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https://www.readbyqxmd.com/read/27804953/structural-roles-of-guide-rnas-in-the-nuclease-activity-of-cas9-endonuclease
#1
Youngbin Lim, So Young Bak, Keewon Sung, Euihwan Jeong, Seung Hwan Lee, Jin-Soo Kim, Sangsu Bae, Seong Keun Kim
The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex...
November 2, 2016: Nature Communications
https://www.readbyqxmd.com/read/27616359/crispr-as-a-strong-gene-editing-tool
#2
Shengfu Shen, Tiing Jen Loh, Hongling Shen, Xuexiu Zheng, Haihong Shen
Clustered regularly-interspaced short palindromic repeats (CRISPR) is a new and effective genetic editing tool. CRISPR was initially found in bacteria to protect it from virus invasions. In the first step, specific DNA strands of virus are identified by guide RNA that is composed of crRNA and tracrRNA. Then RNAse III is required for producing crRNA from pre-crRNA. In The second step, a crRNA:tracrRNA:Cas9 complex guides RNase III to cleave target DNA. After cleavage of DNA by CRISPR-Cas9, DNA can be fixed by Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR)...
September 12, 2016: BMB Reports
https://www.readbyqxmd.com/read/27594566/the-crispr-cas-system-from-bacterial-immunity-to-genome-engineering
#3
Maria Czarnek, Joanna Bereta
Precise and efficient genome modifications present a great value in attempts to comprehend the roles of particular genes and other genetic elements in biological processes as well as in various pathologies. In recent years novel methods of genome modification known as genome editing, which utilize so called "programmable" nucleases, came into use. A true revolution in genome editing has been brought about by the introduction of the CRISP-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) system, in which one of such nucleases, i...
2016: Postȩpy Higieny i Medycyny Doświadczalnej
https://www.readbyqxmd.com/read/27545104/increasing-the-performance-of-pooled-crispr-cas9-drop-out-screening
#4
Benedict C S Cross, Steffen Lawo, Caroline R Archer, Jessica R Hunt, Joanne L Yarker, Alessandro Riccombeni, Annette S Little, Nicola J McCarthy, Jonathan D Moore
Components of the type II CRISPR-Cas complex in bacteria have been used successfully in eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation and functional genomic screens. Such developments are providing new opportunities for drug target identification and validation, particularly with the application of pooled genetic screening. As CRISPR-Cas is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27441384/conjugation-and-evaluation-of-triazole-linked-single-guide-rna-for-crispr-cas9-gene-editing
#5
Kaizhang He, Eldon T Chou, Shawn Begay, Emily M Anderson, Anja van Brabant Smith
The CRISPR-Cas9 gene editing system requires Cas9 endonuclease and guide RNAs (either the natural dual RNA consisting of crRNA and tracrRNA or a chimeric single guide RNA) that direct site-specific double-stranded DNA cleavage. This communication describes a click ligation approach that uses alkyne-azide cycloaddition to generate a triazole-linked single guide RNA (sgRNA). The conjugated sgRNA shows efficient and comparable genome editing activity to natural dual RNA and unmodified sgRNA constructs.
October 4, 2016: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/27374403/versatility-of-chemically-synthesized-guide-rnas-for-crispr-cas9-genome-editing
#6
REVIEW
Melissa L Kelley, Žaklina Strezoska, Kaizhang He, Annaleen Vermeulen, Anja van Brabant Smith
The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments. This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications...
September 10, 2016: Journal of Biotechnology
https://www.readbyqxmd.com/read/27371605/guide-rnas-a-glimpse-at-the-sequences-that-drive-crispr-cas-systems
#7
Alexandra E Briner, Rodolphe Barrangou
CRISPR-Cas systems provide adaptive immunity in bacteria and archaea. Although there are two main classes of CRISPR-Cas systems defined by gene content, interfering RNA biogenesis, and effector proteins, Type II systems have recently been exploited on a broad scale to develop next-generation genetic engineering and genome-editing tools. Conveniently, Type II systems are streamlined and rely on a single protein, Cas9, and a guide RNA molecule, comprised of a CRISPR RNA (crRNA) and trans-acting CRISPR RNA (tracrRNA), to achieve effective and programmable nucleic acid targeting and cleavage...
