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https://www.readbyqxmd.com/read/28040780/highly-efficient-cpf1-mediated-gene-targeting-in-mice-following-high-concentration-pronuclear-injection
#1
Dawn E Watkins-Chow, Gaurav K Varshney, Lisa J Garrett, Zelin Chen, Erin A Jimenez, Cecilia Rivas, Kevin S Bishop, Raman Sood, Ursula L Harper, William J Pavan, Shawn M Burgess
Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet or even surpass Cas9 targeting rates (approaching 100% targeting) but require higher concentrations of mRNA and guide. We also demonstrate that co-injecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.
December 30, 2016: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28000776/cpf1-is-a-versatile-tool-for-crispr-genome-editing-across-diverse-species-of-cyanobacteria
#2
Justin Ungerer, Himadri B Pakrasi
Cyanobacteria are the ideal organisms for the production of a wide range of bioproducts as they can convert CO2 directly into the desired end product using solar energy. Unfortunately, the engineering of cyanobacteria to create efficient cell factories has been impaired by the cumbersome genetic tools that are currently available for these organisms; especially when trying to accumulate multiple modifications. We sought to construct an efficient and precise tool for generating numerous markerless modifications in cyanobacteria using CRISPR technology and the alternative nuclease, Cpf1...
December 21, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27992409/in-vivo-high-throughput-profiling-of-crispr-cpf1-activity
#3
Hui K Kim, Myungjae Song, Jinu Lee, A Vipin Menon, Soobin Jung, Young-Mook Kang, Jae W Choi, Euijeon Woo, Hyun C Koh, Jin-Wu Nam, Hyongbum Kim
CRISPR from Prevotella and Francisella 1 (Cpf1) is an effector endonuclease of the class 2 CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) gene editing system. We developed a method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells, in a high-throughput manner. A library of >11,000 target sequence and guide RNA pairs was delivered into human cells using lentiviral vectors. Subsequent delivery of Cpf1 into this cell library induced insertions and deletions (indels) at the integrated synthetic target sequences, which allowed en masse evaluation of Cpf1 activity by using deep sequencing...
December 19, 2016: Nature Methods
https://www.readbyqxmd.com/read/27989439/c2c1-sgrna-complex-structure-reveals-rna-guided-dna-cleavage-mechanism
#4
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
https://www.readbyqxmd.com/read/27984729/pam-dependent-target-dna-recognition-and-cleavage-by-c2c1-crispr-cas-endonuclease
#5
Hui Yang, Pu Gao, Kanagalaghatta R Rajashankar, Dinshaw J Patel
C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA...
December 15, 2016: Cell
https://www.readbyqxmd.com/read/27918548/multiplex-gene-editing-by-crispr-cpf1-using-a-single-crrna-array
#6
Bernd Zetsche, Matthias Heidenreich, Prarthana Mohanraju, Iana Fedorova, Jeroen Kneppers, Ellen M DeGennaro, Nerges Winblad, Sourav R Choudhury, Omar O Abudayyeh, Jonathan S Gootenberg, Wen Y Wu, David A Scott, Konstantin Severinov, John van der Oost, Feng Zhang
Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.
January 2017: Nature Biotechnology
https://www.readbyqxmd.com/read/27905529/efficient-targeted-mutagenesis-of-rice-and-tobacco-genomes-using-cpf1-from-francisella-novicida
#7
Akira Endo, Mikami Masafumi, Hidetaka Kaya, Seiichi Toki
CRISPR/Cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. CRISPR from Prevotella and Francisella 1 (Cpf1) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. First, Cpf1 utilizes a thymidine-rich protospacer adjacent motif (PAM) while Cas9 prefers a guanidine-rich PAM. Cpf1 could be used as a sequence-specific nuclease to target AT-rich regions of a genome that Cas9 had difficulty accessing. Second, Cpf1 generates DNA ends with a 5' overhang, whereas Cas9 creates blunt DNA ends after cleavage...
December 1, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27895652/rapid-evolution-of-manifold-crispr-systems-for-plant-genome-editing
#8
REVIEW
Levi Lowder, Aimee Malzahn, Yiping Qi
Advanced CRISPR-Cas9 based technologies first validated in mammalian cell systems are quickly being adapted for use in plants. These new technologies increase CRISPR-Cas9's utility and effectiveness by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. Here, we review the current state of advanced CRISPR-Cas9 and Cpf1 capabilities in plants and cover the rapid evolution of these tools from first generation inducers of double strand breaks for basic genetic manipulations to second and third generation multiplexed systems with myriad functionalities, capabilities, and specialized applications...
