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The CRISPR gene-editing technology is taking the scientific world by storm, but researchers are still uncovering the platform’s potential and pitfalls.
The “Democratization” of Gene Editing
The idea of modifying human genomes using homology directed repair (HDR) has been around for decades. HDR is a widely-used repair mechanism to fix double-strand breaks in the cell’s DNA. By supplying an exogenous, homologous piece of DNA to the cell and increasing the probability of HDR occurring, changes in the DNA sequence can be introduced to the targeted area.
Some of the first gene editing platforms taking advantage of HDR used engineered endonucleases such as zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). Both ZFNs and TALENs required a custom protein to target a specific DNA sequence, making them pricey and very difficult to engineer. The more recently developed CRISPR/Cas9 platform works differently: instead of using a protein, the Cas enzyme uses a small guide RNA to locate the targeted DNA, which is then cut. CRISPR is more low tech and “user-friendly” than other platforms, as users are no longer required to do the onerous chore of protein engineering. Now, the ability to modify genomes can be done without extensive training or expensive equipment. CRISPR can be used for knocking out genes, creating reporter or selection genes, or modifying disease-specific mutations—by practically anyone with basic knowledge of molecular biology.
The CRISPR Revolution
Because of its relative simplicity and accessibility, it is no wonder that life scientists from all over the world are eager to incorporate CRISPR into their research. One of the most remarkable things about CRISPR technology is how quickly its popularity has allowed the platform to evolve. The discovery revealing that CRISPR can be used for RNA-programmable genome editing was first published in 2012 (1). Since then, about 2,000 manuscripts have been published including this technology and millions of dollars have been invested into CRISPR-related research and start-up companies. Scientists have developed a myriad of applications for CRISPR, which can be used in practically any organism under the sun. The popularity and progress of gene editing promises revolutionary advancements in virtually every scientific field: from eradicating disease-carrying mosquitoes to creating hypoallergenic eggs and even curing genetic diseases in humans.
There is no doubt that CRISPR has enormous potential – the widespread interest and rapid progress are evidence of that. The main reason CRISPR has been so widely adapted is because development and customization is way less labor-intensive and time-consuming than previous methods. But material preparation is a miniscule part of the CRISPR platform. Contrary to popular belief, just because CRISPR is easier to use than other methods of gene editing, it is not “easy.”
Hitting the Bull’s-Eye
To understand the limitations of this gene editing technology, it’s important to understand more about how CRISPR works. The CRISPR/Cas9 system functions by inducing double-strand breaks at a specific target and allowing the host DNA repair system to fix the site of interest. Cells have two major repair pathways to fix these types of breaks, Non-Homologous End Joining (NHEJ) and homology directed repair (HDR). NHEJ is faster and more active than HDR and does not require a repair template, so NHEJ is the principle means by which CRISPR/Cas9-induced breaks are repaired. If the editing goal is to induce insertions or deletions through NHEJ to cut out a part of a gene, then using the CRISPR platform is less complicated. But directing CRISPR to correct a gene with exogenous DNA does not work very well, as the rate of HDR occurring at the site of interest is often very low, sometimes less than 1%. For precise genome-editing through CRISPR, it is essential to have HDR while minimizing damaging NHEJ events.
Increasing the rate of HDR is not trivial. Recent research has shown that the conditions for the two repair pathways vary based on the cell type, location of the gene, and the nuclease used (2). Besides, optimizing CRISPR to better control HDR versus NHEJ events is only half the battle. One of the greatest challenges in using CRISPR is to be able to quickly and accurately detect different genome-editing events. Many scientists measure changes by sequencing. A more sophisticated method is to use droplet digital PCR (ddPCR) (3), but many labs have limited accessibility to ddPCR systems. After screening multiple clones and detecting the desired genetic edit comes what is usually the most laborious and time-consuming step: pure clonal isolation. Because each individual cell is affected by CRISPR independently, isogenic cell lines must be established.
CRISPR has become the gold standard for many scientists now that the potential to perform gene editing has become so universal. Top journals expect isogenic lines for characterizing genes and mutations, and many researchers feel pressured to include such experiments to stay competitive in grant proposals. Moreover, a federal biosafety committee has recently approved the first study in patients using this genome-editing technology. To keep up with the pace of this rapidly moving field, significant improvements in CRISPR technology need to be made.
First, a better grasp of the basic principles behind CRISPR is likely to lead to improvements in targeting and efficiency. For example, understanding how the cell type, locus, and genomic landscape affect the targeting and cutting of Cas9 could lead to higher efficiencies of HDR. Similarly, engineering Cas9 and the guide RNA to maximize on-target activity could also accelerate the technology’s success. Screening the ability of the guide RNA to target the intended sequence can be performed in vitro by assessing the Cas9-mediated cuts on a PCR-amplified fragment of DNA; this method often gives a good indication of what to expect in cells. Another hindrance to CRISPR technology is that currently, all methods to detect genome changes require destruction of the cells of interest. Development of an assay to detect chromosomal changes in live cells – reminiscent of live-cell RNA detection – would immensely improve the processes of clonal screening and isolation.
