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Konrad Schwefel, Stefanie Spiegler, Sabine Ameling, Christiane D Much, Robin A Pilz, Oliver Otto, Uwe Völker, Ute Felbor, Matthias Rath
CCM3, originally described as PDCD10, regulates blood-brain barrier integrity and vascular maturation in vivo. CCM3 loss-of-function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral- and plasmid-free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss-of-function CCM3 variants into human ECs and studied the molecular and functional effects of long-term CCM3 inactivation...
December 13, 2018: Journal of Cellular and Molecular Medicine
Winston X Yan, Pratyusha Hunnewell, Lauren E Alfonse, Jason M Carte, Elise Keston-Smith, Shanmugapriya Sothiselvam, Anthony J Garrity, Shaorong Chong, Kira S Makarova, Eugene V Koonin, David R Cheng, David A Scott
Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, g, h, and i. Cas12c, h, and i demonstrate RNA-guided double-stranded (ds) DNA interference activity. Cas12i exhibits markedly different efficiencies of crRNA spacer complementary and non-complementary strand cleavage resulting in predominant dsDNA nicking...
December 6, 2018: Science
Daniel O'Reilly, Zachary J Kartje, Eman A Ageely, Elise Malek-Adamian, Maryam Habibian, Annabelle Schofield, Christopher L Barkau, Kushal J Rohilla, Lauren B DeRossett, Austin T Weigle, Masad J Damha, Keith T Gagnon
CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity...
December 4, 2018: Nucleic Acids Research
Chensi Miao, Huiwei Zhao, Long Qian, Chunbo Lou
With a unique crRNA processing capability, the CRISPR associated Cpf1 protein holds great potential for multiplex gene regulation. Unlike the well-studied Cas9 protein, however, conversion of Cpf1 to a transcription regulator and its related properties have not been systematically explored yet. In this study, we investigated the mutation schemes and crRNA requirements for the DNase deactivated Cpf1 (dCpf1). By shortening the direct repeat sequence, we obtained genetically stable crRNA co-transcripts and improved gene repression with multiplex targeting...
March 2019: Synthetic and Systems Biotechnology
Ning Jia, Charlie Y Mo, Chongyuan Wang, Edward T Eng, Luciano A Marraffini, Dinshaw J Patel
Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on cryo-EM structures of Thermococcus onnurineus CsmcrRNA binary, CsmcrRNA -target RNA and CsmcrRNA -target RNAanti-tag ternary complexes in the 3.1 Å range. The topological features of the crRNA 5'-repeat tag explains the 5'-ruler mechanism for defining target cleavage sites, with accessibility of positions -2 to -5 within the 5'-repeat serving as sensors for avoidance of autoimmunity...
November 27, 2018: Molecular Cell
Lilan You, Jun Ma, Jiuyu Wang, Daria Artamonova, Min Wang, Liang Liu, Hua Xiang, Konstantin Severinov, Xinzheng Zhang, Yanli Wang
Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex...
November 26, 2018: Cell
Stefano Stella, Pablo Mesa, Johannes Thomsen, Bijoya Paul, Pablo Alcón, Simon B Jensen, Bhargav Saligram, Matias E Moses, Nikos S Hatzakis, Guillermo Montoya
Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation...
November 15, 2018: Cell
Zongliang Gao, Elena Herrera-Carrillo, Ben Berkhout
The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system expands the genome editing toolbox. This system exhibits several distinct features compared to the widely used CRISPR-Cas9 system, but has reduced gene editing efficiency. To optimize the CRISPR-Cpf1 (Cas12a) system, we report the inclusion of self-cleaving ribozymes that facilitate processing of the crRNA transcript to produce the precise guide molecule. Insertion of the 3'-terminal HDV ribozyme boosted the gene editing activity of the CRISPR-Cpf1 system ranging from 1...
November 23, 2018: RNA Biology
Ahsen Özcan, Patrick Pausch, Andreas Linden, Alexander Wulf, Karola Schühle, Johann Heider, Henning Urlaub, Thomas Heimerl, Gert Bange, Lennart Randau
Type IV CRISPR-Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR-Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome...
