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

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.

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

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

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

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.

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

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.

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

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

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

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

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