Besides the technical challenges that come with maximizing on-target edits is that Cas9 frequently has off-target effects, producing insertions or deletions at unintended sites. Online algorithms can predict where some of these cuts are likely to occur, but currently, there is no efficient method to identify all possible off-target sites (4). To complicate things further, no two human genomes are identical. Due to genetic variation, predicting off-target effects based on reference genomes remains a challenge. Because so much of the hype around CRISPR surrounds the potential to treat human diseases, it is imperative to make sure that CRISPR does not introduce detrimental changes elsewhere in the genome before therapeutic use in humans. Standard screening methods and biological assays need to be established to robustly assess potential damage done to other sites in the genome and to measure its impact on cell function and mutagenesis.
Despite its shortcomings, the hub of activity surrounding CRISPR has been astounding. And with the force of thousands of scientists working on this technology, the possibilities for the future of CRISPR are boundless.
- Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21.
- Miyaoka, Y., et al., Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep, 2016. 6: p. 23549.
- Hindson, B.J., et al., High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem, 2011. 83(22): p. 8604-10.
- Stella, S. and G. Montoya, The genome editing revolution: A CRISPR-Cas TALE off-target story. Bioessays, 2016. 38 Suppl 1: p. S4-s13.
Single-domain nano antibodies have a broad range of applications in biochemistry due to their small size, high affinity, and high specificity. Now, a team of researchers from the University of Basel and the University of Zurich has demonstrated that nano antibodies can be used for research in complex living organisms such as Drosophila, uncovering another new and exciting application for nano antibodies.
The team used nano antibodies to develop an assay for studying morphogens, molecules that regulate the pattern of tissue growth and the positions of various cell types within tissue. Morphogens form long-range concentration gradients from a localized source, ultimately determining the fate and arrangement of cells that respond to that gradient. Drosophila is a classic model system for understanding how morphogens regulate organ development. One morphogen called Dpp controls uniform proliferation and growth of the wing imaginal disc. Yet because Dpp is an extracellular, diffusible protein, it is difficult to immobilize in situ. Therefore, despite over 20 years of studying the role of Dpp as a morphogen, the lack of a dynamic system for controlling Dpp gradients has prevented researchers from understanding precisely how Dpp governs development of the wing disc.
By developing a novel synthetic system using nano antibodies, the researchers were able to modulate the concentration gradient of Dpp at the protein level. Their system—coined “morphotrap”—uses a membrane-bound GFP nano antibody to “trap” GFP-tagged Dpp at different locations along the wing imaginal disc. By tethering Dpp in a controlled spatial manner, researchers were able to determine how Dpp gradients affect wing disc development. They discovered that the gradient of Dpp is required for the patterning of the wing disc but not for lateral growth, disproving one of the field’s popular theories that address the role of Dpp. In addition to resolving the controversy with respect to the role of Dpp as a morphogen, this study pioneers a new method for using nano antibodies in situ.
“Dpp spreading is required for medial but not for lateral wing disc growth.”
Harmansa S., Hamaratoglu F., Affolter M., Caussinus E.
Nature. 2015 Nov 19;527(7578):317-22. doi: 10.1038/nature15712. Epub 2015 Nov 9.
Allele to Generate Human iPS Cells under Good Manufacturing Practice for Private Individuals for Potential Therapeutic Use and Future diagnostics; Cell Banking Scientific Advisory Board formed
October 06, 2015 11:25 AM Eastern Daylight Time
SAN DIEGO–(BUSINESS WIRE)–Allele Biotechnology & Pharmaceuticals, Inc. (“Allele”), a leader in the development of specialized cells for regenerative medicine and pharmaceutical drug discovery, today announced plans to form a commercial business for the banking of human induced pluripotent stem cells (iPSCs) by private individuals.
Allele is pleased to have Drs. Mahendra Rao and Joseph Paulauskis as the first members of its Scientific Advisory Board (SAB) for iPSC banking and cGMP production. Dr. Rao is a world-renowned scientist in the fields of stem cells and medicine, having served as the VP of Regenerative Medicine at Invitrogen, founding Director of the NIH Center for Regenerative Medicine, Chair of the Biological Response Modifiers Committee (BRMAC, now CTAGT) of the FDA, and he is currently Chief Strategy Officer at Q Therapeutics. Dr. Paulauskis is the Chief Operating Officer of Paradigm Dx as well as the Vice-President of Research and Biobanking for the International Genomics Consortium, and has previously held senior positions in pharmacogenomics at Pfizer. Allele plans to make additional appointments to the SAB as individuals with world-class experience and expertise are identified.
