Putting the spotlight on: CRISPR

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Abstract

Rounding up the results from our recent spotlight on CRISPR – are we still just at the beginning for our CRISPR journey?

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Guidelines for optimized gene knockout using CRISPR/Cas9

Claude Campenhout, Pauline Cabochette, Anne-Clémence Veillard … (2019)

CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.

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Removal of extra sequences with I- Sce I in combination with CRISPR/Cas9 technique for precise gene editing in Drosophila

Nikolay Zolotarev, Pavel Georgiev, Oksana Maksimenko (2019)

The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies and has been adopted for many organisms, including Drosophila . Previously, an efficient two-step strategy was developed to engineer the fly genome by combining CRISPR/Cas9 with recombinase-mediated cassette exchange (RMCE). This strategy allows the introduction of designed mutations into a gene of interest in vivo . However, the loxP or frt site remains in the edited locus. Here, we propose a modification of this approach for rapid and efficient seamless genome editing with CRISPR/Cas9 and site-specific recombinase-mediated integration (SSRMI) combined with recombination between homologous sequences induced by the rare-cutting endonuclease I- Sce I. The induced homological recombination leads to the removal of the remaining extraneous sequences from the target locus.

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Combining CRISPR/Cas9-mediated knockout with genetic complementation for in-depth mechanistic studies in human ES cells

Zheng Wang, Yan Yan Zhang, Yu-Wei Lee … (2019)

Gene regulatory networks that control pluripotency of human embryonic stem cells (hESCs) are of considerable interest for regenerative medicine. RNAi and CRISPR/Cas9 technologies have allowed the identification of hESC regulators on a genome-wide scale. However, these technologies are ill-suited for mechanistic studies because knockdown/knockout clones of essential genes do not grow in culture. We have developed a genetic rescue strategy that combines CRISPR/Cas9-mediated knockout with TALEN-mediated integration of a doxycycline-inducible rescue transgene into a constitutive AASV1 locus. The resulting rescue clones are stable in culture, allow modulation of the rescue transgene dosage by titration of doxycycline in the media and can be combined with various molecular assays, thus providing mechanistic insights into gene function in a variety of cellular contexts.

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Journal

Journal ID (publisher-id): BTN

Title: BioTechniques

Publisher: Future Science Ltd (London, UK )

ISSN (Print): 0736-6205

ISSN (Electronic): 1940-9818

Publication date (Electronic, pub): 17 September 2019

Publication date (Print, pub): October 2019

Volume: 67

Issue: 4

Pages: 150-152

Affiliations

¹Future Science Group, Unitec House, 2 Albert Place, London, N3 1QB, UK

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[* ]Author for correspondence: flake@ 123456biotechniques.com

Article

DOI: 10.2144/btn-2019-0099

SO-VID: c5db47f1-e9ae-4227-a91b-f554497e3b27

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This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License

Putting the spotlight on: CRISPR

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Abstract

Most cited references 3

Guidelines for optimized gene knockout using CRISPR/Cas9

Removal of extra sequences with I- Sce I in combination with CRISPR/Cas9 technique for precise gene editing in Drosophila

Combining CRISPR/Cas9-mediated knockout with genetic complementation for in-depth mechanistic studies in human ES cells

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