CRISPR-Blog

Protocol : Improved vectors and genome-wide libraries for CRISPR screening

2014-12-28T09:54:00.001-08:00

Article summary

Genome-wide, targeted loss-of-function pooled screens using the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 in human and mouse cells provide an alternative screening system to RNA interference (RNAi). Previously, we used a genome-scale CRISPR knockout (GeCKO) library to identify loss-of-function mutations conferring vemurafenib resistance in a melanoma model1. However, initial lentiviral delivery systems for CRISPR screening had low viral titer or required a cell line already expressing Cas9, thereby limiting the range of biological systems amenable to screening.

Reference: Sanjana, Neville E., Ophir Shalem, and Feng Zhang. "Improved vectors and genome-wide libraries for CRISPR screening." Nature methods 11.8 (2014): 783-784.

Genome-engineering with CRISPR-Cas9 in the mosquito Aedes aegypti

2014-12-28T09:34:00.001-08:00

Article summary

The mosquito Aedes aegypti is a potent vector of the Chikungunya, yellow fever, and Dengue viruses, which result in hundreds of millions of infections and over 50,000 human deaths per year. Loss-of-function mutagenesis in Ae. aegypti has been established with TALENs, ZFNs, and homing endonucleases, which require the engineering of DNA-binding protein domains to generate target specificity for a particular stretch of genomic DNA. Here, we describe the first use of the CRISPR-Cas9 system to generate targeted, site-specific mutations in Ae. aegypti. CRISPR-Cas9 relies on RNA-DNA base-pairing to generate targeting specificity, resulting in cheaper, faster, and more flexible genome-editing reagents. We investigate the efficiency of reagent concentrations and compositions, demonstrate the ability of CRISPR-Cas9 to generate several different types of mutations via disparate repair mechanisms, and show that stable germ-line mutations can be readily generated at the vast majority of genomic loci tested. This work offers a detailed exploration into the optimal use of CRISPR-Cas9 in Ae. aegypti that should be applicable to non-model organisms previously out of reach of genetic modification.

Reference: Kistler et al Genome-engineering with CRISPR-Cas9 in the mosquito Aedes aegypti Here

Protocol : A CRISPR/Cas9 toolkit for multiplex genome editing in plants

2014-12-18T07:09:00.000-08:00

Article summary

Background

To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.

Results

We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.

Conclusions

We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

Reference:Xing, Hui-Li, et al. "A CRISPR/Cas9 toolkit for multiplex genome editing in plants." BMC plant biology 14.1 (2014): 327.

COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites

2014-12-18T06:12:00.004-08:00

Article summary

Precise genome editing using engineered nucleases can significantly facilitate biological studies and disease treatment. In particular, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are a potentially powerful tool for modifying a genome by targeted cleavage of DNA sequences complementary to designed guide strand RNAs. Although CRISPR/Cas systems can have on-target cleavage rates close to the transfection rates, they may also have relatively high off-target cleavage at similar genomic sites that contain one or more base pair mismatches, and insertions or deletions relative to the guide strand. We have developed a bioinformatics-based tool, COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and Deletions) that searches genomes for potential off-target sites (http://crispr.bme.gatech.edu). Based on the user-supplied guide strand and input parameters, COSMID identifies potential off-target sites with the specified number of mismatched bases and insertions or deletions when compared with the guide strand. For each site, amplification primers optimal for the chosen application are also given as output. This ranked-list of potential off-target sites assists the choice and evaluation of intended target sites, thus helping the design of CRISPR/Cas systems with minimal off-target effects, as well as the identification and quantification of CRISPR/Cas induced off-target cleavage in cells.

CRISPR design tools : CLICK HERE

Reference:Cradick, Thomas J., et al. "COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites." Molecular Therapy—Nucleic Acids 3.12 (2014): e214.

An Active Immune Defence with a Minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and Without the Cas6 Protein

2014-12-17T07:00:00.001-08:00

Article summary

The prokaryotic immune system CRISPR-Cas1 is a defence system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III crRNAs are generated by the endonuclease Cas6. We developed a Cas6b2-independent crRNA maturation pathway for the Haloferax type I-B system in vivo, that expresses a functional crRNA that we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analysed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering a interference reaction. The complete 3′ handle could be removed without loss of activity. However manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore we could show that in the presence of an icrRNA a strain without Cas6b (∆cas6b) is still active in interference.

