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Gene Editing

Targeted genome editing has been explosively fueled by the introduction of several novel DNA cleavage (nuclease) technologies including homing endonucleases or meganucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and CRISPR-associated system 9 (Cas9) nucleases1. These nucleases utilize site-specific double-strand DNA break (DSB)-mediated DNA repair mechanisms allowing precise genome editing. Such strategy has enabled rapid, easy and efficient modification of endogenous genes/genomes in a wide variety of cell types and species. Clinically, it is being explored as a novel therapeutic approach for genetic disorders, viral infections and cancer. All ZFN, TALEN and Cas9 techniques have been used to disrupt HIV-1 entry co-receptors (CCR5, CXCR4) and proviral DNA-encoding viral proteins2. CCR5 gene-targeting ZFNs are in phase 2 clinical trials for HIV-1/AIDS treatment3. Also, ZFN and Cas9/gRNA have been recently shown to remove the proviral HIV-1 DNA from host cellular genome, by targeting its highly-conserved 5’ and 3’ long term repeats (LTRs)4. Our recent studies found that LTR-directed Cas9 eradicates the HIV-1 genome and effectively vaccinates target cells against HIV-1 reactivation and infection with high specificity and efficiency5. These properties may provide a viable path toward a permanent or “sterile” HIV-1 cure, and perhaps provide a means to eradicate and vaccinate against other pathogenic viruses.

Traditional homologous recombination-mediated gene targeting has made a great breakthrough in transgenic animal models, but the low efficiency, time-consuming and high costs limit its routine application6. Site-specific DSBs allow precise and efficient genome editing1 with 100-50,000 fold increase in the targeting efficiency (Ellis et al., 2013). In the past three years, ZFN and TALEN have been widely used for such genome editing. However, both require the engineering of custom DNA-binding protein for each target site and both produce uncontrollable off-target effects7. In the past two years, the Cas9 biotechnology has dominated the field of genome editing because of its easy, fast, cheap and versatile features. Cas9, together with small guide RNA (sgRNA), generates DSBs at any site defined by a 20-nucleotide guide (seed) sequence and trinucleotide (NGG or NAG) protospacer adjacent motif (PAM) recognized by Cas98. This method needs only a tiny custom RNA molecule which, like small hairpin RNA (shRNA), can be synthesized or in vitro transcribed for direct RNA transfection or expressed from U6-promoted sgRNA expression vector. In addition to gene targeting, the Cas9/gRNA technology has been expanded to the gene regulation (activation and repression).

Cas9 and sgRNA can be expressed in individual vector or all-in-one vector. Various types of Cas9/gRNA expression vectors are available through Addgene or commercial companies. The Genome Editing Core at CNAC provides various types of consultations including experimental design, gRNA screening, Cas9/gRNA vector selection, genotyping analysis, specificity and off-target evaluation, and data interpretation. This Core also assists investigators with the generation of Cas9 stable cell lines and transgenic animals as well as target gene knockin/knockout cell lines and transgenic animals.

1 (Bedell et al., 2012; Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013; Qi et al., 2013; Wang et al., 2012a)
2 (Manjunath et al., 2013; Stone et al., 2013)
3 (Hofer et al., 2013; Tebas et al., 2014)
4 (Ebina et al., 2013; Qu et al., 2013)
5 (Hu et al. 2014)
6 (Wang et al., 2012b)
7 (Bogdanove and Voytas, 2011; Chen et al., 2013; Lee et al., 2012; Mussolino et al., 2011)
8 (Gasiunas et al., 2012; Jinek et al., 2012)

Support for CRISPR/Cas9 based gene editing approaches targeting viral and cellular genes delivered by transient or stable transfection, viral vector transduction, for in vitro and in vivo approaches. Guidance is provided on target selection, bioinformatics screening, validation of target modification, and assessment of safety ands efficacy