The Power of Cas

Precise engineering of the genomes of higher eukaryotes can enable a variety of biological and medical applications. Targeted gene disruption, editing, and insertion can translate into the much desired freedom to generate cells or organisms bearing a desired genetic change. 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), researchers were able to rescue neurodegenerative phenotypes [1].

Cumbersome reagent development and high costs have been major barriers to targeted genome modification using the current technologies, which include the zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN). Unlike the ZFN and TALEN systems, CRISPR/cas does not require assembly of DNA pieces that encode the functional proteins every time a new sequence is to be targeted. Instead, it uses a guide RNA to direct the traffic of a nuclease complex. Five recent publications of modifying eukaryotic chromosomes showed the importance of the CRISPR/cas system [2-6], they also hinted at the ease of adapting this system in eukaryotes given that the functions of cas and the small guide RNA were described in bacteria merely few months ago [7].

The concern that the bacterial CRISPR/cas system would not access the chromatin structures of eukaryotic genome was muted as a result of recent publications; it also seems that the cas9 protein is as powerful an enzyme as one could have hoped in an endonuclease. As a matter of fact, cas9 from S. pyogenes contains 2 different single-stranded DNAse domains independent of each other, and can be mutated to change from a double-stranded DNA endonuclease to a single-strand cutter, or a non-cutting block. That’s not all, a more recent Nature publication further showed that cas9 (from another species, F. novicida), can bind to yet another small RNA and, instead of cutting chromosomal DNA, it degrades RNA, apparently through a direct cas9/RNA binding mechanism [8]. It may be chromosomal modification and RNAi rolled in one (cas9 from different genera are quite different though). One has to admire the powerful cas!

1. 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.
2. Qi, L.S., et al., Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 2013. 152(5): p. 1173-83.
3. Mali, P., et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6.
4. Cong, L., et al., Multiplex genome engineering using CRISPR/Cas systems. Science, 2013. 339(6121): p. 819-23.
5. Cho, S.W., S. Kim, J.M. Kim, and J.S. Kim, Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol, 2013. 31(3): p. 230-2.
6. Hwang, W.Y., et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol, 2013. 31(3): p. 227-9.
7. Jinek, M., et al., A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 2012.
8. Sampson, T.R., et al., A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature, 2013.

Tags: cas9, choromsome modification, CRISPR/cas, F. novicida, genome engineering, nuclease, RNA interference, RNAi, S. pyogenes, TALE, TALEN, ZFN

The Development of mNeonGreen

This week our most recent publication, “A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum” will be published in Nature Methods. It has already been viewable online for some time now, here is a link. We believe this new protein possesses a great deal of potential to advance the imaging fields through enhanced fluorescent microscopy. mNeonGreen enables numerous super resolution imaging techniques and allows for greater clarity and insight into one’s research. As a result of this we are taking a new approach at Allele for distribution of this protein, and here we will describe the history of the protein and some of the factors that led us down this path.

mNeonGreen was developed by Dr. Nathan Shaner at Allele Biotechnology and the Scintillon Institute through the directed evolution of a yellow fluorescent protein we offer called LanYFP. LanYFP is a super bright yellow fluorescent protein derived from the Lancelet fish species, characterized by its very high quantum yield, however, in its native state LanYFP is tetrameric. Dr. Shaner was able to monomerize the protein and enhance a number of beneficial properties such as photostability and maturation time. The result is a protein that performs very well in a number of applications, but is also backwards compatible with and equipment for GFP imaging.

