Other special issues

Protocols for the CRISPR/Cas Technology

CRISPR/Cas-based technologies witness a growing number of applications in life sciences. These technologies are based on the CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins) adaptive immune system in prokaryotes, which functions by storing small DNA fragments derived from nucleic acid invaders as spacers in genomic CRISPR loci. CRISPR loci are transcribed and processed into CRISPR RNA (crRNA) guides that are utilized by Cas effector proteins, such as Cas9 or Cas12a, to target and degrade invading nucleic acids in a sequence-specific manner. The high specificity of these ribonucleoprotein complexes and their potential to introduce double strand breaks in target DNA have been the foundation for many technical innovations (such as targeted genome engineering, gene silencing, and visualization of genomic regions) that enable us to address important biological questions.

Our CRISPR/Cas special issue presents a comprehensive collection of detailed and peer reviewed protocols focused on CRISPR/Cas applications (Section 1), different approaches for guide RNA design (Section 2), delivery mechanisms of Cas effector proteins and/or guide RNAs into cells and organisms (Section 3), as well as protocols that allow further investigation of the biological role and mechanisms of CRISPR/Cas systems (Section 4). Bio-protocols’ uniquely interactive platform supports communication between scientists – through feedback, Q&A, and protocol updates sections – and will allow you to set up CRISPR/Cas based technologies for your research. Bio-protocol is a living platform and our CRISPR/Cas special issue will grow with the CRISPR/Cas field, giving you access to the latest developments.

The free-access CRISPR protocols provided in this special issue are distinguished in several ways, most notably by a degree of precision that is tremendously useful. The availability of an online 'Ask the Authors' tool means that their usefulness will only grow over time.
Erik Sontheimer, Professor
RNA Therapeutics Institute, University of Massachusetts Medical School
Having just gone through your rigorous review process (twice), I can speak to the high quality of the protocols that ultimately get published. I believe it is the responsibility of every investigator to take a pro-active stance to ensure the reproducibility of their findings, and Bio-protocol provides the perfect outlet to do so. Having students write up their protocols is also an excellent way to cultivate an attention to detail while introducing students to the peer review process.
Asma Hatoum-Aslan, Assistant Professor
Department of Biological Sciences, The University of Alabama

Targeted Gene Mutation in Rice Using a CRISPR-Cas9 System
Authors:  Kabin Xie, Bastian Minkenberg and Yinong Yang, date: 09/05/2014, view: 26800, Q&A: 0

RNA-guided genome editing (RGE) using bacterial type II cluster regularly interspaced short palindromic repeats (CRISPR)–associated nuclease (Cas) has emerged as a simple and versatile tool for genome editing in many organisms including plant and crop species. In RGE based on the Streptococcus pyogenes CRISPR-Cas9 system, the Cas9 ...

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A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9
Authors:  Johan Vad-Nielsen, Lin Lin , Kristopher Torp Jensen, Anders Lade Nielsen and Yonglun Luo, date: 12/05/2016, view: 18959, Q&A: 1

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using ...

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Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli
Authors:  Enrico König, Francesca Zerbini, Ilaria Zanella, Davide Fraccascia and Guido Grandi, date: 01/20/2018, view: 18554, Q&A: 1

With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the ...

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Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9
Authors:  Florian Hahn, Marion Eisenhut, Otho Mantegazza and Andreas P. M. Weber, date: 07/05/2017, view: 16256, Q&A: 1

The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing ...

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Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery
Authors:  Giel Vandemoortele, Delphine De Sutter and Sven Eyckerman, date: 04/05/2017, view: 16200, Q&A: 0

The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair ...

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Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9

CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously ...

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CRISPR/Cas9 Editing of the Bacillus subtilis Genome
Authors:  Peter E. Burby and Lyle A. Simmons, date: 04/20/2017, view: 13926, Q&A: 4

A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 technology to bacterial genetics has allowed researchers to manipulate bacterial genomes with ...

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Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters
Authors:  Tao Zhang, Yangbin Gao, Rongchen Wang and Yunde Zhao, date: 02/20/2017, view: 11860, Q&A: 0

CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct ...