Reference:Maier et al J. Biol. Chem. jbc.M114.617506. doi:10.1074/jbc.M114.617506

Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

2014-12-17T06:54:00.001-08:00

Article summary

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

Reference:Lin et al eLife 2014;10.7554/eLife.04766

Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles

2014-12-15T08:24:00.001-08:00

Article summary

Site-directed genome engineering in higher plants has great potential for basic research and molecular breeding. Here, we describe a method for site-directed mutagenesis of the Arabidopsis nuclear genome that efficiently generates heritable mutations using the RNA-guided endonuclease (RGEN) derived from bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated) protein system. To induce mutagenesis in proliferating tissues during embryogenesis and throughout the plant life cycle, the single guide RNA (sgRNA) and Cas9 DNA endonuclease were expressed from the U6 snRNA and INCURVATA2 promoters, respectively. After Agrobacterium-mediated introduction of T-DNAs encoding RGENs that targets FLOWERING LOCUS T (FT) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 4 genes, somatic mutagenesis at the targeted loci was observed in T1 transformants. In the results of FT-RGEN, T1 plants often showed late flowering indicative of the presence of large somatic sectors in which the FT gene is mutated on both chromosomes. DNA sequencing analysis estimated that about 90 % of independent chromosomal DNA fragments carried mutations in the analyzed tissue of a T1 plant showing late flowering. The most frequently detected somatic polymorphism showed a high rate of inheritance in T2 plants, and inheritance of less frequent polymorphisms was also observed. As a result, late-flowering plants homozygous for novel, heritable null alleles of FT including a 1 bp insertion or short deletions were recovered in the following T2 and T3 generations. Our results demonstrate that dividing tissue-targeted mutagenesis using RGEN provides an efficient heritable genome engineering method in A. thaliana.

Reference:Hyun, Youbong, et al. "Site-directed mutagenesis in Arabidopsis thaliana using dividing tissue-targeted RGEN of the CRISPR/Cas system to generate heritable null alleles." Planta (2014): 1-14.

CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites

2014-12-12T07:58:00.003-08:00

Summary:

CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼20 nt targeted sequence. The target sequence requirements are twofold. First, the 5′-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast.

Reference:Naito, Yuki, et al. "CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites." Bioinformatics (2014): btu743.

CRISPR design tools : CLICK HERE

2nd Annual Symposium on RNA Science and its Applications

2014-12-10T11:31:00.003-08:00

The 2nd Annual Symposium on RNA Science and its Applications entails a full day of scientific presentations, poster session and awards on Friday, March 20th, 2015. Additionally, Sigma-Aldrich will be providing a hands-on CRISPR systems workshop which is tentatively scheduled to begin on March 18th and concludes the evening of March 19th. The workshop is free for those registered for the Symposium, and demonstrating importance of the workshop to their research through submission of an abstract for presentation at the Symposium. Workshop space is limited, but workshop lectures can be attended by more than those accepted for the hands-on experimentation.

Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing

2014-12-09T08:46:00.003-08:00

Article Abstract

Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, SPAK and OSR1. Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full length WNK1 expression from mammalian cells using RNA-guided CRISPR/Cas9 endonucleases. Two clonal cell lines were generated by using a single guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/Cullin-3 E3 ubiquitin ligase complex, and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase (RVI) following hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene editing technology can be used to dissect and analyze WNK signaling networks.

Reference: Roy, Ankita, et al. "Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing." American Journal of Physiology-Renal Physiology (2014): ajprenal-00612.

Optimization of scarless human stem cell genome editing

2014-11-30T19:23:00.001-08:00

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. Here, authors present several improvements: First, they developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. Then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. Authors found Cas9-gRNA achieved 7–8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, they demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.

Reference:Yang, Luhan, et al. "Optimization of scarless human stem cell genome editing." Nucleic acids research 41.19 (2013): 9049-9061.

The CRISPR–Cas system for plant genome editing: advances and opportunities

2014-11-09T18:52:00.000-08:00

Genome editing is an approach in which a specific target DNA sequence of the genome is altered by adding, removing, or replacing DNA bases. Artificially engineered hybrid enzymes, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), and the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated protein) system are being used for genome editing in various organisms including plants. The CRISPR–Cas system has been developed most recently and seems to be more efficient and less time-consuming compared with ZFNs or TALENs. This system employs an RNA-guided nuclease, Cas9, to induce double-strand breaks. The Cas9-mediated breaks are repaired by cellular DNA repair mechanisms and mediate gene/genome modifications. Here, authors provide a detailed overview of the CRISPR–Cas system and its adoption in different organisms, especially plants, for various applications. Important considerations and future opportunities for deployment of the CRISPR–Cas system in plants for numerous applications are also discussed. Recent investigations have revealed the implications of the CRISPR–Cas system as a promising tool for targeted genetic modifications in plants. This technology is likely to be more commonly adopted in plant functional genomics studies and crop improvement in the near future.

