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https://www.readbyqxmd.com/read/28431230/structural-basis-for-guide-rna-processing-and-seed-dependent-dna-targeting-by-crispr-cas12a
#1
Daan C Swarts, John van der Oost, Martin Jinek
The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding...
April 20, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28417998/marker-free-coselection-for-crispr-driven-genome-editing-in-human-cells
#2
Daniel Agudelo, Alexis Duringer, Lusiné Bozoyan, Caroline C Huard, Sophie Carter, Jeremy Loehr, Dafni Synodinou, Mathieu Drouin, Jayme Salsman, Graham Dellaire, Josée Laganière, Yannick Doyon
Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems...
April 17, 2017: Nature Methods
https://www.readbyqxmd.com/read/28387220/targeted-activation-of-diverse-crispr-cas-systems-for-mammalian-genome-editing-via-proximal-crispr-targeting
#3
Fuqiang Chen, Xiao Ding, Yongmei Feng, Timothy Seebeck, Yanfang Jiang, Gregory D Davis
Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner...
April 7, 2017: Nature Communications
https://www.readbyqxmd.com/read/28375596/efficient-transcriptional-gene-repression-by-type-v-a-crispr-cpf1-from-eubacterium-eligens
#4
Seong Keun Kim, Haseong Kim, Woo-Chan Ahn, Kwang-Hyun Park, Eui-Jeon Woo, Dae-Hee Lee, Seung-Goo Lee
Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is an emerging technology for artificial gene regulation. Type II CRISPR-Cas endonuclease Cas9 is the most widely used protein for gene regulation with CRISPRi. Here, we present type V-A CRISPR-Cas endonuclease Cpf1-based CRISPRi. We constructed an l-rhamnose-inducible CRISPRi system with DNase-deactivated Cpf1 from Eubacterium eligens (EedCpf1) and compared its performance with catalytically deactivated Cas9 from Streptococcus pyogenes (SpdCas9)...
April 11, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28371222/new-variants-of-crispr-rna-guided-genome-editing-enzymes
#5
REVIEW
Jana Murovec, Žan Pirc, Bing Yang
CRISPR-mediated genome editing using the Streptococcus pyogenes Cas9 enzyme is revolutionizing life science by providing new, precise, facile and high throughput tools for genetic modification by the specific targeting of double-strand breaks in the genome of hosts. Plant biotechnologists have extensively used the S. pyogenes Cas9-based system since its inception in 2013. However, there are still some limitations to its even broader usage in plants. Major restrictions, especially in agricultural biotechnology, are the currently unclear regulatory status of plants modified with CRISPR/Cas9 and the lack of suitable delivery methods for some plant species...
April 1, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28349358/crispr-cas9-and-crispr-cpf1-mediated-targeting-of-a-stomatal-developmental-gene-epfl9-in-rice
#6
Xiaojia Yin, Akshaya K Biswal, Jacqueline Dionora, Kristel M Perdigon, Christian P Balahadia, Shamik Mazumdar, Caspar Chater, Hsiang-Chun Lin, Robert A Coe, Tobias Kretzschmar, Julie E Gray, Paul W Quick, Anindya Bandyopadhyay
CRISPR-Cas9/Cpf1 system with its unique gene targeting efficiency, could be an important tool for functional study of early developmental genes through the generation of successful knockout plants. The introduction and utilization of systems biology approaches have identified several genes that are involved in early development of a plant and with such knowledge a robust tool is required for the functional validation of putative candidate genes thus obtained. The development of the CRISPR-Cas9/Cpf1 genome editing system has provided a convenient tool for creating loss of function mutants for genes of interest...
March 27, 2017: Plant Cell Reports
https://www.readbyqxmd.com/read/28261237/progress-in-genome-editing-technology-and-its-application-in-plants
#7
REVIEW
Kai Zhang, Nadia Raboanatahiry, Bin Zhu, Maoteng Li
Genome editing technology (GET) is a versatile approach that has progressed rapidly as a mechanism to alter the genotype and phenotype of organisms. However, conventional genome modification using GET cannot satisfy current demand for high-efficiency and site-directed mutagenesis, retrofitting of artificial nucleases has developed into a new avenue within this field. Based on mechanisms to recognize target genes, newly-developed GETs can generally be subdivided into three cleavage systems, protein-dependent DNA cleavage systems (i...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28260792/genome-editing-the-efficient-tool-crispr-cpf1
#8
Magdy M Mahfouz
No abstract text is available yet for this article.