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In utero Electroporation of Mouse Cerebellar Purkinje Cells
Author:  Yukari H. Takeo, date: 06/05/2016, view: 11630, Q&A: 0

In utero electroporation (IUE) of mouse cerebellar Purkinje cells allows high expression levels of transgenes without toxicity (Nishiyama et al., 2012). This technique is suitable for co-transfection of multiple plasmid genes. Therefore, it is useful to express various sets of genes such as drug-inducible Cre/loxP constructs and ...

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Identification of Proteins Interacting with Genomic Regions of Interest in vivo Using Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (enChIP)
Authors:  Toshitsugu Fujita and Hodaka Fujii, date: 05/20/2014, view: 11278, Q&A: 0

Elucidation of molecular mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. To this end, it is useful to isolate the target regions retaining molecular interactions. We established locus-specific chromatin immunoprecipitation (ChIP) technologies consisting of ...

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Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9

Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended ...

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Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9
Authors:  Marie-Laurence Lemay, Ariane C. Renaud, Geneviève M. Rousseau and Sylvain Moineau, date: 01/05/2018, view: 10308, Q&A: 1

This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is ...

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Targeted Nucleotide Substitution in Mammalian Cell by Target-AID
Authors:  Takayuki Arazoe, Keiji Nishida and Akihiko Kondo, date: 06/05/2017, view: 10207, Q&A: 0

Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have ...

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Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
Authors:  Noelia Lander, Miguel A. Chiurillo, Aníbal E. Vercesi and Roberto Docampo, date: 05/20/2017, view: 10119, Q&A: 0

To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then ...

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Use of Geminivirus for Delivery of CRISPR/Cas9 Components to Tobacco by Agro-infiltration
Authors:  Kangquan Yin, Ting Han and Yule Liu, date: 04/05/2017, view: 9932, Q&A: 0

CRISPR/Cas9 system is a recently developed genome editing tool, and its power has been demonstrated in many organisms, including some plant species (Wang et al., 2016). In eukaryotes, the Cas9/gRNA complexes target genome sites specifically and cleave them to produce double-strand breaks (DSBs), which can be repaired by non-homologous end ...

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Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii
Authors:  Kevin M. Brown, Shaojun Long and L. David Sibley, date: 02/20/2018, view: 9854, Q&A: 0

Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii’s 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to ...

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DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis
Authors:  Jihyeon Yu, Kwangryul Baek, EonSeon Jin and Sangsu Bae, date: 06/05/2017, view: 9750, Q&A: 0

We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a ...

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Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library
Authors:  Brooke A Napier and Denise M Monack, date: 05/20/2017, view: 8918, Q&A: 0

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of ...

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Construction of a Single Transcriptional Unit for Expression of Cas9 and Single-guide RNAs for Genome Editing in Plants
Authors:  Xu Tang, Zhaohui Zhong, Xuelian Zheng and Yong Zhang, date: 09/05/2017, view: 8684, Q&A: 0

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein9 (Cas9) is a simple and efficient tool for genome editing in many organisms including plant and crop species. The sgRNAs of the CRISPR/Cas9 system are typically expressed from RNA polymerase III promoters, such as U6 and U3. In many transformation events, ...

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Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae
Authors:  Huanhuan Hao, Jing Huang, Tongtong Liu, Hui Tang and Liping Zhang, date: 07/20/2017, view: 8595, Q&A: 0

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very ...

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Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles
Authors:  Rubul Mout and Vincent M. Rotello, date: 10/20/2017, view: 8426, Q&A: 1

In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in ...

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CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum

The establishment of the CRISPR/Cas9 technology in diatoms (Hopes et al., 2016; Nymark et al., 2016) enables a simple, inexpensive and effective way of introducing targeted alterations in the genomic DNA of this highly important group of eukaryotic phytoplankton. Diatoms are of interest as model microorganisms in a variety of ...

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Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions
Authors:  Amanda Hopes, Vladimir Nekrasov, Nigel Belshaw, Irina Grouneva, Sophien Kamoun and Thomas Mock, date: 12/05/2017, view: 8214, Q&A: 1

Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include ...

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Targeted Mutagenesis Using RNA-guided Endonucleases in Mosses
Authors:  Toshihisa Nomura and Hitoshi Sakakibara, date: 06/20/2017, view: 8190, Q&A: 0

RNA-guided endonucleases (RGENs) have been used for genome editing in various organisms. Here, we demonstrate a simple method for performing targeted mutagenesis and genotyping in a model moss species, Physcomitrella patens, using RGENs. We also performed targeted mutagenesis in a non-model moss, Scopelophilla cataractae, using a ...