Reference: Vinay Kumar and Mukesh Jain. The CRISPR–Cas system for plant genome editing: advances and opportunities J. Exp. Bot. first published online November 4, 2014 doi:10.1093/jxb/eru429

Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9

2014-11-02T10:47:00.003-08:00

The CRISPR/Cas9 system has ushered in a new era of targeted genetic manipulations. Here, authors report the use of CRISPR/Cas9 to induce double-stranded breaks in the genome of the sea squirt Ciona intestinalis. They use electroporation to deliver CRISPR/Cas9 components for tissue-specific disruption of the Ebf (Collier/Olf/EBF) gene in hundreds of synchronized Ciona embryos. Phenotyping of transfected embryos in the ‘F0’ generation revealed that endogenous Ebf function is required for specification of Islet-expressing motor ganglion neurons and atrial siphon muscles. They demonstrate that CRISPR/Cas9 is sufficiently effective and specific to generate large numbers of embryos carrying mutations in a targeted gene of interest, which should allow for rapid screening of gene function in Ciona.

Reference:Stolfi, Alberto, et al. "Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9." Development 141.21 (2014): 4115-4120.

Enhanced Specificity and Efficiency of the CRISPR/Cas9 System with Optimized sgRNA Parameters in Drosophila

2014-10-25T17:20:00.002-07:00

Summary

The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, authors found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, they document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration they determined, authors could efficiently generate mutations in four genes in one step. Finally, they generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. This work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.

Reference:Ren et al., 2014, Enhanced Specificity and Efficiency of the CRISPR/Cas9 System with Optimized sgRNA Parameters in Drosophila. Cell Reports 9, 1–12

A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification

2014-10-25T17:13:00.000-07:00

Summary

Gene activation by the CRISPR/Cas9 system has the potential to enable new approaches to science and medicine, but the technology must be enhanced to robustly control cell behavior. Authors show that the fusion of two transactivation domains to Cas9 dramatically enhances gene activation to a level that is necessary to reprogram cell phenotype. Targeted activation of the endogenous Myod1 gene locus with this system led to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. The levels of myogenic marker expression obtained by the activation of endogenous Myod1 gene were comparable to that achieved by overexpression of lentivirally delivered MYOD1 transcription factor.

Reference:Chakraborty et al., A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification, Stem Cell Reports (2014), http://dx.doi.org/10.1016/j.stemcr.2014.09.013

Gene editing: how to stay on-target with CRISPR

2014-10-21T19:46:00.001-07:00

Abstract

Efficiently cutting a target sequence to effect a desired change in the genome is one gene-editing task. Knowing where else in the genome a tool might have made its mark is quite another.

Table: CRISPR off target prediction tools

CasOT	PKU Zebrafish Functional Genomics group, Peking University
CHOPCHOP	Harvard University
CRISPR Design	Feng Zhang, Massachusetts Institute of Technology
CRISPR Design tool	The Broad Institute of Harvard and MIT
CRISPR/Cas9 gRNA finder	Jack Lin, University of Colorado
CRISPRfinder	Christine Pourcel, Université Paris-Sud 11
E-CRISP	DKFZ German Cancer Research Center
CRISPR gRNA Design tool	DNA 2.0
PROGNOS	Gang Bao, Emory University/Georgia Institute of Technology
ZiFiT	Keith Joung, Massachusetts General Hospital

Reference:Vivien Marx. Gene editing: how to stay on-target with CRISPR. Nature Methods 11, 1021–1026 (2014)

Mouse Genome Editing Using the CRISPR/Cas System.

2014-10-21T19:42:00.003-07:00

Abstract

The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much more quickly than the previously used techniques, and, more importantly, multiple mutations can be created in a single experiment. Here authors provide protocols for preparation of CRISPR/Cas reagents and microinjection into one-cell mouse embryos to create knockout or knock-in mouse models

Reference:Harms, Donald W., et al. "Mouse Genome Editing Using the CRISPR/Cas System." Current Protocols in Human Genetics (2014): 15-7.

MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems

2014-10-19T19:58:00.000-07:00

Motivation

Biologists often wish to use their knowledge on a few experimental models of a given molecular system to identify homologs in genomic data. We developed a generic tool for this purpose.

Results

Macromolecular System Finder (MacSyFinder) provides a flexible framework to model the properties of molecular systems (cellular machinery or pathway) including their components, evolutionary associations with other systems and genetic architecture. Modelled features also include functional analogs, and the multiple uses of a same component by different systems. Models are used to search for molecular systems in complete genomes or in unstructured data like metagenomes. The components of the systems are searched by sequence similarity using Hidden Markov model (HMM) protein profiles. The assignment of hits to a given system is decided based on compliance with the content and organization of the system model. A graphical interface, MacSyView, facilitates the analysis of the results by showing overviews of component content and genomic context. To exemplify the use of MacSyFinder we built models to detect and class CRISPR-Cas systems following a previously established classification. Authors show that MacSyFinder allows to easily define an accurate “Cas-finder” using publicly available protein profiles.

