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Sushil Devkota
Non-homologous end joining (NHEJ), and to a lesser extent, the error-free pathway known as homology-directed repair (HDR) are cellular mechanisms for recovery from double-strand DNA breaks (DSB) induced by RNA-guided programmable nuclease CRISPR/Cas. Since NHEJ is equivalent to using a duck tape to stick two pieces of metals together, the outcome of this repair mechanism is prone to error. Any out-of-frame mutations or premature stop codons resulting from NHEJ repair mechanism are extremely handy for loss-of-function studies...
August 14, 2018: BMB Reports
Anna Buchman, Omar S Akbari
Despite the importance of Y-chromosomes in evolution and sex determination, their heterochromatic, repeat-rich nature makes them difficult to sequence (due, in part, to ambiguities in sequence alignment and assembly) and to genetically manipulate, and therefore they generally remain poorly understood. For example, the D. melanogaster Y-chromosome, one of the most extensively studied Y-chromosomes, is widely heterochromatic and composed mainly of highly repetitive sequences, with only a handful of expressed genes scattered throughout its length...
August 6, 2018: Insect Molecular Biology
Xiaoyun Xu, Dongbing Gao, Ping Wang, Jian Chen, Jinxue Ruan, Jie Xu, Xiaofeng Xia
CRISPR/Cas9 efficiently generates gene knock-out via nonhomologous end joining (NHEJ), but the efficiency of precise homology-directed repair (HDR) is substantially lower, especially in the hard-to-transfect human stem cells and primary cells. Herein we report a tube electroporation method that can effectively transfect human stem cells and primary cells with minimal cytotoxicity. When applied to genome editing using CRISPR/Cas9 along with single stranded DNA oligonucleotide (ssODN) template in human induced pluripotent stem cells (iPSCs), up to 42...
August 3, 2018: Scientific Reports
Lupeng Ye, Chengkun Wang, Lingjuan Hong, Ninghe Sun, Danyang Chen, Sidi Chen, Feng Han
CRISPR systems have been proven as versatile tools for site-specific genome engineering in mammalian species. During the gene editing processes, these RNA-guide nucleases introduce DNA double strand breaks (DSBs), in which non-homologous DNA end joining (NHEJ) dominates the DNA repair pathway, limiting the efficiency of homology-directed repair (HDR), the alternative pathway essential for precise gene targeting. Multiple approaches have been developed to enhance HDR, including chemical compound or RNA interference-mediated inhibition of NHEJ factors, small molecule activation of HDR enzymes, or cell cycle timed delivery of CRISPR complex...
2018: Cell Discovery
Lei Li, Keke Wei, Guosong Zheng, Xiaocao Liu, Shaoxin Chen, Weihong Jiang, Yinhua Lu
Streptomyces has strong capability to produce a large number of bioactive natural products and remains invaluable sources for the discovery of novel drug leads. Although the Streptococcus pyogenes ( Sp ) CRISPR-Cas9-assisted genome editing tool has been developed for rapid genetic engineering in Streptomyces , it has a number of limitations, including the toxicity of Sp Cas9 expression in some important industrial Streptomyces strains and the need for complex expression constructs when targeting multiple genomic loci...
July 6, 2018: Applied and Environmental Microbiology
Jun Bo Tang, Hao Wei Cao, Rui Xu, Dan Dan Zhang, Juan Huang
Multiple genes work together to maintain the normal functions of the reproductive system. However, for many of these genes, little is known about their specific functions and mechanisms. In the present study, eight Drosophila genes, including CG4161, CG11475, CG2921, CG10541, CG7276, CG3800, CG8117 and CG16779, were selected for detailed studies based on their testis expression, undefined functions, and having highly homologous and conserved genes in humans (Homo sapiens) and mouse (Mus musculus). We analyzed their expression levels in different tissues, and determined their probably functions in male reproduction...
