genome sequencing

Genome Modification—a Practical Approach

The ability to modify genomes has always been fervidly sought after by molecular, developmental biologists and geneticists as it would provide them with the means for finding out what a particular piece of the genome may do in the biological process they are studying. The discovery of naturally existing P-element helped a generation of Drosophila geneticists and made the fruit fly a prime model system for gene function studies in the 80’s and 90’s. But P-elements inserted at uncontrolled sites, making it essentially a gene transfer vehicle without much control. The introduction of prokaryotic recombination systems, e.g. LoxP and Cre, provided researchers with tools to obtain more control of the inserted genes in a host chromosome during a biological process such as development. Transposons like Sleeping Beauty, Piggybac, or Tol2 made similar experiments possible in mammalian cells.

Still, the randomness of transposon-type elements’ insertion, much like retrovirus or lentivirus, could cause trouble if they land in an undesirable spot. Methods of inserting transgenes only in well-known, harmless, and transcriptionally active regions, so called “safe harbors”, were subjects of interest of researchers and NIH grant topics in the past couple of years under “directed genome editing”. Gene knock-out or knock-in can be achieved through vector-mediated homologous recombination such as the rAAV genome engineering system and the “TARGATT” system, which are commercially available as kits or services.

However, instead of inserting an exogenous gene, it is often highly desirable to modify an endogenous genome sequence, which requires the modification apparatus to first recognize the target sequence. ZFN and TALEN both recognize DNA targets through specific nucleotide binding protein domains, with TALEN having more flexibility if assembled in a “Lego”-like format because each domain can specifically recognize a “C”, “G”, “T”, or “A” base. The description of using CRISPR/cas system in a recent burst of publications opened up new ways of binding to specific DNA sequences and nicking or severing the dsDNA. This system does not require engineeredDNA binding domain assembly; instead, it uses a guide RNA to find the target DNA sequence to direct endonuclease, in a sense quite like RNAi. However, the enthusiasm about CRISPR/cas was somewhat dampened by a report last month in Nature Biotechnology that reported off-target effects of CRISPR/cas was much higher than ZFN and TALEN. Particularly, if mismatches are located in the 5’ portion of the guide RNA targeting sequence, they can be well tolerated up to 3 or 4, even 5 mismatches. Unfortunately this is also similar to the tolerance of the RNAi matching region outside the core 12-base region. The difference is: for RNAi, the off-target damage is temporary and ignorable if the extent is insignificant compared to the effects on the intended target while for CRISPR/cas, an off-target cut on the chromosome is permanent.

On the positive side, in an even more recent publication in Nature Methods, mutant strains of C. elegans were obtained using the CRISPR/cas system and no evidence was found for off-target changes, at least not in an overwhelming fashion. Much value of the estimates of off-target effects relates to the methods used for analysis. Currently, most of the studies looked at potential off-target sides by searching for partial matches. In the future, whole genome sequencing will be increasingly required for submitting such publications.

On a practical note, if you intend to take a dive and try to use any one of these methods, your number one problem will be that none of the methods will result in 100% modification even if you can ignore the off-target problems for now. Therefore, many of our customers ask about a screening strategy. One could use traditional drug selection and fluorescent protein (FP)-based sorting, but these can only help you find cells that are successfully transfected with the ZFN, TALEN, or CRISPR/cas expressing DNA molecules, not necessarily having the genome modification result. We have formulated the idea of inserting the target site into an FP-bearing plasmid as a surrogate target cutting indicator, and use another FP to track transfection of the TALEN plasmid. Nonetheless, in the end, PCR-amplifying the target region of the chromosome and doing either an enzymatic mismatch detection assay (e.g. T7 endonuclease) or sequencing is the only way to know for sure whether genome editing has occurred.

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Wednesday, July 10th, 2013 Synthetic biology, Viruses and cells 1 Comment

Solving the Big Problems of the World

Science, by nature, is something you do without knowing for sure that it will work. By doing an experiment, testing a theory, or tabulating large data sets to find statistical significance, researchers make small discoveries or incremental improvements on technologies. It is easy for any researcher to get buried under the enormous amount of experimental details while trying to complete a project that lasts for months and years. For a team or an organization, however, it is critical to create a level of alertness of the big questions we try to answer – why are we doing this line of research? Is the technology or theory being developed going to be disruptive in terms of changing the ways of thinking in its field or solving a big challenge that faces the world?

The world does not lack for challenges: there may not be any ice left at the North Pole as early as 2015, there are still a billion people who need reliable electric energy while the carbon fuels may run out on all of us in just a few decades, during which time usable land may not be able to provide enough food for the growing population, cancer or dementia will strike almost everybody if we all live long enough. Well, we have sent humans to the moon; we have completely eradicated smallpox and almost done with polio, can technologies once again enable us to do big things if we all aim high and pull together?

The success stories of future technology companies should not be only the types of Facebook or Twitter, which are nice stories on their own values, but success stories should also include those that deal with big, material, and imminent challenges, provide tools that help people in desperate need. Examples in our biomedical field could include diagnostic kits based on genomic information that will one day be put into each household, so that everybody will be able to decide and receive the most suitable treatment when having an ailment. New businesses will merge because of the technology advancements of deep sequencing, information storage and analysis, biosensors, and stem cell-derived assays and delivery vehicles.

Technologies will continue to develop at a faster pace than most people’s imagination as long as there is a culture that encourages it and a system that allows those with the extraordinary ambition and brains to take their risks. As an example in one of our specific fields, the barriers to making induced pluripotent stem cells (iPSCs) have been dramatically lowered through several generations of method revolution only 6 years after the Nobel Prize-winning discovery was first published in 2006 because researchers believe that there will be new opportunities if reprogramming can be done more efficiently and “cleanly”. We have contributed our share of innovation in 2012 and our ambition is to provide everybody with his or her own pluripotent stem cells ready for medical use and to find a solution to most diseases with each individual’s own tissue-derived cells, in another term, point-of-care autologous treatment. It’s unproven, it’s futuristic, but it’s exciting and feasible and we will put every effort to make it happen. Theodore Roosevelt once said that “Far and away the best prize that life has to offer is the chance to work hard at work worth doing.” We are the lucky few.

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Saturday, December 29th, 2012 Open Forum No Comments