2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27371591/prediction-and-validation-of-native-and-engineered-cas9-guide-sequences
#8
Alexandra E Briner, Emily D Henriksen, Rodolphe Barrangou
Cas9-based technologies rely on native elements of Type II CRISPR-Cas bacterial immune systems, including the trans-activating CRISPR RNA (tracrRNA), CRISPR RNA (crRNA), Cas9 protein, and protospacer-adjacent motif (PAM). The tracrRNA and crRNA form an RNA duplex that guides the Cas9 endonuclease to complementary nucleic acid sequences. Mechanistically, Cas9 initiates interactions by binding to the target PAM sequence and interrogating the target DNA in a 3'-to-5' manner. Complementarity between the guide RNA and the target DNA is key...
2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27140916/characterizing-cas9-protospacer-adjacent-motifs-with-high-throughput-sequencing-of-library-depletion-experiments
#9
Jonathan L Braff, Stephanie J Yaung, Kevin M Esvelt, George M Church
This protocol outlines a general approach for characterizing the protospacer-adjacent motifs (PAMs) of Cas9 orthologs. It uses a three-plasmid system: One plasmid carries Cas9 and its tracrRNA, a second targeting vector contains the spacer and repeat, and the third plasmid encodes the targeted sequence (as the protospacer) with varying PAM sequences. It leverages the Cas9 nuclease activity to cleave and destroy plasmids that bear a compatible PAM. The level of depletion of a library of targeted plasmids after Cas9-mediated selection can then be assessed by deep sequencing to reveal candidate PAMs for downstream validation...
2016: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/27096362/the-crispr-associated-dna-cleaving-enzyme-cpf1-also-processes-precursor-crispr-rna
#10
Ines Fonfara, Hagen Richter, Majda Bratovič, Anaïs Le Rhun, Emmanuelle Charpentier
CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA. The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA) and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA...
April 28, 2016: Nature
https://www.readbyqxmd.com/read/26972821/utilization-of-the-crispr-cas9-system-for-the-efficient-production-of-mutant-mice-using-crrna-tracrrna-with-cas9-nickase-and-foki-dcas9
#11
Miho Terao, Moe Tamano, Satoshi Hara, Tomoko Kato, Masato Kinoshita, Shuji Takada
The CRISPR/Cas9 system is a powerful genome editing tool for the production of genetically modified animals. To produce mutant mice, chimeric single-guide RNA (sgRNA) is cloned in a plasmid vector and a mixture of sgRNA and Cas9 are microinjected into the fertilized eggs. An issue associated with gene manipulation using the CRISPR/Cas9 system is that there can be off-target effects. To simplify the production of mutant mice with low risks of off-target effects caused by the CRISPR/Cas9 system, we demonstrated that genetically modified mice can be efficiently obtained using chemically synthesized CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and modified Cas9s, such as the nickase version and FokI-fused catalytically inactive Cas9, by microinjection into fertilized eggs...
July 29, 2016: Experimental Animals
https://www.readbyqxmd.com/read/26939437/-advances-in-crispr-cas9-mediated-gene-editing
#12
REVIEW
Cong Li, Wenguang Cao
Clustered regulatory interspaced short palindromic repeats (CRISPR) found in bacteria and archaea genome that contains multiple short repeats loci, provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage. The first inkling of this hot new genetic engineering tool turned up in 1987, when a research team observed an oddly repetitive sequence at one end of a bacterial gene. Now three types of CRISPR/Cas system have been identified: types I, II and III. In the type II CRISPR/Cas9 system, short segments of foreign DNA termed 'spacers' are integrated within the CRISPR genomic loci, transcribed and processed into short CRISPR RNA (crRNA)...
November 2015: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/26782639/the-neisseria-meningitidis-crispr-cas9-system-enables-specific-genome-editing-in-mammalian-cells
#13
Ciaran M Lee, Thomas J Cradick, Gang Bao
The clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence...
