CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo

Moreno-Mateos, Miguel A.; Vejnar, Charles E.; Beaudoin, Jean-Denis; Fernández, Juan Pablo; Mis, Emily K; Khokha, Mustafa K.; Giráldez, Antonio J.

doi:10.1038/nmeth.3543

Cited by 1,113 publications

(1,109 citation statements)

References 51 publications

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“…In particular, knockin efficiency depends critically on the activity of the sgRNA used for genomic cleavage. Different sgRNA sequences can vary widely in term of potency, and prediction algorithms have been developed to overcome this issue (37,38). However, because HDR knockin requires genomic cleavage close to the site of tag integration, for some genes the choice of sgRNAs to pick from might be scarce.…”

Section: Discussionmentioning

confidence: 99%

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

Leonetti

Sekine

Kamiyama

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

228

201

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A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. CRISPR/Cas9 | GFP library | genome engineering M ore than a decade after the completion of the Human Genome Project (1), over 30% of human genes still lack clear functional annotation (2, 3). Functional tagging is a powerful strategy to characterize the cellular role of proteins. In particular, tags allow access to two key features of protein function: localization (using fluorescent tags) and interaction partners (using epitope tags and immunoprecipitation). Hence, by tagging proteins in a systematic manner, a comprehensive functional description of an organism's proteome can be achieved. The power of systematic tagging approaches is best illustrated by studies conducted in the budding yeast Saccharomyces cerevisiae (4). In particular, a genome-wide collection of GFP-tagged yeast strains enabled the systematic study of protein localization in live cells (5), whereas libraries of strains expressing TAP epitope-fusion proteins paved the way for the large-scale isolation and proteomic analysis of protein complexes (6, 7). One of the great advantages of yeast genetics (especially in S. cerevisiae) is the efficiency and relative simplicity of PCR-based homologous recombination (8). As a result, functional tags can be easily inserted in a gene locus of interest, preserving endogenous expression levels and minimizing genomic disruption. Together, these genome-wide tagged libraries helped provide a comprehensive snapshot of the yeast protein landscape under near-native conditions (4, 5, 9-11).The development of clustered regularly interspersed short palindromic repeat associated protein 9 (CRISPR/Cas9)-based methods has profoundly transformed our ability to dir...

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Section: Discussionmentioning

confidence: 99%

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

Leonetti

Sekine

Kamiyama

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

228

201

View full text Add to dashboard Cite

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“…The online CRISPRscan tool was used to design gRNAs targeting the zebrafish 3' UTR region in the rab13 locus (Moreno-Mateos et al, 2015). Oligonucleotides comprising the target sequences flanked by the T7 promoter and the Tail annealing sequence (Moreno-Mateos et al, 2015) were annealed to the Tail primer in a T100 thermal cycler (BioRad).…”

Section: Grna Generation and In Vitro Translationmentioning

confidence: 99%

“…Oligonucleotides comprising the target sequences flanked by the T7 promoter and the Tail annealing sequence (Moreno-Mateos et al, 2015) were annealed to the Tail primer in a T100 thermal cycler (BioRad).…”

Section: Grna Generation and In Vitro Translationmentioning

confidence: 99%

mRNA compartmentalisation spatially orients tissue morphogenesis

Costa

Bradbury

Tarannum

et al. 2018

Preprint

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SUMMARYThe subcellular distribution of mRNAs is a fundamental biological mechanism implicated in the spatial regulation of gene expression in many cellular contexts. However, whether mRNA localisation contributes to complex morphogenetic processes underpinning tissue development remains unknown.We focused on a model transcript, RAB13, and demonstrated how it is preferentially localised to and locally translated at the leading edge of migrating endothelial cells. We generated a novel MS2-GFPnls zebrafish reporter strain and use the 3' UTR of RAB13 to show, for the first time, tissuespecific visualisation of a localised RNA in migratory cell processes in a living vertebrate organism.Moreover, after mutating the endogenous 3' UTR of RAB13 to disrupt transcript localisation in-vivo, we report altered formation of endothelial cell protrusions within sprouting vessels. Hence, we demonstrate the involvement of mRNA localisation in endothelial cell migration during angiogenesis and are the first to reveal that subcellular transcript distribution modulates tissue morphogenesis.

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“…This may indicate that the sequence context of the targeted site can be a determinant of either the repair mechanism [131] induced by the DNA nick or the activity of the guide. The latter has been demonstrated to be affected by the sequence content [210]. Furthermore, although the Merkle et al [145] study reported a total mean indel frequency for Cas9WT that is higher than Cas9n (10-fold difference for the untargeted allele), the distribution pattern of the data was similar.…”

Section: Screening For the Presence Of Random Integrationsmentioning

confidence: 89%

“…The difference in mutagenic activity of Cas9WT associated with a given guide sequence has been the subject of intense study [91,210,230,231]. Various factors influence nuclease activity such as sgRNA characteristics, Cas9 protein characteristics, and the locus context of the target DNA sequence in the cell type under study.…”

Section: Discussion and Future Directionsmentioning

confidence: 99%

CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

Al-Ateeq¹

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Beta thalassaemia is caused by mutations in the adult haemoglobin gene (HBB) and is one of the most prevalent monogenic blood disorders worldwide. The transplantation of haematopoietic stem cells derived from gene-corrected patient induced pluripotent stem cells (iPSCs) could provide a promising therapeutic strategy. Here, the generation and characterisation of footprint-free iPSCs from dermal fibroblasts of an individual with a homozygous beta thalassaemia-causing mutation affecting splicing is described (Chapter III). Successful gene correction of the disease-causing mutation was achieved by employing a CRISPR/Cas9 dual nickase approach and two donor design strategies aimed at reducing undesired on-target mutagenesis (Chapter IV), followed by the This study combined cutting-edge cellular reprogramming methods to generate beta thalassaemia iPSCs, a double nickase-mediated gene editing approach and piggyBac transposase-aided excision to achieve seamless gene repair, and a CRISPRa approach to model the impact of the diseasecausing mutation and demonstrate restoration of normal transcription following genetic repair.Having established a platform for precise gene correction and for validation of the restoration of the functional allele in this monogenic disease, this strategy may provide a viable avenue for iPSCiii based cell therapy of beta thalassaemic patients provided mature engraftable blood cells can be generated from hiPSCs in the future.iv

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CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo

Cited by 1,113 publications

References 51 publications

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

mRNA compartmentalisation spatially orients tissue morphogenesis

CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

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