June 20, 2018: Yi Chuan, Hereditas
Shaoya Li, Jingying Li, Jiahui Zhang, Wenming Du, Jindong Fu, Suhas Sutar, Yunde Zhao, Lanqin Xia
The recently developed CRISPR/Cpf1 system expands the range of genome editing and is emerging as an alternative powerful tool for both plant functional genomics and crop improvement. Cpf1-crRNA produces double strand DNA breaks (DSBs) with long 5' protruding ends, which may facilitate the pairing and insertion of repair templates through homology-directed repair (HDR) for targeted gene replacement and introduction of the desired DNA elements at specific gene loci for crop improvement. However, the potential mechanism underlying HDR of DSBs generated by Cpf1-crRNA remains to be investigated and the inherent low efficiency of HDR and poor availability of exogenous donor DNA as repair templates strongly impede the use of HDR for precise genome editing in crop plants...
June 28, 2018: Journal of Experimental Botany
Yannik Bollen, Jasmin Post, Bon-Kyoung Koo, Hugo J G Snippert
Model systems with defined genetic modifications are powerful tools for basic research and translational disease modelling. Fortunately, generating state-of-the-art genetic model systems is becoming more accessible to non-geneticists due to advances in genome editing technologies. As a consequence, solely relying on (transient) overexpression of (mutant) effector proteins is no longer recommended since scientific standards increasingly demand genetic modification of endogenous loci. In this review, we provide up-to-date guidelines with respect to homology-directed repair (HDR)-mediated editing of mammalian model systems, aimed at assisting researchers in designing an efficient genome editing strategy...
July 27, 2018: Nucleic Acids Research
Ekaterina Antonova, Olga Glazova, Anna Gaponova, Aykaz Eremyan, Svetlana Zvereva, Natalya Grebenkina, Natalya Volkova, Pavel Volchkov
Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene...
2018: F1000Research
Ben Ewen-Campen, Norbert Perrimon
Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of Drosophila genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, ovoD1 In this system (" ovoD co-selection"), the only functional germ cells in injected females are those that have been edited at the ovoD1 locus, and thus all offspring of these flies have undergone editing of at least one locus...
July 31, 2018: G3: Genes—Genomes—Genetics
Derek M Mason, Cédric R Weber, Cristina Parola, Simon M Meng, Victor Greiff, William J Kelton, Sai T Reddy
Antibody engineering is often performed to improve therapeutic properties by directed evolution, usually by high-throughput screening of phage or yeast display libraries. Engineering antibodies in mammalian cells offer advantages associated with expression in their final therapeutic format (full-length glycosylated IgG); however, the inability to express large and diverse libraries severely limits their potential throughput. To address this limitation, we have developed homology-directed mutagenesis (HDM), a novel method which extends the concept of CRISPR/Cas9-mediated homology-directed repair (HDR)...
June 21, 2018: Nucleic Acids Research
Denise G Lanza, Angelina Gaspero, Isabel Lorenzo, Lan Liao, Ping Zheng, Ying Wang, Yu Deng, Chonghui Cheng, Chuansheng Zhang, John R Seavitt, Francesco J DeMayo, Jianming Xu, Mary E Dickinson, Arthur L Beaudet, Jason D Heaney
BACKGROUND: The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs)...
June 21, 2018: BMC Biology
Max Gerlach, Theresia Kraft, Bernhard Brenner, Björn Petersen, Heiner Niemann, Judith Montag
During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement...
June 13, 2018: Genes
Wenhan Yu, Zhijian Wu
The rapidly evolving CRISPR-based genome editing technology is bringing revolutionary changes to the entirety of the life sciences. In this mini-review, we summarize the recent progress of in vivo applications of CRISPR genome editing in retinal studies. Non-viral and viral vector mediated delivery have been developed for temporary or persistent expression of CRISPR components in retinal cells. Although in theory CRISPR-based genome editing can correct a large number of mutant genes responsible for a variety of inherited retinal disorders (IRDs), precise gene modification relies on homology-directed repair (HDR)-the efficiency of which is not currently high enough for meaningful benefit...