Upon publication there was a question of how distribution should be structured. How would we make this protein available to researchers in a simple manner was a very difficult challenge? We also relied heavily on Dr. Shaner’s knowledge and experience in these matters, as he related his experiences to us from his time in Roger Tsien’s lab at UCSD. When the mFruits was published their lab was inundated with requests. The average waiting period was 3 months to receive a protein and they required a dedicated research technician to handle this process. Eventually the mFruits from the Tsien lab were almost exclusively offered through Clontech. Thus we decided that Allele Biotechnology would handle the protein distribution and take a commercial approach to drastically decrease the turnaround time. The next challenge we faced was how to charge for this protein. Due to the cost of developing this protein, which was fully funded by Allele, there is a necessity to recoup our investment and ideally justify further development of research tools, but we also understand the budget constraints every lab now faces. From this line of thinking we conceived our group licensing model; we wanted to limit the charge to $100 per lab. The way this is fiscally justifiable is having every lab in a department or site license the protein at this charge, including access to all related plasmids made by us as well as those generated by other licensed users (Click here for our licensing page). The benefit we see to this is that the protein is licensed for full use at a low cost, and collaboration amongst one’s colleagues is not only permissible, it’s encouraged. We saw this as a win-win situation. We would recoup our cost and invest in further fluorescent protein research, and our protein costs would not be a barrier to research and innovation.

The granting of a license to use but not distribute material is not unique to commercial sources. Although academic material transfer agreements typically contain specific language forbidding distribution of received material beyond the recipient laboratory, some researchers choose to disregard these provisions. Unfortunately through this action they are disrespecting the intellectual property rights of the original researchers as well as violating the terms of the legal contract they signed in order to receive the material. We believe most researchers choose to respect the great deal of effort that goes into the creation of research tools for biology and do not distribute any material received from other labs without their express permission. However for a company that funds its own basic research our focus is often on the former example rather than the latter. We believe that this focus artificially drives up the costs of licensing a fluorescent protein and obtaining the plasmid, thus we have chosen to believe researchers will respect our intellectual property as long as we are reasonable in our distribution which is something we have truly striven for.

Additionally we believe the broad-range usage of a superior, new generation FP is an opportunity to advocate newer technologies that can be enabled by mNeonGreen, together with a number of Allele’s other fluorescent proteins (such as the photoconvertible mClavGR2, and mMaple). These new imaging technologies are called super resolution imaging (MRI). They provide researchers with a much finer resolution of cellular structures, protein molecule localizations, and protein-protein interaction information. We have started the construction of a dedicated webpage to provide early adopters with practical and simple guidance, click here to visit our super resolution imaging portal.

Tags: cell imaging, fluorescent protein, FP, GFP, mNeonGreen, Superresolution

Autologous versus Allogeneic iPSCs in Immune Rejection

The enthusiasm of using autologous induced pluripotent stem cells (iPSCs) for cell replacement therapy was dampened by a publication 2 years ago in Nature (Zhao et al, 2011), which suggested that even syngeneic (genetically identical) iPSCs could still invoke strong immune rejection because, as the authors in Yang Xu’s lab at UCSD explained, the iPSCs overexpress a number of tumor antigens possibly linked to genomic mistakes acquired during reprogramming. Embryonic stem cells (ESCs), on the other hand, did not show similar rejection problems in the same studies, indicating that the immune responses were due to somatic reprogramming.

If proven true, the iPSC-specific immune rejection would have been the biggest hurdle for any iPSC-inspired clinical plans. Naturally, a number of labs performed series of experiments that were aimed at addressing the concerns raised by Zhao et al. This month in Cell Stem Cell, researchers from Ashleigh Boyd’s lab at Boston University demonstrated that autologous (self) or syngeneic iPSCs or their derivatives were not rejected (Guha et al. 2013). These iPSCs behaved essentially the same as ESCs in transplantation settings. When immunogenicity was measured in vitro by monitoring T cell responses in co-culture, no immune response was observed either. In contrast, cells and tissues from allogeneic (genetically different) iPSCs were rejected immediately.

In light of this new publication and an earlier Nature paper (Araki et al. 2013), Kaneko and Yamanaka have commented that autologous iPSCs still seem to have a very good chance of being used in cell replacement therapy, pending, of course, additional research and trial results. In their Preview article in Cell Stem Cell (Kaneko and Yamanaka 2013) two points were particularly emphasized: 1) autologous iPSCs are preferred because of the lack of immune rejection; 2) iPSCs generated with footprint-free reprogramming technologies are preferred because the problems reported by Zhao et al 2011 might be correlated with the use of retroviral vectors (even though they also used episomal plasmid-reprogrammed iPSCs). We strongly support both of these points and believe that they point out the direction of future stem cell therapies.