Human iPSCs are cells that can be grown to virtually infinite numbers and can become any cell in the human body, features which hold great promise for therapies that can alleviate or cure human disease. Allele’s business model recognizes that an individual can have their own cells ready for future therapeutic use via the generation and storage of iPSCs from that individual’s skin cells. While this is similar to what is done with newborn cord blood, iPSCs can be generated from humans of any age. The banking of human iPSCs for potential future therapeutic use is a relatively new industry with unprecedented potential, and Allele is benefitting from expert opinions internally and from current and future SAB members, ensuring a solid scientific and ethical foundation for this business.
In addition to serving its customers, Allele’s iPSC bank will be an unparalleled resource for biomedical research. Proper consent and privacy guardianship will allow thousands of iPSC lines with accompanying sequence database and health information to be made available from the bank to scientists and clinicians. Currently, iPSC banks are funded by government agencies at multi-million dollar costs per project; Allele’s model does not rely on tax dollars and provides potentially a larger bank of iPSCs of higher quality to aid research and treatment efforts. To this end, NIH Director Dr. Francis Collins recently announced the implementation of the Precision Medicine Initiative (PMI), the goal of which is for health care professionals to have the resources to take into account individual differences in genes, environments, and lifestyles that contribute to disease when providing treatments in the new era.
“We are happy to have the guidance from world leading experts in stem cells, biobanking, and cell therapy fields such as Drs. Rao and Paulauskis”, said Jiwu Wang Ph.D., President and CEO of Allele. “We believe that setting the bar high will be ultimately beneficial to future customers, fellow researchers, industry partners, and regulatory agencies alike. We are happy to see the recent release by the International Society for Stem Cell Research of a draft of ‘Guidelines for the Clinical Translation of Stem Cells’, whose principals we plan to follow closely. We also intend to obtain certification by the cord blood banking association AABB, if possible, and abide by other regulatory rules as they become public, such as the “Stem Cell Clinical Research Management (tentative)” by the Health Commission of China, if and when we move to operate under that jurisdiction”.
Towards the establishment of this business effort, Allele has recently purchased an 18,000 square-foot facility, located near its headquarters in San Diego, California. This new facility will be the center of cGMP-production of human iPSCs using Allele’s proprietary synthetic mRNA platform, a technology that generates cells with neither the random integration of foreign DNA nor the use of viruses or virus-based elements, drawbacks common to other technologies for making hiPSCs; thus, the “footprint-free” cells generated by Allele’s synthetic mRNA platform are optimally suited for therapeutic use, and Allele’s technology has been licensed for clinical trials by companies such as Ocata Therapeutics (formerly ACT). This effort received strong support from Yuan Capital and Yifang Ventures.
About Allele Biotechnology & Pharmaceuticals, Inc.
Allele Biotechnology and Pharmaceuticals, Inc. is a private, San Diego-based company that explores the mechanisms of biological processes to develop technologies and products for biomedical researchers. Allele utilizes proprietary non-integrating cellular reprogramming methods to generate human and non-human primate iPS cells, GMP-grade human iPS cells and their derivatives, and differentiated cell types. With additional expertise in genome modification and cell-based sensors/reporters, Allele provides advanced cell-based assays for drug discovery. Allele also has developed a wide variety of reagents including superior fluorescent proteins and camelid antibodies. The company has also been a leader in the RNAi field with its patents in Pol III promoter-driven siRNA, shRNA, and miRNA.
Allele Biotechnology & Pharmaceuticals, Inc.
Matthew A. Singer, Ph. D.
Director of Business Development and Strategic Alliances
+1 858-587-6645, ext. 1
Press Release: Ocata Therapeutics Licenses Induced Pluripotent Stem Cell Technology from Allele Biotechnology and Extends Leadership Position in Cell Therapy Capabilities
Ocata Announces Proof-of-Concept Results in Restoring Vision and Prevention of Blindness
MARLBOROUGH, Mass., Mar 24, 2015 (BUSINESS WIRE) — Ocata Therapeutics, Inc. (“Ocata” or “the Company”; NASDAQ: OCAT), a leader in the field of Regenerative Ophthalmology™, today announced that it has entered into a definitive agreement with Allele Biotechnology & Pharmaceuticals, Inc. of San Diego, CA (“Allele”) to access Allele’s proprietary technology for generating human induced pluripotent stem cells (“iPSCs”).
“This agreement with Allele is part of our strategy to broaden our technology platform and increase our leadership in regenerative ophthalmology,” said Paul Wotton Ph.D., President and CEO of Ocata. “Ocata can now take advantage of induced and embryonic pluripotent stem cells to produce commercially viable human tissue for transplantation. We recently confirmed proof of concept in creating photoreceptors capable of preventing blindness and restoring vision in established animal models. Data from these studies will be published later this year.”