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Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9
Authors:  Julia Schumacher, Kerstin Kaufmann and Wenhao Yan, date: 03/05/2017, view: 8188, Q&A: 0

Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional ...

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Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci
Authors:  Forrest C. Walker and Asma Hatoum-Aslan, date: 05/05/2017, view: 8092, Q&A: 0

CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between ...

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Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells
Authors:  Benjamin E. Housden, Hilary E. Nicholson and Norbert Perrimon, date: 02/05/2017, view: 7828, Q&A: 0

A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. ...

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Using CRISPR-ERA Webserver for sgRNA Design
Authors:  Honglei Liu, Xiaowo Wang and Lei S. Qi, date: 09/05/2017, view: 7814, Q&A: 0

The CRISPR-Cas9 system is emerging as a powerful technology for gene editing (modifying the genome sequence) and gene regulation (without modifying the genome sequence). Designing sgRNAs for specific genes or regions of interest is indispensable to CRISPR-based applications. CRISPR-ERA (http://crispr-era.stanford.edu/ ...

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Generation of Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors
Authors:  Chin-kai Chuang, Ching-Fu Tu and Chien-Hong Chen, date: 06/05/2017, view: 7647, Q&A: 0

A set of Cas9 and single guide CRISPR RNA expression vectors was constructed. Only a very simple procedure was needed to prepare specific single-guide RNA expression vectors with high target accuracy. Since the de novo zygotic transcription had been detected in mouse embryo at the 1-cell stage, the plasmid DNA vectors encoding Cas9 and ...

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A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9
Authors:  Satoshi Hara, Miho Terao and Shuji Takada, date: 06/05/2017, view: 7351, Q&A: 0

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific ...

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Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts
Authors:  Andrew M. Tidball, Preethi Swaminathan, Louis T. Dang and Jack M. Parent, date: 04/05/2018, view: 7350, Q&A: 1

For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector ...

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Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System
Authors:  Miriam Manor and Udi Qimron, date: 08/05/2017, view: 7315, Q&A: 0

We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous ...

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Multiplex Gene Editing via CRISPR/Cas9 System in Sheep
Authors:  Yiyuan Niu, Yi Ding, Xiaolong Wang and Yulin Chen, date: 07/05/2017, view: 7310, Q&A: 0

Sheep is a major large animal model for studying development and disease in biomedical research. We utilized CRISPR/Cas9 system successfully to modify multiple genes in sheep. Here we provide a detailed protocol for one-cell-stage embryo manipulation by co-injecting Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and ...

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CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii
Authors:  Shaojun Long, Kevin M. Brown and L. David Sibley, date: 03/20/2018, view: 7270, Q&A: 0

Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii. When combined with CRISPR/Cas9 based tagging, this ...

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Method for CRISPR/Cas9 Mutagenesis in Candida albicans
Authors:  Neta Dean and Henry Ng, date: 04/20/2018, view: 7183, Q&A: 0

Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that ...

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Expression and Purification of the Cas10-Csm Complex from Staphylococci
Authors:  Lucy Chou-Zheng and Asma Hatoum-Aslan, date: 06/05/2017, view: 7100, Q&A: 0

CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a class of prokaryotic immune systems that degrade foreign nucleic acids in a sequence-specific manner. These systems rely upon ribonucleoprotein complexes composed of Cas nucleases and small CRISPR RNAs (crRNAs). Staphylococcus epidermidis ...

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Dense sgRNA Library Construction Using a Molecular Chipper Approach
Authors:  Jijun Cheng, Wen Pan and Jun Lu, date: 06/20/2017, view: 7064, Q&A: 0

Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for ...

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Knock-in Blunt Ligation Utilizing CRISPR/Cas9
Authors:  Jonathan M. Geisinger and Michele P. Calos, date: 03/05/2017, view: 7051, Q&A: 0

The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to ...

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CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae
Authors:  Yu Sasano and Satoshi Harashima, date: 05/20/2017, view: 6942, Q&A: 0

Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high ...

