Publication Date:
2017-09-20
Description:
Despite its explosive applications in genome engineering, CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) has been developed into a versatile tool beyond its well-known nuclease function. In this prospect article, we summarize a few exciting "off-label" applications of CRISPR including manipulating DNA sequences, visualizing chromosomal loci in living cells, and modulating transcription and chromatin structures. These novel applications will likely elevate CRISPR tools into yet another level of sophistication and diversity, leading to many more exciting cell biological discoveries. Since its inception as an exciting new genome-engineering platform, the CRISPR/Cas (CRISPR associated protein) system has evolved to be more than just a tool for targeted mutagenesis. Adapted from its natural role as part of the bacterial adaptive immune system, the CRISPR system has been simplified to consist of a single-guide RNA (sgRNA) and Cas nuclease (most commonly spCas9 derived from Streptococcus pyogenes ). Comprised of nuclease binding stem-loop structures and a ∼20 bp 5'- overhang for sequence-specific DNA recognition, a Cas9-loaded sgRNA scans the DNA for its complementary target sequence. Novel applications for the CRISPR/Cas system have emerged with the disabling of the nuclease domains of the Cas9 protein, resulting in a catalytically-inactive Cas9 nuclease (dCas9). This was done by introducing two point mutations, H840A to the HNH domain and D10A to the RUVC domain, which eliminated its ability to cleave either strand of DNA while maintaining its sequence-specific DNA recognition capability (Jinek et al., 2012). Acting as a programmable DNA binding protein, the CRISPR gene-editing system may now be repurposed to carry out new functions such as manipulating gene expression and labeling loci for dynamic cell imaging. In this prospect article, we discuss a few of the new and exciting uses of the CRISPR system, beyond its most popular application as a designer nuclease. This article is protected by copyright. All rights reserved
Electronic ISSN:
0091-7419
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
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