Since Dr. Yamanaka discovered how to generate iPSCs in 2007 there has been tremendous enthusiasm about the potential to use these pluripotent cells to develop commercially viable therapies. Despite many efforts to develop iPSC derived therapies in the same scalable and reliable way as embryonic stem cells, many of those efforts have been unsuccessful due to issues relating to the growth capacity, differentiation potentials and epigenetic properties of iPSCs. The “footprint-free” reprogramming technology developed by Allele potentially offers a reliable and scalable process for producing iPSCs with superior properties and is a major step for translation of iPSC technology to practical clinical use. These iPSCs can potentially be used to manufacture millions of treatment doses as off-the-shelf therapies for any patient.
“We have had a strong leadership role in this area,” said Robert Lanza, M.D., Chief Scientific Officer of Ocata. “Ocata has extensive experience and patent rights in generating both ocular and non-ocular cell types from human iPSCs. We have painstakingly and patiently evaluated many different iPSC technologies and selected the Allele technology only after we were satisfied and confident that this represented the best of all approaches and could permit us to generate transplantable tissues that would be potentially safe in human patients. In our hands, the iPSCs we are generating are comparable to our embryonic stem cells in those features required for use in potential human therapies.”
“It is particularly rewarding to us that Ocata, a company whose understanding of the science and regulatory requirements in this space is unparalleled, has selected the iPSC technology developed at Allele for application in its own pipeline,” said Jiwu Wang Ph.D., President and CEO of Allele. “It only serves to confirm our belief that our iPSC platform is a solution to what otherwise have been unresolved issues associated with the maturation of iPSCs to a fully functional state. The ability to predictably derive stable iPSC lines without using any viral element or foreign DNA enables both fundamental scientific research and clinical applications, which has been the mission of Allele Biotechnology from its inception.”
For Contact at Allele:
P 858-587-6645, 800-991-RNAi(7624)
For full release with Ocata contact, see MarketWatch
Precise engineering of the genomes of mammalian cells enabled biological and medical applications researchers had dreamed of for decades. Recent developments in the stem cell field have created even more excitement for genetically modifying genomes because it enables delivering more beneficial stem cell-derived therapeutic cells to patients . For instance, by correcting a gene mutation known to be critical to Parkinson’s disease, LRRK2 G2019S, in patient-specific iPSCs (induced pluripotent stem cells), it appeared possible to rescue neurodegenerative phenotypes .
Significant amount of fund and energy had been invested in technologies such as ZFN and TALEN, however, judging from the explosion of publications and business activities in just about 2 years since the illustration of its mechanism (just today, Jan 8th, 2015, Novartis announced CRISPR collaborations with Intellia, Caribou, applying it in CAR T cell and HSCs), the CRISPR/cas system is the rising star. This system uses a guide RNA to direct the traffic of a single nuclease towards different targets on a chromosome to alter DNA sequence through cutting. The nuclease, cas9, can be mutated from a double-stranded DNA endonuclease to a single-strand cutter or a non-cutting block, or further fused to various functional domains such as a transcription activation domain. This system can also be used to edit RNA molecules.
A weak spot on the sharp blade of CRISPR is, like any methods for creating loss-of-function effects (RNAi if you remember), the potential of off-target effects. While they can never be completely avoided, with the ever growing popularity of deep sequencing, at least we can know all unintended changes on the edited genome. Almost a perfect storm! As an interesting side story, when we at Allele Biotech first saw the paper in Science describing the CIRPSR/cas system , we immediately wrote an SBIR grant application for applying the bacterial system to mammalian cells. The first round of review in December 2012 concluded that it would not work due to eukaryotes’ compact chromatin structures. Of course, the flurry of publication in early 2013, while our application was being resubmitted, proved otherwise. The good news is, Allele Biotech still received an SBIR grant from NIGMS in 2014. Unlike most of the genome editing platforms known in the literature, our goal was to build an all-RNA CRISPR/cas system, thereby with higher potency, less off-target effects, and, as a footprint-free platform, more suitable for therapeutic applications. This system will be combined with our strengths in iPSC and stem cell differentiation, fluorescent protein markers, and deep sequencing based bioinformatics to improve cell therapy and cell based assays.
1 Urnov, F.D., et al., Genome editing with engineered zinc finger nucleases. Nat Rev Genet, 2010. 11(9): p. 636-46.
2 Reinhardt, P., et al., Genetic Correction of a LRRK2 Mutation in Human iPSCs Links Parkinsonian Neurodegeneration to ERK-Dependent Changes in Gene Expression. Cell Stem Cell, 2013. 12(3): p. 354-67.
3 Jinek, M., et al., A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 2012.
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