In recent years, CRISPR-Cas9 system has garnered increasing attention as an effective and simple genome-engineering tool and has revolutionized the life sciences. The ability to use nucleic-acid hybridization rules to reprogram Cas9 specificity significantly simplifies gene editing applications particularly given that sgRNAs are easily synthesized and introduced into cells to facilitate targeted genome modifications. The CRISPR-Cas9 system has been used on mammalian cells since early 2013. It has been recognized for its potentially transformative applications in transcriptional modulation, epigenetic regulations, base editing, high-throughput genetic screening, generation of animal or cell models of diseases and gene therapy.
Despite being a nascent technology, Cas9 has been successfully used to a variety of basic research as well as biotechnology applications. Because CRISPR-Cas9 systems are highly programmable, Cas9/sgRNA complexes can be utilized for gene editing or catalytically inactive Cas9 (dCas9)/sgRNA complexes can be used for gene regulation. The dCas9 protein is a Cas9 variant which is capable of binding to the target sequence but unable to cleave its target. dCas9 has been adopted as a DNA-binding platform for transcription modulation and epigenetic editing, and engineered by using a variety of effector domains. Combined with activate or repression domain (such as VP64 and KRAB), dCas9 can activate or repress, respectively, DNA transcription without changing DNA sequence. The transcription modulation of CRISPR-Cas9 system requires the design of sgRNAs that are efﬁcient and speciﬁc.
Fig 1. Applications overview of CRISPR-Cas9 system.
Fig 2. Transcription modulation of CRISPR-Cas9 system.
In the previous studies, dCas9 is usually converted into a synthetic transcriptional activator by fusing with conventional transcriptional activators such as VP16/VP64, p65 or a subunit of RNA polymerase. However, the dCas9-VP64 system was not very effective, targeting dCas9 activators with a single sgRNA to a particular endogenous gene promoter leads to only modest transcriptional upregulation. Thus, fusion or recruitment of multiple transcriptional activation domains to the dCas9/gRNA complex, synergistic activation mediator (SAM) or dCas9-Suntag were performed to improve the activation capacity and expand the range of applications. These systems could induce very high activation by using several sgRNAs, or even one sgRNA for each target gene, enabling high levels of activation in vitro.
This CRISPR-based interference, or CRISPRi, works efficiently in prokaryotic genomes but is less effective in eukaryotic cells. dCas9 can block target gene transcription by fusing a repressive effector domain such as the Kruppel-associated box (KRAB) or SID effectors, which promote epigenetic silencing. KRAB repression is mediated by repressive histone modiﬁcations such as H3K9me3. By utilizing epigenome-modifying repressors, including Lys-speciﬁc histone demethylase1 (LSD1), histone deacetylase (HDAC), DNA methyltransferases DNMT3A and MQ1, and mSin3 interaction domains, the scope of applying CRISPR repression has been extended to epigenetic editing. Similarly, epigenome editing approaches can also be used for targeted transcriptional activation, such as dCas9 fused with a DNA demethylase or a histone acetyltransferase.
CRISPR-Cas9 has many potential clinical applications. The initial focus has been on cancer immunotherapy and correction of single gene disorders. For example, researchers have used the CRISPR-Cas9 system to correct pathogenic variants underlying beta thalassemia, a hemoglobinopathy. CRISPR-Cas9 offers multiple options to correct such defects, including changing the genetic code at the locus containing the pathogenic variant or creating an alternate hemoglobin product that can reduce severity of disease. With the advent of CRISPR-Cas9 come new considerations of when and how this technology should be applied in the clinical setting. Conversations about the ethics of clinical applications of gene editing and its potential impacts on minorities have been happening for years. These conversations should continue to move into the public sphere.
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