However, we do not agree with the last statement by Kaneko and Yamanaka in that article stating that as a result of the cost and time required to generate iPSC lines from each patient in GMP facilities, iPSC lines from HLA homologous donors will be the choice going forward to clinical applications. First of all, HLA-matched iPSCs should be closer to allogeneic than to autologous iPSCs. From what we just learned in the last round of debates, the field should certainly go with autologous. Second, generating foot-print free iPSCs may already not be the rate-limiting step, even in cGMP protocols, compared to downstream differentiations that are required using any pluripotent stem cells. We have shown that human fibroblasts can be reprogrammed in a completely feeder-free, xeno-free, passage-free process, using only mRNAs, in just over a week, achieving sometimes “bulk conversion”—converting nearly all cells within a well into iPSCs (Warren et al. 2012). We have drawn up a plan to establish cGMP protocols and to quickly apply autologous, footprint-free iPSCs to clinical programs through partnerships. The field can move at a faster speed, with all due scientific vigor and caution, if the best technology available is chosen for building the foundation.

Zhao, T., Z.N. Zhang, Z. Rong, and Y. Xu, Immunogenicity of induced pluripotent stem cells. Nature, 2011. 474(7350): p. 212-5.

Guha, P., et al., Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells. Cell Stem Cell, 2013. 12(4): p. 407-1

Kaneko, S. and S. Yamanaka, To Be Immunogenic, or Not to Be: That’s the iPSC Question. Cell Stem Cell, 2013. 12(4): p. 385-6.

Araki, R., et al., Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells. Nature, 2013. 494(7435): p. 100-4.

Warren, L., Y. Ni, J. Wang, and X. Guo, Feeder-free derivation of human induced pluripotent stem cells with messenger RNA. Sci Rep, 2012. 2: p. 657.

Tags: allogeneic, autologous, cell banking, cell therapy, GMP iPSCs, Guha et al. 2013, HLA homologous, iPSCs, Kaneko and Yamanaka, pluripotent stem cells, warren et al. 2012, zhao et al 2011

Allele Publishes mNeonGreen as the Brightest Monomeric Fluorescent Protein for Super-resolution Imaging

SAN DIEGO–(BUSINESS WIRE, Yahoo! Finance)–

This week scientists from Allele Biotechnology and its partner non-profit research institute, the Scintillon Institute, present their latest fluorescent protein, mNeonGreen, in the journal Nature Methods (Nature Publishing Group). In the paper, entitled “A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum,” the scientists describe the development of the brightest monomeric fluorescent protein to date.

The scientific efforts to develop this novel fluorescent protein were led by Dr. Nathan Shaner, a leader in the field of fluorescent protein engineering. Fluorescent proteins are highly valuable research tools that allow the labeling and imaging of individual proteins within a living cell, and tracking of their movements and localization in real time through a microscope. However, since the discovery of the original green fluorescent protein in 1993, imaging technology has advanced rapidly beyond the capability of most fluorescent proteins. The newly described fluorescent protein, mNeonGreen, allows researchers to take full advantage of modern super-resolution optical microscopy techniques that enable visualization of structures in living and fixed cells at much smaller scales than are possible using traditional optical microscopy. This improvement will lead to countless new insights into human health and a greater understanding of protein interactions at very small distance scales within living cells. According to Dr. Jiwu Wang, the CEO of Allele Biotechnology, “Super-resolution imaging will become the standard for publication in a short period of time, and mNeonGreen allows researchers to meet this standard while still being compatible with the equipment and methods they already use.”