2016: Frontiers in Plant Science
https://www.readbyqxmd.com/read/27875019/generation-of-targeted-mutant-rice-using-a-crispr-cpf1-system
#9
Rongfang Xu, Ruiying Qin, Hao Li, Dongdong Li, Li Li, Pengcheng Wei, Jianbo Yang
CRISPR-Cpf1 is a newly identified CRISPR-Cas system, and Cpf1 was recently engineered as a molecular tool for targeted genome editing in mammalian cells. To test whether the engineered CRISPR-Cpf1 system could induce the production of rice mutants, we selected two genome targets in the OsPDS and OsBEL genes. Our results show that both targets could be efficiently mutated in transgenic rice plants using CRISPR-Cpf1. We found that pre-crRNAs with a full-length direct repeat sequence exhibited considerably increased efficiencies compared with mature crRNAs...
November 22, 2016: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/27760213/inulin-supplementation-lowered-the-metabolic-defects-of-prolonged-exposure-to-chlorpyrifos-from-gestation-to-young-adult-stage-in-offspring-rats
#10
Julie Reygner, Lydia Lichtenberger, Ghada Elmhiri, Samir Dou, Narges Bahi-Jaber, Larbi Rhazi, Flore Depeint, Veronique Bach, Hafida Khorsi-Cauet, Latifa Abdennebi-Najar
Increasing evidence indicates that chlorpyrifos (CPF), an organophosphorus insecticide, is involved in metabolic disorders. We assess the hypothesis whether supplementation with prebiotics from gestation to adulthood, through a modulation of microbiota composition and fermentative activity, alleviates CPF induced metabolic disorders of 60 days old offspring. 5 groups of Wistar rats, from gestation until weaning, received two doses of CPF pesticide: 1 mg/kg/day (CPF1) or 3.5 mg/kg/day (CPF3.5) with free access to inulin (10g/L in drinking water)...
2016: PloS One
https://www.readbyqxmd.com/read/27669025/two-distinct-rnase-activities-of-crispr-c2c2-enable-guide-rna-processing-and-rna-detection
#11
Alexandra East-Seletsky, Mitchell R O'Connell, Spencer C Knight, David Burstein, Jamie H D Cate, Robert Tjian, Jennifer A Doudna
Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear...
October 13, 2016: Nature
https://www.readbyqxmd.com/read/27630115/cpf1-nucleases-demonstrate-robust-activity-to-induce-dna-modification-by-exploiting-homology-directed-repair-pathways-in-mammalian-cells
#12
Eszter Tóth, Nóra Weinhardt, Petra Bencsura, Krisztina Huszár, Péter I Kulcsár, András Tálas, Elfrieda Fodor, Ervin Welker
BACKGROUND: Cpf1 nucleases have recently been repurposed for site-specific genome modification. Two members of the Cpf1 family, the AsCpf1 from Acidaminococcus sp. and the LbCpf1 from Lachnospiraceae bacterium were shown to induce higher indel frequencies than SpCas9 when examining four randomly-selected target sequences for each type of nuclease. Whether they are a real match for Cas9 nucleases, however, remains to be verified. RESULTS: Here, we used AsCpf1 and LbCpf1 to induce homology directed repair, either single strand annealing (SSA) or homologous recombination (HR), in N2a mouse neuroblastoma cells...
2016: Biology Direct
https://www.readbyqxmd.com/read/27595403/augmenting-crispr-applications-in-drosophila-with-trna-flanked-sgrnas
#13
Fillip Port, Simon L Bullock
We present tRNA-based vectors for producing multiple clustered regularly interspaced short palindromic repeats (CRISPR) single guide RNAs (sgRNAs) from a single RNA polymerase II or III transcript in Drosophila. The system, which is based on liberation of sgRNAs by processing flanking tRNAs, permits highly efficient multiplexing of Cas9-based mutagenesis. We also demonstrate that the tRNA-sgRNA system markedly increases the efficacy of conditional gene disruption by Cas9 and can promote editing by the recently discovered RNA-guided endonuclease Cpf1...