Availability and Implementation

MacSyFinder is a standalone application implemented in Python. It requires Python 2.7, Hmmer and makeblastdb (version 2.2.28 or higher). It is freely available with its source code under a GPLv3 license at https://github.com/gem-pasteur/macsyfinder. It is compatible with all platforms supporting Python and Hmmer/makeblastdb. The “Cas-finder” (models and HMM profiles) is distributed as a compressed tarball archive as Supporting Information.

Reference:

Abby SS, Néron B, Ménager H, Touchon M, Rocha EPC (2014) MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems. PLoS ONE 9(10): e110726. doi:10.1371/journal.pone.0110726

Solutions for Genome Editing: TALEN & CRISPR-Cas9,& Safe Harbor Knock-in ORF clones

2014-10-18T19:56:00.000-07:00

Abstract

Application Scientist will discuss the advantages and disadvantages of each genome editing system, and provide information on GeneCopoeia's powerful suite of genome editing products and services. There will be a Q&A session at the end of the webinar in which you can ask questions.

Wednesday, October 22nd, 2014, 1:30 PM-2:30 PM EST

Genome Editing – Transfecting ZFNs, TALENs or CRISPR/Cas Using Nucleofector™ Technology

2014-10-18T19:42:00.004-07:00

Abstract

Specialists from Lonza and Sigma-Aldrich will discuss the following topics

Brief introduction to genome editing tools
Use of Nucleofector™ Technology for transfection of ZFN, TALEN or CRISPR/Cas9 system
Application examples
Technical tips for ZFN/CRISPR usage and Nucleofector™ Technology

Speakers

Dr. Nazim El-Andaloussi Scientific Support Specialist, Lonza

Erika Holroyd ZFN and CRISPR Technical Specialist, Sigma-Aldrich
Dr. Claudia Schwartz Scientific Support Specialist, Lonza
Erika Holroyd ZFN and CRISPR Technical Specialist, Sigma-Aldrich

Session 1 Tuesday, 28 October 2014 11 AM PDT (Los Angeles) 2 PM EDT(New York)	Session 2 Wednesday, 29 October 2014 1 PM GMT (London) 2 PM CET (Berlin) 9 AM EDT (New York)

Genome modification by CRISPR/Cas9 : CRISPR Review

2014-10-16T13:15:00.001-07:00

Abstract

CRISPR/Cas9-mediated genome modification enables us to edit the genomes of a variety of organisms rapidly and efficiently. The advantages of the CRISPR/Cas9 system have made it an increasingly popular genetic engineering tool for biological and therapeutic applications. Moreover, CRISPR/Cas9 has been employed to recruit functional domains that repress/activate gene expression or label specific genomic loci in living cells or organisms, in order to explore the developmental mechanisms, gene expression regulation and animal behavior. One major concern about this system is its specificity; although the CRISPR/Cas9-mediated off-target mutation has been broadly studied, more efforts are required to further improve the specificity of CRISPR/Cas9.

Reference:

Genome modification by CRISPR/Cas9 Ma et al. 2014 FEBS letter accepted article doi: 10.1111/febs.13110

Upcoming Webinar –Design high specificity CRISPR/Cas9 gRNAs: principles and tools

2014-10-15T19:47:00.001-07:00

Abstract

The CRISPR/Cas9 system has recently emerged as the most powerful gene editing method to study gene function. This new technology has made gene editing easy to do in any lab. To perform CRISPR/Cas9-mediated gene editing, the first step is to design guide RNA sequences for the target gene. In this webinar, we will present detailed gRNA design principles and provide step-by-step guidance on how to design high specificity gRNAs that avoid off-target effects. We will also introduce the online gRNA design tool and other resources offered by GenScript to help you start doing CRISPR-mediated gene editing with the knowledge and tools you need to be successful.

Speaker:

Heidi Huang, Ph.D.

CRISPR Webinar Details::

November 5, 2014/8:00 am	November 5, 2014/2:00 pm

CRISPRs: Ushering in a New Age of Gene Editing

2014-10-12T07:00:00.004-07:00

Development and Applications of CRISPR Cas9 for Genome Editing

2014-10-12T06:58:00.000-07:00

CRISPR Cas Technology and its Applications

2014-10-12T06:56:00.000-07:00