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A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism
Author:  Hiroshi Arakawa, date: 05/20/2017, view: 6752, Q&A: 0

While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in ...

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Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for Escherichia coli
Authors:  Xin-tian Li, Cindy Sou and Suckjoon Jun, date: 10/05/2017, view: 6603, Q&A: 0

We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and ...

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Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting
Authors:  Fuqiang Chen, Xiao Ding, Yongmei Feng, Timothy Seebeck, Yanfang Jiang and Gregory D Davis, date: 08/05/2017, view: 6327, Q&A: 0

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous ...

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In vitro Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells

The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating ...

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Synthetic Genetic Interaction (CRISPR-SGI) Profiling in Caenorhabditis elegans
Authors:  John A. Calarco and Adam D. Norris, date: 03/05/2018, view: 5166, Q&A: 0

Genetic interaction screens are a powerful methodology to establish novel roles for genes and elucidate functional connections between genes. Such studies have been performed to great effect in single-cell organisms such as yeast and E. coli (Schuldiner et al., 2005; Butland et al., 2008; Costanzo et al., 2010), ...

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CRISPR-Cas9 Mediated Genome Editing in Drosophila
Authors:  Ping Peng, Xia Wang, Da Shen, Jin Sun, Yu Jia, Rong-Gang Xu, Li-Fei Zhu and Jian-Quan Ni , date: 01/20/2019, view: 5137, Q&A: 0

In recent years, great progress has been made in the research of genome editing systems, one of which is the CRISPR-Cas9 system, a powerful technology that is applied to edit animal genome. Here, we describe a CRISPR-Cas9 mediated mutation protocol for efficiently and specifically editing genes in Drosophila. In this optimized system, the ...

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CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus

Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of ...

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High Resolution Melting Temperature Analysis to Identify CRISPR/Cas9 Mutants from Arabidopsis
Authors:  Cynthia Denbow, Sonia Carole Ehivet and Sakiko Okumoto, date: 07/20/2018, view: 4807, Q&A: 0

CRISPR/Cas9 made targeted mutagenesis and genome editing possible for many plant species. One of the ways that the endonuclease is used for plant genetics is the creation of loss-of-function mutants, which typically result from erroneous DNA repair through non-homologous end joining (NHEJ) pathway. The majority of erroneous repair events results ...

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Single and Multiplexed Gene Editing in Ustilago maydis Using CRISPR-Cas9

The smut fungus Ustilago maydis is an established model organism for elucidating how biotrophic pathogens colonize plants and how gene families contribute to virulence. Here we describe a step by step protocol for the generation of CRISPR plasmids for single and multiplexed gene editing in U. maydis. Furthermore, we describe the ...

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Genotyping-free Selection of Double Allelic Gene Edited Medaka Using Two Different Fluorescent Proteins
Authors:  Yu Murakami, Satoshi Ansai, Akari Yonemura and Masato Kinoshita, date: 12/20/2017, view: 4440, Q&A: 0

This protocol describes a simple genotyping using two different colors of fluorescent protein genes inserted at the target locus. This method makes it possible to determine the genotype of each individual simply by observing the fluorescence later than F1 generation.

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flySAM Transgenic CRISPRa System Manual
Authors:  Yu Jia, Da Shen, Xia Wang, Jin Sun, Ping Peng, Rong-Gang Xu, Bowen Xu and Jian-Quan Ni , date: 01/20/2019, view: 4161, Q&A: 0

Powerful and general methods that can enhance gene expression are useful to systematically study gene function. To date, compared with the methods in generating loss-of-function mutants, methods to achieve gain-of-function are limited. The entire field in Drosophila has relied heavily on the Gal4/UAS:cDNA overexpression system developed ...

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CRISPR/Cas9-mediated ssDNA Recombineering in Corynebacterium glutamicum
Authors:  Jiao Liu, Yu Wang, Ping Zheng and Jibin Sun, date: 10/05/2018, view: 3725, Q&A: 0

Corynebacterium glutamicum is a versatile workhorse for industrial bioproduction of many kinds of chemicals and fuels, notably amino acids. Development of advanced genetic engineering tools is urgently demanded for systems metabolic engineering of C. glutamicum. Recently unveiled clustered regularly interspaced short palindromic ...

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