Read by QxMD icon Read


Zachary J Kartje, Christopher L Barkau, Kushal J Rohilla, Eman A Ageely, Keith T Gagnon
DNA substitutions in RNA can probe the importance of A-form struc-ture, 2'-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. Using this approach, we found that Cas9 bio-chemical activity tolerated significant substitution with DNA nucleo-tides in the CRISPR RNA (crRNA). Only minimal RNA content was needed in or near the seed region. Simultaneous substitution at all positions with predicted crRNA-Cas9 2'-hydroxyl contacts had no effect on enzyme activity. The trans-activating crRNA (tracrRNA) also tolerated over 50% substitution with DNA...
May 10, 2018: Biochemistry
Gaetan Burgio
In the recent Research Highlight [1], it has been highlighted that part b of Fig. 1 was incorrectly labelled as "sgRNA + tracrRNA" instead of "sgRNA (crRNA + tracrRNA)". An updated Figure 1, including also the amended figure legend has therefore been provided below.
March 26, 2018: Genome Biology
Gaurav Dugar, Ryan T Leenay, Sara K Eisenbart, Thorsten Bischler, Belinda U Aul, Chase L Beisel, Cynthia M Sharma
Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Campylobacter jejuni Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs...
March 1, 2018: Molecular Cell
Juliane Behler, Kundan Sharma, Viktoria Reimann, Annegret Wilde, Henning Urlaub, Wolfgang R Hess
Specialized RNA endonucleases for the maturation of clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNAs (crRNAs) are critical in CRISPR-CRISPR-associated protein (Cas) defence mechanisms. The Cas6 and Cas5d enzymes are the RNA endonucleases in many class 1 CRISPR-Cas systems. In some class 2 systems, maturation and effector functions are combined within a single enzyme or maturation proceeds through the combined actions of RNase III and trans-activating CRISPR RNAs (tracrRNAs). Three separate CRISPR-Cas systems exist in the cyanobacterium Synechocystis sp...
March 2018: Nature Microbiology
Emily M Anderson, Shawn McClelland, Elena Maksimova, Žaklina Strezoska, Megan Basila, Alexandra E Briner, Rodolphe Barrangou, Anja van Brabant Smith
While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L. gasseri and found that it has modest activity in a cell-free lysate assay but no activity in mammalian cells even when altering promoter, position of tag sequences and NLS, and length of crRNA:tracrRNA. In the lysate assay we tested over 400 sequential crRNA target sequences and found that the Lga Cas9 PAM is NNGA/NDRA, different than NTAA predicted from the native bacterial host...
2018: PloS One
Yukiko U Inoue, Yuki Morimoto, Mikio Hoshino, Takayoshi Inoue
Pax6 encodes a transcription factor that plays pivotal roles in eye development, early brain patterning, neocortical arealization, and so forth. Visualization of Pax6 expression dynamics in these events could offer numerous advantages to neurodevelopmental studies. While CRISPR/Cas9 system has dramatically accelerated one-step generation of knock-out mouse, establishment of gene-cassette knock-in mouse via zygote injection has been considered insufficient due to its low efficiency. Recently, an improved CRISPR/Cas9 system for effective gene-cassette knock-in has been reported, where the native form of guide RNAs (crRNA and tracrRNA) assembled with recombinant Cas9 protein are directly delivered into mouse fertilized eggs...
January 31, 2018: Neuroscience Research
Jianyong Xu, Wei Lian, Yuning Jia, Lingyun Li, Zhong Huang
The genome editing tool Cas9-gRNA (guide RNA) has been successfully applied in different cell types and organisms with high efficiency. However, more efforts need to be made to enhance both efficiency and specificity. In the current study, we optimized the guide RNA structure of Streptococcus pyogenes CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system to improve its genome editing efficiency. Comparing with the original functional structure of guide RNA, which is composed of crRNA and tracrRNA, the widely used chimeric gRNA has shorter crRNA and tracrRNA sequence...
November 7, 2017: Oncotarget
Megan Basila, Melissa L Kelley, Anja van Brabant Smith
Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications...
2017: PloS One
Rie Sawamura, Natsumi Osafune, Takahiro Murakami, Fumiya Furukawa, Takeshi Kitano
Several animal models generated by genome editing methods develop somatic mosaic mutations including wild-type genome sequence in F0 generation because it is difficult to use editing tools at the one-cell stage. Producing complete knockout animals quickly is a great advantage in determining the function of target genes. This study investigated the generation of F0 knockout medaka using the CRISPR/Cas9 system. To determine whether this editing system induced mutations in the medaka genome at the one-cell stage, recombinant Cas9 protein, tracrRNA and crRNA for dead end (dnd), which is essential for germ cell development, were injected into one-cell stage embryos of olvas-DsRedExpress transgenic medaka...
August 2017: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Rolen M Quadros, Hiromi Miura, Donald W Harms, Hisako Akatsuka, Takehito Sato, Tomomi Aida, Ronald Redder, Guy P Richardson, Yutaka Inagaki, Daisuke Sakai, Shannon M Buckley, Parthasarathy Seshacharyulu, Surinder K Batra, Mark A Behlke, Sarah A Zeiner, Ashley M Jacobi, Yayoi Izu, Wallace B Thoreson, Lisa D Urness, Suzanne L Mansour, Masato Ohtsuka, Channabasavaiah B Gurumurthy
BACKGROUND: Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models...
May 17, 2017: Genome Biology
Žaklina Strezoska, Matthew R Perkett, Eldon T Chou, Elena Maksimova, Emily M Anderson, Shawn McClelland, Melissa L Kelley, Annaleen Vermeulen, Anja van Brabant Smith
The CRISPR-Cas9 system has been utilized for large-scale, loss-of-function screens mainly using lentiviral pooled formats and cell-survival phenotypic assays. Screening in an arrayed format expands the types of phenotypic readouts that can be used to now include high-content, morphology-based assays, and with the recent availability of synthetic crRNA libraries, new studies are emerging. Here, we use a cell cycle reporter cell line to perform an arrayed, synthetic crRNA:tracrRNA screen targeting 169 genes (>600 crRNAs) and used high content analysis (HCA) to identify genes that regulate the cell cycle...
June 10, 2017: Journal of Biotechnology
Fuguo Jiang, Jennifer A Doudna
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes...
May 22, 2017: Annual Review of Biophysics
Kumiko Ui-Tei, Shohei Maruyama, Yuko Nakano
Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein...
June 2017: Genome Génome / Conseil National de Recherches Canada
Xiaolong Ma, Chao Chen, Jennifer Veevers, XinMin Zhou, Robert S Ross, Wei Feng, Ju Chen
Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations...
February 8, 2017: Scientific Reports
Liang Liu, Peng Chen, Min Wang, Xueyan Li, Jiuyu Wang, Maolu Yin, Yanli Wang
C2c1 is a type V-B CRISPR-Cas system dual-RNA-guided DNA endonuclease. Here, we report the crystal structure of Alicyclobacillus acidoterrestris C2c1 in complex with a chimeric single-molecule guide RNA (sgRNA). AacC2c1 exhibits a bi-lobed architecture consisting of a REC and NUC lobe. The sgRNA scaffold forms a tetra-helical structure, distinct from previous predictions. The crRNA is located in the central channel of C2c1, and the tracrRNA resides in an external surface groove. Although AacC2c1 lacks a PAM-interacting domain, our analysis revealed that the PAM duplex has a similar binding position found in Cpf1...
January 19, 2017: Molecular Cell
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
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)...
January 2017: BMB Reports
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...
September 1, 2016: Postȩpy Higieny i Medycyny Doświadczalnej
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
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
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"