CRISPR/Cas9: From Defending Bacteria to Powerful Genome and Epigenome Editing

Harness the power of the CRISPR/Cas9 system as a precise gene and epigenome editing tool.

The CRISPR/Cas9 system is a genome editing tool that promises to transform the biomedical research field. Applications of CRISPR/Cas9 extend well beyond its use in basic science research as a tool for editing and study of genes functional roles. It also has potential in the medical field not only for treating but possibly even curing disease by “correcting” genes involved in the pathogenesis of numerous diseases.

CRISPR/Cas9 as well as other genome-editing systems, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases, are all based on the use of programmable DNA nucleases to cut DNA at specific locations introducing double strand breaks (DSBs) in the genome, which can then be repaired by non-homologous end joining (NHEJ) or the high fidelity homology-directed repair (HDR), leading to disruption in the DNA sequence. These systems can cut out, insert or modify specific gene sequences, thereby harnessing the potential to permanently “edit” the genome.

Easily quantify the levels of Cas9 nuclease in extracts from cells and tissues of a variety of species using the EpiQuik CRISPR/Cas9 Assay ELISA Kit. Plus, use a CRISPR/Cas9 monoclonal antibody to confirm transfection, detect Cas9 expression levels, and verify nuclear localization of Cas9 protein for your gene editing research.

The CRISPR/Cas9 system is derived from an adaptive immunity mechanism used by bacteria as defense against viral and other foreign DNA (Figure 1). Upon infection, bacteria store sequences of the invading DNA between repeats in the CRISPR (Clustered regularly interspaced short palindromic repeat) locus. After transcription, the pre-crRNAs are processed to small crRNAs, which contain a CRISPR repeat, and a portion of the sequence of the invading DNA. The crRNA associates with a trans-activating crRNA (tracrRNA) and a Cas9 nuclease. This complex binds by sequence complementarity to the target DNA, and the Cas9 cleaves the invader DNA. This system has later been simplified by researchers to include the crRNA and the tracrRNA in just a single guide RNA (sgRNA).

Figure 1. CRISPR/Cas9 mediated bacterial immune defense. Zhao Y, Ying Y, Wang Y (2014) Developing CRISPR/Cas9 Technologies for Research and Medicine. MOJ Cell Sci Report 1(1): 00006.

One of the advantages of the RNA-guided CRISPR/Cas9 system over the other genome-editing tools is its simplicity, just modifying the specificity region (~20 bp sequence that’s complementary to the target DNA) in the guide RNA is all that’s needed to change gene target, and the simplest system just requires Cas and the sgRNA. Although different CRISPR systems exist that use various forms of Cas nuclease, the most widely used CRISPR/Cas system derives from the Streptococcus pyogenes bacteria and uses the Cas9 nuclease. Cas9 wild-type has full nuclease activity and cuts at both strands of the DNA. Another type of Cas9, nuclease deficient-Cas9 (dCas9), lacks nuclease activity but retains DNA binding activity. dCas9 can be fused to regulatory effector proteins to be used for genome regulatory (gene activation or repression) or visualization (e.g. fused to eGFP) purposes but not for editing the DNA sequence per se.

The CRISPR/Cas9 system has been used in more than 40 species for targeted gene editing in cells, tissues and even whole organisms. CRISPR/Cas9 has been successful in correcting certain genes implicated in disease etiology, such as the dystrophin gene involved in Duchenne muscular dystrophy (Dmd), leading to restoration of skeletal muscle function in a Dmd mouse model. The first clinical trial in humans began in October 2016 in China, where researchers edited the PD-1 gene in T-cells and transplanted cells back to induce the patients’ immune system to fight against lung cancer cells. Clinical trials are arising in the US as well, where researchers aim to simultaneously edit three T-cell genes to boost a patient’s immune system as a therapeutic approach against several types of cancers.

In the epigenetics field, the CRISPR/Cas9 system has been used as an epigenome editing tool for targeting histone modifications and DNA methylation. These approaches use the nuclease deficient Cas9 fused to different epigenetic effectors aimed at activating or repressing transcription and gene expression. In several studies, dCas9 fused to the catalytic domains of DNA methyltransferase 3A (DNMT3A) or Ten-Eleven Translocation dioxygenase 1 (TET1), has successfully induced DNA methylation and demethylation, respectively, in specific DNA loci. This has led to repression or activation of target genes, including CDKN2A, ARF, BRCA1, MMP2, and others. Specific histone modifications have also been targeted by using dCas9 fused to diverse histone modifiers, such as the acetyltransferase domain of the HAT p300, with concomitant acetylation and activation of repressed genes. These findings highlight the potential of CRISPR/Cas9 in reversing epigenetic alterations involved in pathological processes, and may have profound therapeutic implications for certain diseases, including cancer.

To facilitate genome and epigenome editing research, EpiGentek offers the EpiQuik CRISPR/Cas9 Assay ELISA Kit to quantify the levels of Cas9 nuclease in extracts from cells and tissues of a variety of species. This Cas9 detection tool can be useful in measuring Cas9 or dCas9 levels to verify transfection efficiency during screening and selection of Cas9 transfected clones. It can also be used to measure Cas9 when following the kinetics of expression of Cas9 in transient expression systems. It is a fast and easy assay, that only requires a microplate reader for colorimetric detection, and the high-throughput 96-well format allows for quantification of Cas9 protein in multiple samples without the need of time-consuming western blots.

References:

Sander JD and Joung JK, CRISPR-Cas systems for genome editing, regulation and targeting. Nat Biotechnol. 2014 April ; 32(4): 347–355.
Zhao Y, et al., Developing CRISPR/Cas9 Technologies for Research and Medicine. MOJ Cell Sci Report (2014) 1(1): 00006
Yin H, et al., Delivery technologies for genome editing. Nat Rev Drug Discov. 2017 Mar 24.
Mout R, et al., In Vivo Delivery of CRISPR/Cas9 for Therapeutic Gene Editing: Progress and Challenges. Bioconjug Chem. 2017 Mar 17.
Enriquez P, CRISPR-Mediated Epigenome Editing. Yale J Biol Med. 2016 Dec 23;89(4):471-486.
Cyranoski D, CRISPR gene-editing tested in a person for the first time. Nature. 2016 Nov 24;539(7630):479.

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