A Guide RNA Sequence Design Platform for the CRISPR/Cas9 System for Model Organism Genomes

Ma, Ming; Ye, Adam Yongxin; Zheng, Weiguo; Kong, Lei

doi:10.1155/2013/270805

Cited by 62 publications

(45 citation statements)

References 21 publications

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“…. Cas9 guide RNA Design, developed by the Kong lab, is hosted at the Center for Bioinformatics, Peking University, Beijing and searches the Drosophila genome, among others [77]. FlyCas9 is hosted by the National Institute of Genetics in Kyoto, Japan, and hosts both a target finder, and protocols for following the methods of Kondo and Ueda [78].…”

Section: Designing and Engineering Talens And Crispr Reagentsmentioning

confidence: 99%

Targeted genome engineering techniques in Drosophila

Beumer

Carroll

2014

Methods

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For a century, Drosophila has been a favored organism for genetic research. However, the array of materials and methods available to the Drosophila worker has expanded dramatically in the last decade. The most common gene targeting tools, zinc finger nucleases, TALENs, and RNA-guided CRISPR/Cas9, have all been adapted for use in Drosophila, both for simple mutagenesis and for gene editing via homologous recombination. For each tool, there exist a number of web sites, design applications, and delivery methods. The successful application of any of these tools also requires an understanding of methods for detecting successful genome modifications. This article provides an overview of the available gene targeting tools and their application in Drosophila. In lieu of simply providing a protocol for gene targeting, we direct the researcher to resources that will allow access to the latest research in this rapidly evolving field.

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Section: Designing and Engineering Talens And Crispr Reagentsmentioning

confidence: 99%

Targeted genome engineering techniques in Drosophila

Beumer

Carroll

2014

Methods

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“…To help researchers to select the best gRNAs for input sequences, it is essential to identify gRNAs and their potential off-targets, and accurately predict their relative cleavage rates. To facilitate gRNA design, many computational tools have been developed Ma et al, 2013;Doench et al, 2014;Heigwer et al, 2014;Xiao et al, 2014;Zhu et al, 2014;Prykhozhij et al, 2015), and a few representative ones are summarized in Table 1.…”

Section: Overview Of Grna Design Toolsmentioning

confidence: 99%

Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology

Zhu

2015

Front. Biol.

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CRISPR-Cas (Clustered, Regularly Interspaced, Short Palindromic Repeats -CRISPR-associated (Cas)) RNA guided endonuclease has emerged as the most effective and widely used genome editing technology, which has become the most exciting and rapidly advancing research field. Efficient genome editing by the CRISPR-Cas9 system has been demonstrated in many species, and several laboratories have established CRISPR-Cas9 as a screening tool for systematic genetic analysis, similar to shRNA screening. At least three companies have been founded to leverage this technology for therapeutic uses. To facilitate the implementation of this technology, many software tools have been developed to identify guide RNAs that effectively target a desired genomic region. Here, I provide an overview of the technology, focusing on guide RNA design principles, available software tools and their strengths and weaknesses.

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“…The CRISPR/Cas9 system is part of the adaptive immune system within certain bacteria (Chakraborty et al 2009; Karginov and Hannon 2010) and uses short RNA sequences to guide a DNA endonuclease (Cas9), resulting in a DSB (Sander and Joung 2014; Bassett and Liu 2014; Liu and Fan 2014; Mali et al 2013b). The CRISPR/Cas9 system has been further adapted to use synthetic guide RNAs, further optimizing the process (Bassett et al 2013; Ma et al 2013; Upadhyay and Sharma 2014; Bae et al 2014). Re-engineering new target sites is as simple as synthesizing a new small RNA molecule; this ease of use explains why Cas9-based editing has so rapidly supplanted other technologies.…”

Section: Mosquito-borne Diseasementioning

confidence: 99%

“…aegypti Ku70, a homology-based search (tblastn) using the Drosophila or Anopheles Ku70 protein sequences indicates that a likely ortholog is located on scaffold 1.240. The kinase DNA-PKcs tethers the Ku complexes and activates multiple proteins associated with the pathway (Williams et al 2014), primarily the endonuclease Artemis, which is responsible for removing nucleotides from the DSB (MA et al 2002, 2013). Subsequently, the polymerases μ and λ play a role in adding any additional nucleotides needed for ligation.…”

Section: Mosquito-borne Diseasementioning

confidence: 99%

Understanding the DNA damage response in order to achieve desired gene editing outcomes in mosquitoes

et al. 2015

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Mosquitoes are high-impact disease vectors with the capacity to transmit pathogenic agents that cause diseases such as malaria, yellow fever, chikungunya, and dengue. Continued growth in knowledge of genetic, molecular, and physiological pathways in mosquitoes allows for the development of novel control methods and for the continued optimization of existing ones. The emergence of site-specific nucleases as genomic engineering tools promises to expedite research of crucial biological pathways in these disease vectors. The utilization of these nucleases in a more precise and efficient manner is dependent upon knowledge and manipulation of the DNA repair pathways utilized by the mosquito. While progress has been made in deciphering DNA repair pathways in some model systems, research into the nature of the hierarchy of mosquito DNA repair pathways, as well as in mechanistic differences that may exist, is needed. In this review, we will describe progress in the use of site-specific nucleases in mosquitoes, along with the hierarchy of DNA repair in the context of mosquito chromosomal organization and structure, and how this knowledge may be manipulated to achieve precise chromosomal engineering in mosquitoes.

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A Guide RNA Sequence Design Platform for the CRISPR/Cas9 System for Model Organism Genomes

Cited by 62 publications

References 21 publications

Targeted genome engineering techniques in Drosophila

Targeted genome engineering techniques in Drosophila

Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology

Understanding the DNA damage response in order to achieve desired gene editing outcomes in mosquitoes

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