Due to its robustness and ﬂexibility, CRISPR applications are becoming more and more widespread. CRISPR has been a versatile tool with applications that are transforming not only gene editing studies, but also many other genome and chromatin manipulation efforts. These application areas are largely because of the programmable targeting capacity of catalytically inactive dead Cas9 (dCas9), which cannot cleave DNA but can still be guided to the target sequence. While native Cas9 enables gene editing through its guidable DNA cleavage activity, catalytically impaired Cas9 enzymes have been repurposed to achieve targeted gene regulation, epigenome editing, chromatin imaging, and chromatin topology manipulations.
Furthermore, the catalytically impaired nickase Cas9 enzyme has been used as a platform for base editing without double strand breaks (DSBs). Gene editing has already broadened our ability to investigate the contribution of specific genes and mutations to disease by facilitating the creation of the accurate cellular and animal models. For the past few years, gene therapy applications have also been demonstrated, e.g. by repairing the cftr gene in cultured cells from human cystic ﬁbrosis patients, by curing dominant cataract disorder and Duchenne muscular dystrophy by altering DNA in mouse germ-line cells, and by curing hereditary tyrosinemia in adult mice.
Another important milestone was the ﬁrst primate with precise genetic modiﬁcations, a result of gene editing in embryos. The ﬁnding allows for development of disease models in animals very similar to humans. A similar approach could be used to alter DNA in human embryos to prevent non-complex hereditary diseases, but also to attempt alteration of complex traits, which has triggered extensive ethical discussion. CRISPR is becoming an indispensable tool in biological research. The CRISPR-based technologies will undoubtedly continue to transform basic as well as clinical and biotechnological research. However, the road ahead is not free of obstacles. Many more in-depth studies are needed to be done in the future.
Fig 1. Major application areas of CRISPR-Cas-based technologies.
Imaging offers a direct approach for studying the spatial and temporal behavior of the genome in living cells. The ability of Cas protein (e.g. Cas9) to target speciﬁc sequences in the genome makes it a promising imaging tool for directly observing genomic organization and dynamics in living cells. Historically, ﬂuorescent in-situ hybridization (FISH) methods have been fundamental in determining the precise nuclear positions of speciﬁc genetic loci. Zinc ﬁngers (ZNF) and TALE proteins, which are developed later, have also been used in this application. However, the advances in the dCas9 platform technology have substantially improved both the efﬁciency and scope of genome targeting for live cell chromatin imaging. Researchers used ﬂuorescently labeled dCas9 to target repetitive regions of the genome to achieve the goal.
The ﬁrst work fused the S. pyogenes dCas9 to EGFP and used the fusion protein to visualize the dynamics of coding or noncoding sequences in living human cell lines. Scientists tracked the dynamics of telomeres, and the repetitive and non-repetitive sequences of coding genes in a short time frame (minutes) and throughout the whole cell cycle. In addition, dCas9 fused to EGFP has been used to label endogenous centromeres and telomeres loci in live mouse embryonic stem cells. A similar approach has been utilized to target repetitive natures of telomeres and centromeres by co-expression of dCas9 orthologs fused to different ﬂuorescent proteins and dual-color chromatin imaging of these repetitive regions. Targeting dCas9 to a non-repetitive genomic locus is more challenging because of the background ﬂuorescence signals due to free-ﬂoating ﬂuorescently labeled dCas9 proteins.
The simple and efﬁcient gene-targeting capacity of CRISPR has been harnessed to achieve large-scale functional screenings. The oligo libraries encoding hundreds of thousands of sgRNAs can be computationally designed and chemically synthesized to target a broad set of genome sequences. By pairing with Cas9 or dCas9 fusion proteins, this provides an approach to systematically knock out, repress, or activate genes on a large scale. Although the approach requires a number of technical and analytical considerations, once established, such an approach becomes a powerful high-throughput assay to functionally screen a large number of genes at the same time. Researchers have already applied dCas9-based epigenome-editing tools for a number of exciting purposes including high-throughput screenings to characterize functional distal enhancers, generation of induced pluripotent stem cells, and reversal of HIV latency.
The technique requires a delicate delivery method that ensures that every cell only receives a single sgRNA, usually via lentiviral or retroviral delivery into mammalian cells. The screens are frequently performed in a pooled manner, because cells transduced with the lentiviral library as a mixed population are cultured together. Since each sgRNA is stably integrated into the genome during viral infection, the guiding sequences of each sgRNA can be used as a unique 'barcode'. Via deep sequencing and analysis of the sgRNA features in the pooled cells, genes causing changes in cell growth and death can be inferred with bioinformatics. Indeed, CRISPR screens can easily identify genes, their regulatory elements, and protein domains in the mammalian genome responsible for cell growth and drug resistance.
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