2018: Frontiers in Cell and Developmental Biology
Mert Yanik, Surya Prakash Goud Ponnam, Tobias Wimmer, Lennart Trimborn, Carina Müller, Isabel Gambert, Johanna Ginsberg, Annabella Janise, Janina Domicke, Wolfgang Wende, Birgit Lorenz, Knut Stieger
Common genome-editing strategies are either based on non-homologous end joining (NHEJ) or, in the presence of a template DNA, based on homologous recombination with long (homology-directed repair [HDR]) or short (microhomology-mediated end joining [MMEJ]) homologous sequences. In the current study, we aim to develop a model system to test the activity of MMEJ after CRISPR/Cas9-mediated cleavage in cell culture. Following successful proof of concept in an episomally based reporter system, we tested template plasmids containing a promoter-less luciferase gene flanked by microhomologous sequences (mhs) of different length (5, 10, 15, 20, 30, and 50 bp) that are complementary to the mouse retinitis pigmentosa GTPase regulator (RPGR)-ORF15, which is under the control of a CMV promoter stably integrated into a HEK293 cell line...
June 1, 2018: Molecular Therapy. Nucleic Acids
Bowen Wang, Jiahui Zuo, Wenzhen Kang, Qianqi Wei, Jianhui Li, Chunfu Wang, Zhihui Liu, Yuanan Lu, Yan Zhuang, Bianli Dang, Qing Liu, Wen Kang, Yongtao Sun
The ability of monocytes to travel through the bloodstream, traverse tissue barriers, and aggregate at disease sites endows these cells with the attractive potential to carry therapeutic genes into the nervous system. However, gene editing in primary human monocytes has long been a challenge. Here, we applied the CRISPR/Cas9 system to deliver the large functional Hutat2:Fc DNA fragment into the genome of primary monocytes to neutralize HIV-1 transactivator of transcription (Tat), an essential neurotoxic factor that causes HIV-associated neurocognitive disorder (HAND) in the nervous system...
June 1, 2018: Molecular Therapy. Nucleic Acids
Birgit Koch, Bianca Nijmeijer, Moritz Kueblbeck, Yin Cai, Nike Walther, Jan Ellenberg
Gene tagging with fluorescent proteins is essential for investigations of the dynamic properties of cellular proteins. CRISPR-Cas9 technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest (GOI) and allows functionality and physiological expression of the fusion protein. It is essential to evaluate such genome-edited cell lines carefully in order to preclude off-target effects caused by (i) incorrect insertion of the fluorescent protein, (ii) perturbation of the fusion protein by the fluorescent proteins or (iii) nonspecific genomic DNA damage by CRISPR-Cas9...
June 2018: Nature Protocols
Yuanming Wang, Kaiwen Ivy Liu, Norfala-Aliah Binte Sutrisnoh, Harini Srinivasan, Junyi Zhang, Jia Li, Fan Zhang, Charles Richard John Lalith, Heyun Xing, Raghuvaran Shanmugam, Jia Nee Foo, Hwee Ting Yeo, Kean Hean Ooi, Tore Bleckwehl, Yi Yun Rachel Par, Shi Mun Lee, Nur Nadiah Binte Ismail, Nur Aidah Binti Sanwari, Si Ting Vanessa Lee, Jan Lew, Meng How Tan
BACKGROUND: While CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing. RESULTS: We systematically compare five different CRISPR-Cas systems in human cells by targeting 90 sites in genes with varying expression levels. For a fair comparison, we select sites that are either perfectly matched or have overlapping seed regions for Cas9 and Cpf1...
May 29, 2018: Genome Biology
Harriet Prior, Lauren MacConnachie, Jose L Martinez, Georgina C B Nicholl, Asim A Beg
The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals...
April 30, 2018: Journal of Visualized Experiments: JoVE
Rajeswari Jayavaradhan, Devin M Pillis, Punam Malik
The efficient site-specific DNA double-strand breaks (DSB) created by CRISPR/Cas9 has revolutionized genome engineering and has great potential for editing hematopoietic stem/progenitor cells (HSPCs). However, detailed understanding of the variables that influence choice of DNA-DSB repair (DDR) pathways by HSPC is required for therapeutic levels of editing in these clinically relevant cells. We developed a hematopoietic-reporter system that rapidly quantifies the three major DDR pathways utilized at the individual DSB created by CRISPR/Cas9-NHEJ, MMEJ, and HDR-and show its applicability in evaluating the different DDR outcomes utilized by human hematopoietic cell lines and primary human HSPC...
May 9, 2018: Journal of Molecular Biology
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