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https://www.readbyqxmd.com/read/28720252/the-cryptochrome-photolyase-protein-family-in-diatoms
#1
Sarah König, Matthias Juhas, Stefanie Jäger, Tilman Kottke, Claudia Büchel
The cryptochrome - photolyase family (CPF) consists of homologous flavoproteins having completely different functions involving DNA repair, circadian rhythm and/or photoreception. From the original photolyases, working either as (6-4) or cyclobutane pyrimidine dimer photolyases, the animal- and plant-type cryptochromes, respectively, evolved and also the more intermediate DASH cryptochromes. Whereas animal cryptochromes work mostly in clock-related functions, plant cryptochromes are also directly involved in developmental processes such as hypocotyl elongation or flower induction...
July 8, 2017: Journal of Plant Physiology
https://www.readbyqxmd.com/read/28712502/gene-editing-with-talen-and-crispr-cas-in-rice
#2
Honghao Bi, Bing Yang
Engineered, site-specific nucleases induce genomic double-strand DNA breaks and break repair processes enable genome editing in a plethora of eukaryotic genomes. TALENs (transcription activator-like effector nucleases) and CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) are potent biotechnological tools used for genome editing. In rice, species-tailored editing tools have proven to be efficient and easy to use. Both tools are capable of generating DNA double-strand breaks (DSBs) in vivo and such breaks can be repaired either by error-prone NHEJ (nonhomologous end joining) that leads to nucleotide insertions or deletions or by HDR (homology-directed repair) if an appropriate exogenous DNA template is provided...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/28695850/assembly-of-francisella-novicida-cpf1-endonuclease-in-complex-with-guide-rna-and-target-dna
#3
Pablo Alcón, Guillermo Montoya, Stefano Stella
Bacteria and archaea use the CRISPR-Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR-Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target DNA preceded by a 5'-TTN-3' protospacer-adjacent motif (PAM) complementary to the RNA guide...
July 1, 2017: Acta Crystallographica. Section F, Structural Biology Communications
https://www.readbyqxmd.com/read/28678773/erratum-structure-of-the-cpf1-endonuclease-r-loop-complex-after-target-dna-cleavage
#4
Stefano Stella, Pablo Alcón, Guillermo Montoya
This corrects the article DOI: 10.1038/nature22398.
July 5, 2017: Nature
https://www.readbyqxmd.com/read/28654071/protocols-for-c-brick-dna-standard-assembly-using-cpf1
#5
Shi-Yuan Li, Guo-Ping Zhao, Jin Wang
CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites - the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) - flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites...
June 15, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28646112/crispr-cas12a-assisted-recombineering-in-bacteria
#6
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
#7
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
#8
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
#9
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
#10
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
#11
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...
July 6, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28581492/engineered-cpf1-variants-with-altered-pam-specificities
#12
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
#13
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...
June 22, 2017: Nature
https://www.readbyqxmd.com/read/28532598/crispr-cpf1-a-new-tool-for-plant-genome-editing
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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