Genetic Engineering & Biotechnology News

NOV15 2017

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18 | NOVEMBER 15, 2017 | | Genetic Engineering & Biotechnology News These advantages have led to it quickly becoming the gene- editing method of choice, leading to its widespread use in research, medicine, and biotechnology, including for thera- peutic purposes. CRISPR/Cas9 involves introducing a double-strand break (DSB) at a specific DNA site to knockout or delete a gene, or insert or correct a gene. This not only enables the generation of custom cell lines and modification of primary cells for dis- ease modeling, but it can also be used to genetically modify cells for therapeutic purposes. As such, it has exciting poten- tial for studying and treating genetic diseases, being able to alter, replace, or regulate the expression of affected genes to reverse the diseased phenotypic state. However, there are still various challenges concerning the safety and efficacy of CRISPR-based gene editing before it can be widely used for therapeutic applications. Yet, new dis- coveries are constantly helping to overcome these challenges. Below we briefly review recent research developments that could help to realize the great potential of CRISPR-based gene editing in disease research and cell therapy. Minimizing Off-Target Effects while Enhancing Efficacy CRISPR-based gene editing relies on the Cas9 DNA en- donuclease being directed to a specific genomic locus by "guide" RNA (gRNA). Here Cas9 introduces a DSB, which is then repaired via one of two major pathways, nonho- mologous end-joining (NHEJ) or homology-directed repair (HDR), to produce the desired genomic change. HDR uses an undamaged DNA template to repair the DSB, allowing new sequences to be introduced into the gene of interest. As such, gene insertions or corrections can be en- abled by HDR. 2 However, HDR is less likely to occur than NHEJ because the template DNA must be available during repair, so NHEJ inhibitors (e.g., Scr7) 3 or HDR enhancers (e.g., Rad51) 4 have been developed to increase HDR rate. The specificity of CRISPR/Cas9 gene editing is a major concern because DSBs at an unwanted DNA site can lead to off-target effects that could harm patients, potentially limiting clinical applications. As such, recent research has attempted to enhance the specificity of CRISPR/Cas9 while ensuring that on-target activity is not compromised. A Cas9 variant, known as Cas9-nickase or Cas9n, has been developed that cuts just one strand of DNA rather than both, enabling highly specific genome editing via both NHEJ and HDR pathways. 5 Moreover, using nuclease-dead Cas9 fused to the nonspecific endonuclease Fokl (dCas9-Fokl) is unlikely to cut DNA at an off-target site, as Fokl will only cleave DNA when it is dimerized. 6 Other Cas9 variants, in- cluding "enhanced Cas9" (eSpCas9) and "high-fidelity Cas9" (spCas9-HF1), have been found to display reduced off-target cleavage while maintaining robust on-target activity. 7,8 The design of gRNAs can also reduce off-target effects by increasing their specificity to the target genomic site. 9 For example, using truncated gRNAs has been associated with lower off-target effects. 10 Furthermore, double-nicking of DNA using paired gRNAs can reduce off-target activity by 50- to 1500-fold in cell lines 11 and using in silico methods to design CRISPR-based synthetic single guide RNAs (sgRNA) can help to improve specificity. 12 Moreover, chemically modi- fied sgRNA codelivered with Cas9 mRNA or protein can enhance genome-editing efficiency. 13 Additionally, the use of genetic circuits to enable spatio- temporal control of induced Cas9, such as small molecule- regulated approaches for temporal control 14 and tissue- specific promoters for spatial control, 15 can help to balance gRNA on-target activity with off-target effects. The transfection of different gene-editing components can also impact the efficiency and specificity of CRISPR/Cas9 gene editing. Although transfecting plasmid DNA is the con- ventional choice due to it being more stable as well as easy to handle and propagate, it must be transcribed to be effective and as a result, has a prolonged presence in the cell. This prolonged residence time of the plasmid has the potential to introduce off-target effects. Alternatively, with mRNA and a complex of Cas9 pro- tein and sgRNA (referred to as ribonucleoprotein; RNP), the components have to cross only one cellular membrane, giv- ing mRNA and RNP several advantages, including increased transfection efficiency, better dose control, and minimal risk of genomic integration. Thus, they can yield better specificity than plasmid DNA in some contexts, such as when transfect- ing primary cells. For example, a recent study used RNP to correct a gene mutation that causes hypertrophic cardiomy- opathy in human embryos for the first time, reporting mini- mal off-target effects and a high success rate (72%). 16 Optimizing Delivery Methods The delivery methods and vectors used to transfect the CRISPR payload must enable high efficiency while avoiding potentially harmful immune responses in the patient. There- fore, research has focused on how the delivery of CRISPR/ Cas9 components can directly influence genome-editing ef- ficiency as well as safety. CRISPR/Cas9-based disease therapeutics can be achieved both in vivo and ex vivo. In ex vivo therapy, cells are isolated and edited outside of the body using engineered nucleases, after which they are transplanted back into the body (e.g., in cancer immunotherapy; see Figure 1). In in vivo therapy, genetic mate- rials are directly injected into the body (e.g., in genetic disease therapy). Ex vivo editing makes it easier to control the delivery of CRISPR/Cas9 components (such as for variables such as the dose), and more delivery modes are available using this approach. Inhibition of a viral infection and cancer immunotherapy are the main ex vivo applications of CRISPR/Cas9. Cells of Andrea Toell The CRISPR/Cas9 system is a fast-emerging technology that has transformed our ability to precisely target genomic sites. Compared to other gene-editing technologies (e.g., ZFNs and TALENs), it is simpler to re-engineer, relatively inexpensive, easier to use, and has broad versatility, high efficiency, and can target multiple sites. 1 Unlocking the Potential of CRISPR-Based Gene Editing OMICS Figure 1. Cancer immunotherapy is one of the main ex vivo applications of CRISPR/Cas9. Figure 2. An enhanced form of electroporation, Nucleofectorâ„¢ technology (Lonza) is becoming a standard for the nonviral delivery of CRISPR reagents.

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