March 3, 2017: Nature Plants
https://www.readbyqxmd.com/read/28211909/a-crispr-cpf1-system-for-efficient-genome-editing-and-transcriptional-repression-in-plants
#9
Xu Tang, Levi G Lowder, Tao Zhang, Aimee A Malzahn, Xuelian Zheng, Daniel F Voytas, Zhaohui Zhong, Yiyi Chen, Qiurong Ren, Qian Li, Elida R Kirkland, Yong Zhang, Yiping Qi
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 has emerged as an effective genome editing tool in animals. Here we compare the activity of Cpf1 from Acidaminococcus sp. BV3L6 (As) and Lachnospiraceae bacterium ND2006 (Lb) in plants, using a dual RNA polymerase II promoter expression system. LbCpf1 generated biallelic mutations at nearly 100% efficiency at four independent sites in rice T0 transgenic plants. Moreover, we repurposed AsCpf1 and LbCpf1 for efficient transcriptional repression in Arabidopsis, and demonstrated a more than tenfold reduction in miR159b transcription...
February 17, 2017: Nature Plants
https://www.readbyqxmd.com/read/28205546/crispr-cpf1-mediated-dna-free-plant-genome-editing
#10
Hyeran Kim, Sang-Tae Kim, Jahee Ryu, Beum-Chang Kang, Jin-Soo Kim, Sang-Gyu Kim
Cpf1, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif and induces cohesive double-stranded breaks at the target site guided by a single CRISPR RNA (crRNA). Here we show that Cpf1 can be used as a tool for DNA-free editing of plant genomes. We describe the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco. Designed crRNAs are unique and do not have similar sequences (≤3 mismatches) in the entire soybean reference genome...
February 16, 2017: Nature Communications
https://www.readbyqxmd.com/read/28178246/erratum-multiplex-gene-editing-by-crispr-cpf1-using-a-single-crrna-array
#11
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
No abstract text is available yet for this article.
February 8, 2017: Nature Biotechnology
https://www.readbyqxmd.com/read/28140746/cas9-cpf1-and-c2c1-2-3-what-s-next
#12
Shota Nakade, Takashi Yamamoto, Tetsushi Sakuma
Since the rapid emergence of clustered regulatory interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system, developed as a genome engineering tool in 2012-2013, most researchers in the life science field have had a fixated interest in this fascinating technology. CRISPR-Cas9 is an RNA-guided DNA endonuclease system, which consists of Cas9 nuclease defining a few targeting base via protospacer adjacent motif complexed with easily customizable single guide RNA targeting around 20-bp genomic sequence...
January 31, 2017: Bioengineered
https://www.readbyqxmd.com/read/28115632/the-cctl-cpf1-assisted-cutting-and-taq-dna-ligase-assisted-ligation-method-for-efficient-editing-of-large-dna-constructs-in-vitro
#13
Chao Lei, Shi-Yuan Li, Jia-Kun Liu, Xuan Zheng, Guo-Ping Zhao, Jin Wang
As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in DNA assembly. Here, the Cpf1 cleavage sites were precisely characterized, where the cleavage site on the target strand was around the 22nd base relative to the protospacer adjacent motif site, but the cleavage on the non-target strand was affected by the spacer length. When the spacer length was 20 nt or longer, Cpf1 mainly cleaved around the 14th and the 18th bases on the non-target strand; otherwise, with a shorter spacer (i...
January 23, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28111461/diversity-and-evolution-of-class-2-crispr-cas-systems
#14
REVIEW
Sergey Shmakov, Aaron Smargon, David Scott, David Cox, Neena Pyzocha, Winston Yan, Omar O Abudayyeh, Jonathan S Gootenberg, Kira S Makarova, Yuri I Wolf, Konstantin Severinov, Feng Zhang, Eugene V Koonin
Class 2 CRISPR-Cas systems are characterized by effector modules that consist of a single multidomain protein, such as Cas9 or Cpf1. We designed a computational pipeline for the discovery of novel class 2 variants and used it to identify six new CRISPR-Cas subtypes. The diverse properties of these new systems provide potential for the development of versatile tools for genome editing and regulation. In this Analysis article, we present a comprehensive census of class 2 types and class 2 subtypes in complete and draft bacterial and archaeal genomes, outline evolutionary scenarios for the independent origin of different class 2 CRISPR-Cas systems from mobile genetic elements, and propose an amended classification and nomenclature of CRISPR-Cas...
March 2017: Nature Reviews. Microbiology
https://www.readbyqxmd.com/read/28040780/highly-efficient-cpf1-mediated-gene-targeting-in-mice-following-high-concentration-pronuclear-injection
#15
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 coinjecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.
February 9, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28000776/cpf1-is-a-versatile-tool-for-crispr-genome-editing-across-diverse-species-of-cyanobacteria
#16
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
#17
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
#18
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
#19
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
#20
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
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