Prominent researchers within the fluorescent protein field are touting mNeonGreen as a replacement for jellyfish-derived Aequorea GFP, one of the most commonly used fluorescent proteins today. According to lead researcher Dr. Nathan Shaner, “mNeonGreen can be directly substituted for other green fluorescent proteins such as EGFP without the need for any equipment changes,” making the upgrade an attractive prospect for many researchers.

Allele Biotechnology and Pharmaceuticals Inc. is a San Diego-based biotechnology company specializing in the fields of RNAi, stem cells, viral expression, camelid antibodies and fluorescent proteins. The company has co-developed a number of fluorescent proteins and other products for PALM or STORM super-resolution imaging 3D-SIM, and STED imaging. With the arrival of mNeonGreen, Allele plans to collaborate with leading imaging labs, microscope manufacturers, and journals such as Nature Methods to further promote the advantages and capabilities of the latest imaging methods. Additionally, this announcement will coincide with the launch of a new super-resolution imaging web portal and plasmid depository via collaboration with the Scintillon Institute. The Scintillon Institute is a non-profit research institute established in 2012 using seed funding from Allele Biotech. The institute’s researchers are focused on the development of biological tools to improve human health and quality of life, including applications to cancer imaging, regenerative medicine, and sustainable energy and food production.

For details about Allele’s new Superresolution FP distribution method, read our departmental and institutional usage page.

Tags: 3D, fluorescence, in vivo imaging, microscope, PALM, protein label, SIM, STED, STORM, super resolution imaging

Conducting Massively Parallel Sequencing

One of the major breakthroughs in modern biology is the development of massively parallel sequencing, also called next generation sequencing (NGS), which enabled the complete delineation of the human genome more than a decade ago. Since then many more species’ genomes have been sequenced, and the cost per genome has dropped from billions to mere thousands of dollars. New discoveries are being made as a result of the capability many research teams now possess to not only sequence chromosomal DNA, but also to identify which regions a protein of interest specifically binds (Chip-seq), analyze a whole transcriptome of a cell population under investigation (RNA-seq), or find out which RNA regions an RNA binding protein resides (CLIC-seq).

While it is inevitable that many PIs will seriously consider the inclusion of deep sequencing in their next grant proposal, it is not necessarily easy to take the first step and get their feet wet, so to speak. Knowing what format (e.g. 454 for longer reads, HighSeq for higher accuracy, or Ion Torren for bench top convenience) to use and how much to pay requires a vast amount of knowledge and experience. Even when you are done with sample prep, amplification and sequencing, to handle such massive amount of data is not trivial—transporting data alone can be a headache. A database server for storage and analysis requires another layer of expertise. There is no easy solution but to get started somehow. However, be prepared to deal with these issues.

Whether the cost on a type of next generation service is justifiable depends on whether it is required for your purposes. For example, when analyzing a person’s propensity of developing a disease by using known, disease-relevant genetic information, often times exome sequencing is sufficient. This costs anywhere between $1,000 to $3,000 with 100X coverage, significantly less than sequencing a complete genome which typically costs ~$5,000 at ~20x coverage.

High coverage sequencing of maternal blood DNA has been developed into clinically approved prenatal diagnosis of trisomy in Down’s syndrome and other chromosomal abnormalities. Transcriptome analysis helped the understanding of how reprogramming works when iPSCs are. Looking forward, with more routine use of deep sequencing we can predict with much more certainty the “off-target” effects of RNAi or cellular toxicity of chromosomal modifications enabled by ZFN, TALEN, or CRISPR. As a matter of fact, we believe that transcriptome sequencing should be required after each RNAi event to prove a specific linkage between knockdown and functions; similarly, whole genome sequencing results need to be provided after making a site directed chromosomal change in the future for high level publications.

*This blog partially resulted from discussions between Jiwu Wang and his colleagues, who are NGS experts at UCSD’s Cellular and Molecular Medicine, Moore Cancer Center, and BGI Americas.

Tags: CRISPR, deep sequencing, exome sequencing, footprint free, genomic modification, iPSCs, Massively Parallel Sequencing, NSG, TALEN, transcriptome, whole genome sequencing, ZFN