October 2016: Nature Methods
https://www.readbyqxmd.com/read/27545116/impact-of-prenatal-and-postnatal-exposure-to-the-pesticide-chlorpyrifos-on-the-contraction-of-rat-ileal-muscle-strips-involvement-of-an-inducible-nitric-oxide-synthase-dependent-pathway
#14
W Darwiche, S Delanaud, S Dupont, H Ghamlouch, W Ramadan, W Joumaa, V Bach, J Gay-Quéheillard
BACKGROUND: Prenatal/postnatal exposure to insecticides has been linked to developmental disorders in adulthood. Chlorpyrifos (CPF) is a widely used organophosphorus acetylcholinesterase (AChE)-inhibiting insecticide. The present study established whether prenatal and postnatal exposure to CPF is associated with intestinal motor dysfunction in adult rats. METHODS: Three groups of pregnant rats were exposed to either CPF (1 or 5 mg/kg/day; the CPF1 and CPF5 groups) or vehicle (the control group) by gavage from gestational day 1 until weaning...
August 21, 2016: Neurogastroenterology and Motility: the Official Journal of the European Gastrointestinal Motility Society
https://www.readbyqxmd.com/read/27504781/erratum-genome-wide-analysis-reveals-specificities-of-cpf1-endonucleases-in-human-cells
#15
Daesik Kim, Jungeun Kim, Junho K Hur, Kyung Wook Been, Sun-Heui Yoon, Jin-Soo Kim
No abstract text is available yet for this article.
August 9, 2016: Nature Biotechnology
https://www.readbyqxmd.com/read/27444870/type-v-crispr-cas-cpf1-endonuclease-employs-a-unique-mechanism-for-crrna-mediated-target-dna-recognition
#16
Pu Gao, Hui Yang, Kanagalaghatta R Rajashankar, Zhiwei Huang, Dinshaw J Patel
CRISPR-Cas9 and CRISPR-Cpf1 systems have been successfully harnessed for genome editing. In the CRISPR-Cas9 system, the preordered A-form RNA seed sequence and preformed protein PAM-interacting cleft are essential for Cas9 to form a DNA recognition-competent structure. Whether the CRISPR-Cpf1 system employs a similar mechanism for target DNA recognition remains unclear. Here, we have determined the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with crRNA and target DNA. Structural comparison between the AsCpf1-crRNA-DNA ternary complex and the recently reported Lachnospiraceae bacterium Cpf1 (LbCpf1)-crRNA binary complex identifies a unique mechanism employed by Cpf1 for target recognition...
August 2016: Cell Research
https://www.readbyqxmd.com/read/27347757/genome-wide-specificities-of-crispr-cas-cpf1-nucleases-in-human-cells
#17
Benjamin P Kleinstiver, Shengdar Q Tsai, Michelle S Prew, Nhu T Nguyen, Moira M Welch, Jose M Lopez, Zachary R McCaw, Martin J Aryee, J Keith Joung
The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9). We also report that four to six bases at the 3' end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions...
August 2016: Nature Biotechnology
https://www.readbyqxmd.com/read/27294364/c-brick-a-new-standard-for-assembly-of-biological-parts-using-cpf1
#18
Shi-Yuan Li, Guo-Ping Zhao, Jin Wang
So far, several DNA assembly standards have been developed, enabling scientists to conveniently share and modify characterized DNA parts. However, a majority of the restriction endonucleases used in these standards bear short recognition sites (e.g., 6 bps in BioBrick standard), which are widely distributed and need to be removed before further construction, causing much inconvenience. Although homing endonucleases, which recognize long DNA sequences, can be used for DNA assembly (e.g., iBrick standard), long scars will be left between parts, limiting their application...
December 16, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27272387/generation-of-knockout-mice-by-cpf1-mediated-gene-targeting
#19
Yongsub Kim, Seung-A Cheong, Jong Geol Lee, Sang-Wook Lee, Myeong Sup Lee, In-Jeoung Baek, Young Hoon Sung
No abstract text is available yet for this article.
August 2016: Nature Biotechnology
https://www.readbyqxmd.com/read/27272385/targeted-mutagenesis-in-mice-by-electroporation-of-cpf1-ribonucleoproteins
#20
Junho K Hur, Kyoungmi Kim, Kyung Wook Been, Gayoung Baek, Sunghyeok Ye, Junseok W Hur, Seuk-Min Ryu, Youn Su Lee, Jin-Soo Kim
No abstract text is available yet for this article.
August 2016: Nature Biotechnology
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