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https://www.readbyqxmd.com/read/28646112/crispr-cas12a-assisted-recombineering-in-bacteria
#1
Mei-Yi Yan, Hai-Qin Yan, Gai-Xian Ren, Ju-Ping Zhao, Xiao-Peng Guo, Yi-Cheng Sun
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria...
June 23, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28628131/a-crispr-cpf1-system-for-efficient-genome-editing-and-transcriptional-repression-in-plants
#2
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
This corrects the article DOI: 10.1038/nplants.2017.18.
June 19, 2017: Nature Plants
https://www.readbyqxmd.com/read/28628097/cpf1-proteins-excise-crispr-rnas-from-mrna-transcripts-in-mammalian-cells
#3
Guocai Zhong, Haimin Wang, Yujun Li, Mai H Tran, Michael Farzan
Cpf1 is a CRISPR effector protein that has greater specificity than Streptococcus pyogenes Cas9 (SpCas9) in genome-editing applications. Here we show that Lachnospiraceae bacterium (Lb) and Acidaminococus sp. (As) Cpf1 orthologs have RNase activities that can excise multiple CRISPR RNAs (crRNAs) from a single RNA polymerase II-driven RNA transcript expressed in mammalian cells. This property simplifies modification of multiple genomic targets and can be used to increase the efficiency of Cpf1-mediated editing...
June 19, 2017: Nature Chemical Biology
https://www.readbyqxmd.com/read/28619534/cryptochrome-photoreceptors-in-green-algae-unexpected-versatility-of-mechanisms-and-functions
#4
Tilman Kottke, Sabine Oldemeyer, Sandra Wenzel, Yong Zou, Maria Mittag
Green algae have a highly complex and diverse set of cryptochrome photoreceptor candidates including members of the following subfamilies: plant, plant-like, animal-like, DASH and cryptochrome photolyase family 1 (CPF1). While some green algae encode most or all of them, others lack certain members. Here we present an overview about functional analyses of so far investigated cryptochrome photoreceptors from the green algae Chlamydomonas reinhardtii (plant and animal-like cryptochromes) and Ostreococcus tauri (CPF1) with regard to their biological significance and spectroscopic properties...
May 31, 2017: Journal of Plant Physiology
https://www.readbyqxmd.com/read/28607761/multiplex-gene-regulation-by-crispr-ddcpf1
#5
Xiaochun Zhang, Jingman Wang, Qiuxiang Cheng, Xuan Zheng, Guoping Zhao, Jin Wang
The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 system has been widely applied in both transcriptional regulation and epigenetic studies. However, for multiple targets, independent expression of multiple single guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem, we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We demonstrated that ddCpf1 alone could be employed for gene repression in Escherichia coli, and the repression was more effective with CRISPR RNAs (crRNAs) specifically targeting to the template strand of its target genes, which was different from that of dCas9...
2017: Cell Discovery
https://www.readbyqxmd.com/read/28595896/structural-basis-for-the-altered-pam-recognition-by-engineered-crispr-cpf1
#6
Hiroshi Nishimasu, Takashi Yamano, Linyi Gao, Feng Zhang, Ryuichiro Ishitani, Osamu Nureki
The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively...
June 6, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28581492/engineered-cpf1-variants-with-altered-pam-specificities
#7
Linyi Gao, David B T Cox, Winston X Yan, John C Manteiga, Martin W Schneider, Takashi Yamano, Hiroshi Nishimasu, Osamu Nureki, Nicola Crosetto, Feng Zhang
The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells...
June 5, 2017: Nature Biotechnology
https://www.readbyqxmd.com/read/28562584/structure-of-the-cpf1-endonuclease-r-loop-complex-after-target-dna-cleavage
#8
Stefano Stella, Pablo Alcón, Guillermo Montoya
Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA...
