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linear DNA crispr

Fei Ma, Hui Qi, Yufei Hu, Qianru Jiang, Li-Guang Zhang, Peng Xue, Fu-Quan Yang, Rui Wang, Yan Ju, Hidenobu Uchida, Quan Zhang, Sodmergen Sodmergen
Maintaining the appropriate number of mitochondrial DNA (mtDNA) molecules is crucial for supporting mitochondrial metabolism and function in both plant and animal cells. For example, a substantial decrease in mtDNA levels occurs as a key part of pollen development. The molecular mechanisms regulating mtDNA copy number are largely unclear, particularly with regard to those that reduce mtDNA levels. Here, we identified and purified a 20 kD endonuclease, M20, from maize (Zea mays) pollen mitochondria. We found M20 to be an H-N-H/N nuclease that degrades linear and circular DNA in the presence of Mg2+ or Mn2+...
October 9, 2018: Plant Physiology
Wei Deng, Simon Henriet, Daniel Chourrout
Classical non-homologous end joining (c-NHEJ), a fundamental pathway that repairs double-strand breaks in DNA, is almost universal in eukaryotes and involves multiple proteins highly conserved from yeast to human [1]. The genes encoding these proteins were not detected in the genome of Oikopleura dioica, a new model system of tunicate larvaceans known for its very compact and highly rearranged genome [2-4]. After showing their absence in the genomes of six other larvacean species, the present study examined how O...
September 27, 2018: Current Biology: CB
Letian Song, Jean-Paul Ouedraogo, Magdalena Kolbusz, Thi Truc Minh Nguyen, Adrian Tsang
As a powerful tool for fast and precise genome editing, the CRISPR/Cas9 system has been applied in filamentous fungi to improve the efficiency of genome alteration. However, the method of delivering guide RNA (gRNA) remains a bottleneck in performing CRISPR mutagenesis in Aspergillus species. Here we report a gRNA transcription driven by endogenous tRNA promoters which include a tRNA gene plus 100 base pairs of upstream sequence. Co-transformation of a cas9-expressing plasmid with a linear DNA coding for gRNA demonstrated that 36 of the 37 tRNA promoters tested were able to generate the intended mutation in A...
2018: PloS One
Wenwen She, Jing Ni, Ke Shui, Fei Wang, Ruyi He, Jinhui Xue, Manfred T Reetz, Aitao Li, Lixin Ma
The quality and efficiency of any PCR-based mutagenesis technique may not be optimal due to, among other things, amino acid bias, which means that the development of efficient PCR-free methods is desirable. Here, we present a highly efficient in vitro CRISPR/Cas9-mediated mutagenic (ICM) system that allows rapid construction of designed mutants in a PCR-free manner. First, it involves plasmid digestion by utilizing a complex of Cas9 with specific single guide RNA (sgRNA) followed by degradation with T5 exonuclease to generate a 15 nt homologous region...
September 21, 2018: ACS Synthetic Biology
Priya Dharmalingam, Hari Krishna R Rachamalla, Brijesh Lohchania, Bhanuprasad Bandlamudi, Saravanabhavan Thangavel, Mohankumar K Murugesan, Rajkumar Banerjee, Arabinda Chaudhuri, Chandrashekhar Voshavar, Srujan Marepally
Cationic lipid-guided nucleic acid delivery holds great promise in gene therapy and genome-editing applications for treating genetic diseases. However, the major challenge lies in achieving therapeutically relevant efficiencies. Prior findings, including our own, demonstrated that asymmetry in the hydrophobic core of cationic lipids imparted superior transfection efficiencies. To this end, we have developed a lipid nanocarrier system with an asymmetric hydrophobic core ( PS-Lips ) derived from a mixture of fatty acids of food-grade palmstearin and compared its efficiency with symmetric palmitic acid-based nanocarrier system ( P-Lip )...
November 30, 2017: ACS Omega
William Wang, Linlin Zhang, Xiangdong Wang, Yiming Zeng
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system is a prokaryotic immune system that used to resist foreign genetic factors. It rapidly becomes the hot technology in life sciences and is applies for genome editing to solve the problem of genome-derived diseases. Using CRISPR/Cas technique, the biological DNA sequence can be repaired, cut, replaced, or added. It can effectively change the human stem cells and is expected to achieve results in the treatment...
August 25, 2018: Seminars in Cell & Developmental Biology
Soyeong Jun, Hyeonseob Lim, Hoon Jang, Wookjae Lee, Jinwoo Ahn, Ji Hyun Lee, Duhee Bang
CRISPR/Cas9 for genome editing requires delivery of a guide RNA sequence and donor DNA for targeted homologous recombination. Typically, single-stranded oligodeoxynucleotide, serving as the donor template, and a plasmid encoding guide RNA are delivered as two separate components. However, in the multiplexed generation of single nucleotide variants, this two-component delivery system is limited by difficulty of delivering a matched pair of sgRNA and donor DNA to the target cell. Here, we describe a novel codelivery system called "sgR-DNA" that uses a linearized double-stranded DNA consisting of donor DNA component and a component encoding sgRNA...
July 20, 2018: ACS Synthetic Biology
Xiao-Ran Zhang, Jia-Bei He, Yi-Zheng Wang, Li-Lin Du
The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast Schizosaccharomyces pombe by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid...