November 5, 2018: Nature Microbiology
Georg Mohr, Sukrit Silas, Jennifer L Stamos, Kira S Makarova, Laura M Markham, Jun Yao, Patricia Lucas-Elío, Antonio Sanchez-Amat, Andrew Z Fire, Eugene V Koonin, Alan M Lambowitz
Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity...
November 15, 2018: Molecular Cell
Sonali Majumdar, Michael P Terns
Diverse CRISPR-Cas immune systems protect archaea and bacteria from viruses and other mobile genetic elements. All CRISPR-Cas systems ultimately function by sequence-specific destruction of invading complementary nucleic acids. However, each CRISPR system uses compositionally distinct crRNP [CRISPR (cr) RNA/Cas protein] immune effector complexes to recognize and destroy invasive nucleic acids by unique molecular mechanisms. Previously, we found that Type I-A (Csa) effector crRNPs from Pyrococcus furiosus function in vivo to eliminate invader DNA...
October 3, 2018: Extremophiles: Life Under Extreme Conditions
Li Lin, Xiubin He, Tianyuan Zhao, Lingkai Gu, Yeqing Liu, Xiaoyu Liu, Hongyan Liu, Fayu Yang, Mengjun Tu, Lianchao Tang, Xianglian Ge, Changbao Liu, Junzhao Zhao, Zongming Song, Jia Qu, Feng Gu
FnCpf1-mediated genome-editing technologies have enabled a broad range of research and medical applications. Recently, we reported that FnCpf1 possesses activity in human cells and recognizes a more compatible PAM (protospacer adjacent motif, 5'-KYTV-3'), compared with the other two commonly used Cpf1 enzymes (AsCpf1 and LbCpf1), which requires a 5'-TTTN-3' PAM. However, due to the efficiency and fidelity, FnCpf1-based clinical and basic applications remain a challenge. The direct repeat (DR) sequence is one of the key elements for FnCpf1-mediated genome editing...
November 7, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
Mollie S Schubert, Edward Cedrone, Barry Neun, Mark A Behlke, Marina A Dobrovolskaia
Objectives: CRISPR/Cas9 is currently the primary tool used for genome editing in mammalian cells. To cleave and alter genomic DNA, both the Cas9 nuclease and a guide RNA ( gRNA ) must be present in the nucleus. One preferred method of introducing these reagents is direct transfection of a recombinant Cas9 protein complexed with a synthetic gRNA as a ribonucleoprotein (RNP) complex. It is well established from prior work in RNA interference that synthetic RNAs can induce a type I interferon (IFN) response that can limit the application of such methods both in vitro and in vivo...
2018: Journal of cytokine biology
Morito Kurata, Natalie K Wolf, Walker S Lahr, Madison T Weg, Mitchell G Kluesner, Samantha Lee, Kai Hui, Masano Shiraiwa, Beau R Webber, Branden S Moriarity
The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci...
2018: PloS One
Zhen-Hai Li, Min Liu, Xiao-Mei Lyu, Feng-Qing Wang, Dong-Zhi Wei
In this study, we focused on the applicability of CRISPR/Cpf1 in genome simplification of Saccharomyces cerevisiae and established a CRISPR/Cpf1 assisted method for rapid markerless large fragment deletion to facilitate laboratory evolution of geome of S. cerevisiae by rational genetic engineering. This method uses a Cpf1 expression plasmid and a crRNA array expression plasmid. The DNA fragment between two DSBs generated by CRISPR/Cpf1 can be cut off from the chromosome, along with re-ligation of the genomic endpoints of the DSBs...