May 31, 2017: Nature
https://www.readbyqxmd.com/read/28532598/crispr-cpf1-a-new-tool-for-plant-genome-editing
#9
Syed Shan-E-Ali Zaidi, Magdy M Mahfouz, Shahid Mansoor
Clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated proteins (CRISPR-Cas), a groundbreaking genome-engineering tool, has facilitated targeted trait improvement in plants. Recently, CRISPR-CRISPR from Prevotella and Francisella 1 (Cpf1) has emerged as a new tool for efficient genome editing, including DNA-free editing in plants, with higher efficiency, specificity, and potentially wider applications than CRISPR-Cas9.
July 2017: Trends in Plant Science
https://www.readbyqxmd.com/read/28497783/bliss-is-a-versatile-and-quantitative-method-for-genome-wide-profiling-of-dna-double-strand-breaks
#10
Winston X Yan, Reza Mirzazadeh, Silvano Garnerone, David Scott, Martin W Schneider, Tomasz Kallas, Joaquin Custodio, Erik Wernersson, Yinqing Li, Linyi Gao, Yana Federova, Bernd Zetsche, Feng Zhang, Magda Bienko, Nicola Crosetto
Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing...
May 12, 2017: Nature Communications
https://www.readbyqxmd.com/read/28469274/crispr-cpf1-assisted-genome-editing-of-corynebacterium-glutamicum
#11
Yu Jiang, Fenghui Qian, Junjie Yang, Yingmiao Liu, Feng Dong, Chongmao Xu, Bingbing Sun, Biao Chen, Xiaoshu Xu, Yan Li, Renxiao Wang, Sheng Yang
Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86-100%...
May 4, 2017: Nature Communications
https://www.readbyqxmd.com/read/28462777/synthetically-modified-guide-rna-and-donor-dna-are-a-versatile-platform-for-crispr-cas9-engineering
#12
Kunwoo Lee, Vanessa A Mackley, Anirudh Rao, Anthony T Chong, Mark A Dewitt, Jacob E Corn, Niren Murthy
Chemical modification of the gRNA and donor DNA has great potential for improving the gene editing efficiency of Cas9 and Cpf1, but has not been investigated extensively. In this report, we demonstrate that the gRNAs of Cas9 and Cpf1, and donor DNA can be chemically modified at their terminal positions without losing activity. Moreover, we show that 5' fluorescently labeled donor DNA can be used as a marker to enrich HDR edited cells by a factor of two through cell sorting. In addition, we demonstrate that the gRNA and donor DNA can be directly conjugated together into one molecule, and show that this gRNA-donor DNA conjugate is three times better at transfecting cells and inducing HDR, with cationic polymers, than unconjugated gRNA and donor DNA...
May 2, 2017: ELife
https://www.readbyqxmd.com/read/28439558/crispr-cpf1-correction-of-muscular-dystrophy-mutations-in-human-cardiomyocytes-and-mice
#13
Yu Zhang, Chengzu Long, Hui Li, John R McAnally, Kedryn K Baskin, John M Shelton, Rhonda Bassel-Duby, Eric N Olson
Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene (DMD), is characterized by fatal degeneration of striated muscles. Dilated cardiomyopathy is one of the most common lethal features of the disease. We deployed Cpf1, a unique class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effector, to correct DMD mutations in patient-derived induced pluripotent stem cells (iPSCs) and mdx mice, an animal model of DMD. Cpf1-mediated genomic editing of human iPSCs, either by skipping of an out-of-frame DMD exon or by correcting a nonsense mutation, restored dystrophin expression after differentiation to cardiomyocytes and enhanced contractile function...
April 2017: Science Advances
https://www.readbyqxmd.com/read/28431230/structural-basis-for-guide-rna-processing-and-seed-dependent-dna-targeting-by-crispr-cas12a
#14
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
#15
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...
June 2017: Nature Methods
https://www.readbyqxmd.com/read/28387220/targeted-activation-of-diverse-crispr-cas-systems-for-mammalian-genome-editing-via-proximal-crispr-targeting
#16
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
#17
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
#18
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
#19
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
#20
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
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