  
Zhile Wu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Wide Spectrum ◽  
Defense System ◽  
Post Translational Modification ◽  
Defense Systems ◽  
Target Dna ◽  
Short Palindromic Repeat ◽  
Type V

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) system protects bacteria and archaea from the invasion of foreign genetic elements. To cope with the host CRISPR systems, phages have evolved many strategies, including the anti-CRISPR (Acr) proteins, to inactivate the Cas nucleases. Recently, it has been reported that the type V-A Cas12a effector can be acetylated and inactivated by AcrVA5, which is a GNAT-family acetyltransferase. However, it is unclear whether the host has any coping strategies to reactivate the defense system. Here we show that the AcrVA5-acetylated Cas12a can be deacetylated by bacterial deacetylase CobB, reactivating Cas12a for both in vitro cleavage of target DNA sequences and in vivo protection of the host from invasion of foreign nucleic acids. Therefore, this study not only shows the reversible regulation of Cas12a activities by post-translational modification but also reveals CobB as a secondary safeguard to bacterial CRISPR defense systems. In addition, we demonstrate that AcrVA5 is a wide-spectrum acetyltransferase, acetylating a large number of target proteins besides Cas12a, and the AcrVA5-acetylated targets can also be deacetylated by CobB.

Abstract 3056: Development of an integrated CRISPR interference system targeting ΔNp63 to treat lung and esophageal squamous cell carcinoma

2019 ◽  
Author(s):  
Masakazu Yoshida ◽  
Etsuko Yokota ◽  
Tetsushi Sakuma ◽  
Nagio Takigawa ◽  
Toshikazu Ushijima ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Esophageal Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Crispr Interference ◽  
Interference System

scholarly journals Histone H3 K36 Methylation Is Associated with Transcription Elongation in Schizosaccharomyces pombe

2005 ◽  
Vol 4 (8) ◽  
pp. 1446-1454 ◽  
Author(s):  
Stephanie A. Morris ◽  
Yoichiro Shibata ◽  
Ken-ichi Noma ◽  
Yuko Tsukamoto ◽  
Erin Warren ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Budding Yeast ◽  
Transcription Elongation ◽  
Synthetic Medium ◽  
Histone H3 ◽  
Yeast Cells ◽  
Pol Ii ◽  
Elongation Process

ABSTRACT Set2 methylation of histone H3 at lysine 36 (K36) has recently been shown to be associated with RNA polymerase II (Pol II) elongation in Saccharomyces cerevisiae. However, whether this modification is conserved and associated with transcription elongation in other organisms is not known. Here we report the identification and characterization of the Set2 ortholog responsible for K36 methylation in the fission yeast Schizosaccharomyces pombe. We find that similar to the budding yeast enzyme, S. pombe Set2 is also a robust nucleosome-selective H3 methyltransferase that is specific for K36. Deletion of the S. pombe set2 + gene results in complete abolishment of K36 methylation as well as a slow-growth phenotype on plates containing synthetic medium. These results indicate that Set2 is the sole enzyme responsible for this modification in fission yeast and is important for cell growth under stressed conditions. Using the chromatin immunoprecipitation assay, we demonstrate that K36 methylation in S. pombe is associated with the transcribed regions of Pol II-regulated genes and is devoid in regions that are not transcribed by Pol II. Consistent with a role for Set2 in transcription elongation, we find that S. pombe Set2 associates with the hyperphosphorylated form of Pol II and can fully rescue K36 methylation and Pol II interaction in budding yeast cells deleted for Set2. These results, along with our finding that K36 methylation is highly conserved among eukaryotes, imply a conserved role for this modification in the transcription elongation process.

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