Abstract
CRISPR/dCas9 is a versatile tool that can be used to recruit various effectors and fluorescent molecules to defined genome regions where it can modulate genetic and epigenetic markers, or track the chromatin dynamics in live cells. In vivo applications of CRISPR/dCas9 in animals have been challenged by delivery issues. We generate and characterize a mouse strain with dCas9-EGFP ubiquitously expressed in various tissues. Studying telomere dynamics in these animals reveals surprising results different from those observed in cultured cell lines. The CRISPR/dCas9 knock-in mice provide an important and versatile tool to mechanistically study genome functions in live animals.
Highlights
Revolutionary CRISPR/Cas9 technique is becoming one of the most powerful tools in biological and biomedical studies for almost all model organisms [1,2,3,4]
The data were collected from at least two mice for each treatment. d Representative images of telomere aggregations observed in deactivated Cas9 (dCas9)-EGFP mice injected with empty gRNA, gRNAs targeting TRF1, and TRF1 gRNAs plus human TRF1 expression cassette
Aggregation is marked by a red arrow. e Quantification of telomere aggregations in dCas9-EGFP mice injected with different constructs
Summary
Revolutionary CRISPR/Cas technique is becoming one of the most powerful tools in biological and biomedical studies for almost all model organisms [1,2,3,4]. While extensive efforts have been focused on the optimization and implication of targeting and cleavage by CRISPR/ Cas systems for genome editing, recently, the nuclease-deactivated Cas (dCas9) has been developed as a versatile tool to genetically and epigenetically modulate the targeted genomic locus and label the genomic loci in living cells [5,6,7,8,9]. The dCas9/gRNA tools have been mostly developed in cell culture systems where the dCas, gRNA, and effector expression cassettes could be transfected or infected into the cells. For in vivo applications of dCas9/gRNA in live animals, how to efficiently deliver all these components, especially the large dCas expression cassettes, into the cells of various tissues remains to be a major difficulty [13]
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