September 9, 2018: Journal of Basic Microbiology
Su Bin Moon, Jeong Mi Lee, Jeong Gu Kang, Nan-Ee Lee, Dae-In Ha, Do Yon Kim, Sun Hee Kim, Kwangsun Yoo, Daesik Kim, Jeong-Heon Ko, Yong-Sam Kim
Genome editing has been harnessed through the development of CRISPR system, and the CRISPR from Prevotella and Francisella 1 (Cpf1) system has emerged as a promising alternative to CRISPR-Cas9 for use in various circumstances. Despite the inherent multiple advantages of Cpf1 over Cas9, the adoption of Cpf1 has been unsatisfactory because of target-dependent insufficient indel efficiencies. Here, we report an engineered CRISPR RNA (crRNA) for highly efficient genome editing by Cpf1, which includes a 20-base target-complementary sequence and a uridinylate-rich 3'-overhang...
September 7, 2018: Nature Communications
Lisa Nickel, Andrea Ulbricht, Omer S Alkhnbashi, Konrad U Förstner, Liam Cassidy, Katrin Weidenbach, Rolf Backofen, Ruth A Schmitz
The clustered regularly interspaced short palindromic repeat (CRISPR) system is a prokaryotic adaptive defense system against foreign nucleic acids. In the methanoarchaeon Methanosarcina mazei Gö1, two types of CRISPR-Cas systems are present (type I-B and type III-C). Both loci encode a Cas6 endonuclease, Cas6b-IB and Cas6b-IIIC, typically responsible for maturation of functional short CRISPR RNAs (crRNAs). To evaluate potential cross cleavage activity, we biochemically characterized both Cas6b proteins regarding their crRNA binding behavior and their ability to process pre-crRNA from the respective CRISPR array in vivo...
September 13, 2018: RNA Biology
Hyo Min Park, Hui Liu, Joann Wu, Anthony Chong, Vanessa Mackley, Christof Fellmann, Anirudh Rao, Fuguo Jiang, Hunghao Chu, Niren Murthy, Kunwoo Lee
Engineering of the Cpf1 crRNA has the potential to enhance its gene editing efficiency and non-viral delivery to cells. Here, we demonstrate that extending the length of its crRNA at the 5' end can enhance the gene editing efficiency of Cpf1 both in cells and in vivo. Extending the 5' end of the crRNA enhances the gene editing efficiency of the Cpf1 RNP to induce non-homologous end-joining and homology-directed repair using electroporation in cells. Additionally, chemical modifications on the extended 5' end of the crRNA result in enhanced serum stability...
August 17, 2018: Nature Communications
Tim Künne, Yifan Zhu, Fausia da Silva, Nico Konstantinides, Rebecca E McKenzie, Ryan N Jackson, Stan Jj Brouns
Prokaryotes use primed CRISPR adaptation to update their memory bank of spacers against invading genetic elements that have escaped CRISPR interference through mutations in their protospacer target site. We previously observed a trend that nucleotide-dependent mismatches between crRNA and the protospacer strongly influence the efficiency of primed CRISPR adaptation. Here we show that guanine-substitutions in the target strand of the protospacer are highly detrimental to CRISPR interference and interference-dependent priming, while cytosine-substitutions are more readily tolerated...
November 2, 2018: Nucleic Acids Research
Guilhem Faure, Sergey A Shmakov, Kira S Makarova, Yuri I Wolf, Alexandra B Crawley, Rodolphe Barrangou, Eugene V Koonin
Trans-activating CRISPR (tracr) RNA is a distinct RNA species that interacts with the CRISPR (cr) RNA to form the dual guide (g) RNA in type II and subtype V-B CRISPR-Cas systems. The tracrRNA-crRNA interaction is essential for pre-crRNA processing as well as target recognition and cleavage. The tracrRNA consists of an antirepeat, which forms an imperfect hybrid with the repeat in the crRNA, and a distal region containing a Rho-independent terminator. Exhaustive comparative analysis of the sequences and predicted structures of the Class 2 CRISPR guide RNAs shows that all these guide RNAs share distinct structural features, in particular, the nexus stem-loop that separates the repeat-antirepeat hybrid from the distal portion of the tracrRNA and the conserved GU pair at that end of the hybrid...
August 14, 2018: RNA Biology
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