March 2016: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/26671237/optimizing-sgrna-structure-to-improve-crispr-cas9-knockout-efficiency
#14
Ying Dang, Gengxiang Jia, Jennie Choi, Hongming Ma, Edgar Anaya, Chunting Ye, Premlata Shankar, Haoquan Wu
BACKGROUND: Single-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)-transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency. RESULTS: Here, we systematically investigate the effect of these two elements on knockout efficiency and showed that modifying the sgRNA structure by extending the duplex length and mutating the fourth thymine of the continuous sequence of thymines to cytosine or guanine significantly, and sometimes dramatically, improves knockout efficiency in cells...
2015: Genome Biology
https://www.readbyqxmd.com/read/26593719/discovery-and-functional-characterization-of-diverse-class-2-crispr-cas-systems
#15
Sergey Shmakov, Omar O Abudayyeh, Kira S Makarova, Yuri I Wolf, Jonathan S Gootenberg, Ekaterina Semenova, Leonid Minakhin, Julia Joung, Silvana Konermann, Konstantin Severinov, Feng Zhang, Eugene V Koonin
Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent...
November 5, 2015: Molecular Cell
https://www.readbyqxmd.com/read/26589814/synthetic-crispr-rna-cas9-guided-genome-editing-in-human-cells
#16
Meghdad Rahdar, Moira A McMahon, Thazha P Prakash, Eric E Swayze, C Frank Bennett, Don W Cleveland
Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells...
December 22, 2015: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/26474066/dnase-h-activity-of-neisseria-meningitidis-cas9
#17
Yan Zhang, Rakhi Rajan, H Steven Seifert, Alfonso Mondragón, Erik J Sontheimer
Type II CRISPR systems defend against invasive DNA by using Cas9 as an RNA-guided nuclease that creates double-stranded DNA breaks. Dual RNAs (CRISPR RNA [crRNA] and tracrRNA) are required for Cas9's targeting activities observed to date. Targeting requires a protospacer adjacent motif (PAM) and crRNA-DNA complementarity. Cas9 orthologs (including Neisseria meningitidis Cas9 [NmeCas9]) have also been adopted for genome engineering. Here we examine the DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns...
October 15, 2015: Molecular Cell
https://www.readbyqxmd.com/read/26422227/cpf1-is-a-single-rna-guided-endonuclease-of-a-class-2-crispr-cas-system
#18
Bernd Zetsche, Jonathan S Gootenberg, Omar O Abudayyeh, Ian M Slaymaker, Kira S Makarova, Patrick Essletzbichler, Sara E Volz, Julia Joung, John van der Oost, Aviv Regev, Eugene V Koonin, Feng Zhang
The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif...
October 22, 2015: Cell
https://www.readbyqxmd.com/read/26230606/occurrence-and-diversity-of-crispr-cas-systems-in-the-genus-bifidobacterium
#19
Alexandra E Briner, Gabriele Andrea Lugli, Christian Milani, Sabrina Duranti, Francesca Turroni, Miguel Gueimonde, Abelardo Margolles, Douwe van Sinderen, Marco Ventura, Rodolphe Barrangou
CRISPR-Cas systems constitute adaptive immune systems for antiviral defense in bacteria. We investigated the occurrence and diversity of CRISPR-Cas systems in 48 Bifidobacterium genomes to gain insights into the diversity and co-evolution of CRISPR-Cas systems within the genus and investigate CRISPR spacer content. We identified the elements necessary for the successful targeting and inference of foreign DNA in select Type II CRISPR-Cas systems, including the tracrRNA and target PAM sequence. Bifidobacterium species have a very high frequency of CRISPR-Cas occurrence (77%, 37 of 48)...
2015: PloS One
https://www.readbyqxmd.com/read/26187122/high-efficiency-homology-directed-genome-editing-in-caenorhabditis-elegans-using-crispr-cas9-ribonucleoprotein-complexes
#20
Alexandre Paix, Andrew Folkmann, Dominique Rasoloson, Geraldine Seydoux
Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection...
September 2015: Genetics
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