November 15, 2017 (Vol. 37, No. 20)
Andrea Toell Ph.D. Product Manager Lonza Walkersville
The Future of the Gene-Editing Method of Choice
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
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 therapeutic 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 disease modeling, but it can also be used to genetically modify cells for therapeutic purposes. As such, it has exciting potential 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 discoveries 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 endonuclease 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, nonhomologous 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 enabled 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, including “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 lines11 and using in silico methods to design CRISPR-based synthetic single guide RNAs (sgRNA) can help to improve specificity.12 Moreover, chemically modified sgRNA codelivered with Cas9 mRNA or protein can enhance genome-editing efficiency.13
Additionally, the use of genetic circuits to enable spatiotemporal control of induced Cas9, such as small molecule-regulated approaches for temporal control14 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 conventional 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 protein and sgRNA (referred to as ribonucleoprotein; RNP), the components have to cross only one cellular membrane, giving 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 transfecting primary cells. For example, a recent study used RNP to correct a gene mutation that causes hypertrophic cardiomyopathy in human embryos for the first time, reporting minimal 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. Therefore, research has focused on how the delivery of CRISPR/Cas9 components can directly influence genome-editing efficiency 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 materials 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 the hematopoietic system, such as T cells, are particularly suitable for ex vivo modification, as they can be easily isolated from the blood, expanded ex vivo, and transplanted back into a patient with limited immune response.2 For example, Schumann et al. disrupted PD-1 and CXCR4 immune checkpoint genes in human T cells by delivering CRISPR/Cas9 RNPs via electroporation, which successfully prevented the inhibition of T cells from killing cancer cells.17 Moreover, resistance to HIV-1 infection has been obtained through disrupting coreceptors of HIV-1 using CRISPR/Cas9-encoding plasmids, with minimal off-target effects.18
CRISPR/Cas9 components can be introduced via viral or nonviral systems, with the optimal strategy depending on the desired application and target cells/organ. Viral delivery systems are the traditional method for delivering genome-editing components as nucleic acids (DNA and mRNA). Lentivirus, adenovirus, and adeno-associated virus have all been used for delivery of CRISPR/Cas9 components.2 Lentivirus and adenovirus have a large packaging capacity and thus, a high efficiency for in vivo gene transfer, but they have high immunogenicity. Therefore, they may lead to unwanted immune responses and harmful side effects in patients.19 Although adeno-associated viruses have a lower immunogenicity, they have a smaller packaging limit compared to lentivirus and adenovirus.20
In contrast, nonviral methods allow more flexibility, as they can deliver the genome-editing materials either as plasmid DNA, mRNA, or as a RNP complex of Cas9 protein and sgRNA. As proteins can be rapidly degraded in cells, using RNPs is associated with fewer off-target effects compared to using nucleic acids.21
Nonviral methods have much lower immunogenicity and are consequently becoming a preferred transfection approach.1 For example, lipid-mediated delivery enables an easy and simple handling process, and bioreducible lipid nanoparticles can reduce off-target effects compared with viral delivery methods,22 although the efficiency of lipid-mediated delivery depends on the cell type.23
Alternatively, usually electroporation delivers genome-editing materials through nanometer-sized pores in the cell membrane, which are created using an electrical current. This method is less dependent on the cell type and allows high transfection efficiency of nucleases into difficult-to-transfect cells, such as induced pluripotent stem cells.24
Lonza Turns its Focus to CRISPR Gene Editing
CRISPR gene editing is driving forward a new frontier in research, medicine, and biotechnology. As an enhanced form of electroporation, Lonza’s Nucleofector™ technology is becoming crucial for the nonviral delivery of CRISPR reagents, including DNA, mRNA, and RNP, and is thus poised to facilitate the advancement of future CRISPR-based applications (Figure 2).
Nucleofection not only enables high transfection efficiency for a large range of cell types, including primary cells, stem cells, and clinically relevant cell lines, but it also has a low impact on cell viability and functionality. Moreover, its substrate versatility and efficient co-transfection of various substrates means that the same approach can be used for all CRISPR/Cas9 substrates. The Nucleofection portfolio enables the same transfection protocols to be used across different platforms, allowing flexibility of scaling. For example, transfection protocols can be established in smaller scale using the 4D-Nucleofector™ X Unit and smoothly transferred to larger scale using the 4D-Nucleofector™ LV Unit, without the need for reoptimization.
As such, Nucleofector™ Technology is showing great promise for nonviral CRISPR-based genome editing. For example, recent studies have used Nucleofection to improve the therapeutic efficiency of Cas9-edited CAR-T cells in precision cancer immunotherapies,25 and to produce advanced models of acute myeloid leukemia (AML) to gain new insights about AML development and progression.26
With the increasingly important role of Nucleofection in genome editing, leveraging Lonza’s technology can help to develop and improve CRISPR-based gene editing by ensuring the efficient delivery of CRISPR reagents into cells.
CRISPR/Cas9 genome editing is fast emerging as a promising therapeutic approach due to its highly specific, flexible, and easy application to modifying primary cells and producing custom cell lines. Technological advances are driving forward CRISPR-based gene editing applications and research, and these advances promise to continue to open up new therapeutic avenues for studying and combating disease.
It is important, however, to consider various factors when using a CRISPR-based gene-editing approach because these can impact its application to research and therapy. These factors include selecting an approach that best suits the objectives of the research (e.g., ex vivo or in vivo cell editing), ensuring editing is highly specific with minimal off-target effects, and employing efficient transfection methods, such as electroporation via Nucleofection.
Certainly, as research breakthroughs and technological advancements continue, the development of safe and efficient platforms for genetic and epigenetic engineering will transform our ability to therapeutically target human diseases and even potentially engineer disease resistance.
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