May 31, 2018: G3: Genes—Genomes—Genetics
Qing Xiao, Taishan Min, Shuangping Ma, Lingna Hu, Hongyan Chen, Daru Lu
Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis...
August 2018: Molecular Genetics and Genomics: MGG
Beibei Zhang, Qiao Wang, Xinhui Xu, Qiang Xia, Feifei Long, Weiwei Li, Yingchun Shui, Xinyi Xia, Jinke Wang
This study develops a new method for detecting target DNA based on Cas9 nuclease, which was named as CARP, representing CRISPR- or Cas9/sgRNAs-associated reverse PCR. This technique detects target DNA in three steps: (1) cleaving the detected DNA sample with Cas9 in complex with a pair of sgRNAs specific to target DNA; (2) ligating the cleaved DNA with DNA ligase; (3) amplifying target DNA with PCR. In the ligation step, the Cas9-cut target DNA was ligated into intramolecular circular or intermolecular concatenated linear DNA...
May 2018: Analytical and Bioanalytical Chemistry
Brian D Janssen, Yi-Pei Chen, Brenda M Molgora, Shuqi E Wang, Augusto Simoes-Barbosa, Patricia J Johnson
The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T...
January 10, 2018: Scientific Reports
Ryan Marshall, Colin S Maxwell, Scott P Collins, Thomas Jacobsen, Michelle L Luo, Matthew B Begemann, Benjamin N Gray, Emma January, Anna Singer, Yonghua He, Chase L Beisel, Vincent Noireaux
CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells...
January 4, 2018: Molecular Cell
Dongdong Zhao, Xu Feng, Xinna Zhu, Tao Wu, Xueli Zhang, Changhao Bi
The CRISPR/Cas9 system is a powerful, revolutionary tool for genome editing. However, it is not without limitations. There are PAM-free and CRISPR-tolerant regions that cannot be modified by the standard CRISPR/Cas9 system, and off-target activity impedes its broader applications. To avoid these drawbacks, we developed a very simple CRISPR/Cas9-assisted gRNA-free one-step (CAGO) genome editing technique which does not require the construction of a plasmid to express a specific gRNA. Instead, a universal N20 sequence with a very high targeting efficiency is inserted into the E...
November 30, 2017: Scientific Reports
René Verwaal, Nathalie Buiting-Wiessenhaan, Sacha Dalhuijsen, Johannes A Roubos
Cpf1 represents a novel single RNA-guided CRISPR/Cas endonuclease system suitable for genome editing with distinct features compared with Cas9. We demonstrate the functionality of three Cpf1 orthologues - Acidaminococcus spp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1) and Francisella novicida U112 (FnCpf1) - for genome editing of Saccharomyces cerevisiae. These Cpf1-based systems enable fast and reliable introduction of donor DNA on the genome using a two-plasmid-based editing approach together with linear donor DNA...
February 2018: Yeast
Katrin Weidenbach, Lisa Nickel, Horst Neve, Omer S Alkhnbashi, Sven Künzel, Anne Kupczok, Thorsten Bauersachs, Liam Cassidy, Andreas Tholey, Rolf Backofen, Ruth A Schmitz
A novel archaeal lytic virus targeting species of the genus Methanosarcina was isolated using Methanosarcina mazei strain Gö1 as the host. Due to its spherical morphology, the virus was designated <u>Met</u>hanosarcina <u>s</u>pherical <u>v</u>irus (MetSV). Molecular analysis demonstrated that MetSV contains double-stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs), all oriented in the same direction. Functions were predicted for some of these ORFs, i...
November 15, 2017: Journal of Virology
Cory Schwartz, Keith Frogue, Adithya Ramesh, Joshua Misa, Ian Wheeldon
In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical-producing yeast Yarrowia lipolytica...
December 2017: Biotechnology and Bioengineering
András Tálas, Péter István Kulcsár, Nóra Weinhardt, Adrienn Borsy, Eszter Tóth, Kornélia Szebényi, Sarah Laura Krausz, Krisztina Huszár, István Vida, Ádám Sturm, Bianka Gordos, Orsolya Ivett Hoffmann, Petra Bencsura, Antal Nyeste, Zoltán Ligeti, Elfrieda Fodor, Ervin Welker
The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell...
December 1, 2017: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes
Fei Song, Knut Stieger
The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) technology enables rapid and precise genome editing at any desired genomic position in almost all cells and organisms. In this study, we analyzed the impact of different repair templates on the frequency of homology-directed repair (HDR) and non-homologous end joining (NHEJ). We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to quantify HDR and NHEJ events following transfection with Cas9, eight different guide RNAs, and a 1,000 bp donor template generated either as circular plasmid, as linearized plasmid with long 3' or 5' backbone overhang, or as PCR product...
June 16, 2017: Molecular Therapy. Nucleic Acids
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
Alexandre Paix, Andrew Folkmann, Geraldine Seydoux
The ability to introduce targeted edits in the genome of model organisms is revolutionizing the field of genetics. State-of-the-art methods for precision genome editing use RNA-guided endonucleases to create double-strand breaks (DSBs) and DNA templates containing the edits to repair the DSBs. Following this strategy, we have developed a protocol to create precise edits in the C. elegans genome. The protocol takes advantage of two innovations to improve editing efficiency: direct injection of CRISPR-Cas9 ribonucleoprotein complexes and use of linear DNAs with short homology arms as repair templates...
May 15, 2017: Methods: a Companion to Methods in Enzymology
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