A new tool for genome editing

Bacteria defense system takes its place in human genome editing

        An RNA based nuclear acids degradation system found in bacteria and archaea was tamed for human genome editing.

        Genomes store information of life and human genome editing holds promise to improve human’ health and overcome diseases. Some available genome editing tools attract wide interests. Zinc finger nuclease (ZFN) technology and Transcription activator-like effector nucleases (TALENs), both of which were invented recently for genome editing, won the Method of the Year 2011, awarded by the journal Nature Methods. Lately, a new genome editing tool was released in Science¹.

        This new technology is based on an exogenous nuclear acid degradation system in bacteria and archaea. During evolution, bacteria and archaea face frequent and tremendous threats from environment. Exogenous nuclear acid constitute a main jeopardy because genomes of bacteria and archaea are not protected as well as that of advanced life. Bacteria and archaea thus develop a mechanism to combat exogenous nuclear acid. This system adopts RNA, which is characteristic of short repeats, and proteins to degrade invading nuclear acid. Shorted and repeated RNA is mainly responsible for the recognition of threatening exogenous nuclear acids whereas the RNA associated protein takes care of the degradation step. Although this nuclear acid degradation aims at invading DNA or RNA, they may be modified to target the human genome.

        Scientists in Havard Medical School engineer this nuclear acids degradation system in order to obtain targeted degradation of DNA sequence in human genome. The designed DNA sequence targeting interested DNA in human genome was linked to a DNA scaffold that facilitates the access of the protein responsible for degradation. The protein for degradation was also engineered to ensure location and high expression in human cells. The artificial DNA and protein are sent to human cells together. The designed DNA will recognize its corresponding DNA in the genome. Then the protein will function to cut the DNA in the genome. All mentioned above is what this bacteria originated system does. Other steps were finished by DNA repair system in human cells. Realizing a DNA damage (here it is broken DNA), the DNA repair system will try to repair it based on a similar DNA sequence, which is the special designed DNA in the artificial DNA donor in this case. The desired mutations, insertions, deletions will be thus possible.

        What are the advantages of this new genome editing tool compared to other tools? This system is easier and more efficient compared to ZFNs and TALENs. Both ZFNs and TALENs adopt specific proteins to recognize interested DNA sequence. It is tricky to design appropriate protein scaffold to match DNA in genome. The bacteria originated gene editing system uses DNA to recognize DNA in genome so it is easier to design DNA sequence. Actually, the authors have designed DNA sequences that able to cover about 40.5% exons in human genome. This bacteria originated DNA editing tool is also effective. In the Science paper, this new gene editing system achieved at most about 8% gene editing rate whereas TALENs resulted in not more than 1% gene editing rate.  

        A key standard to evaluate a gene editing tool is its specificity. That means it will favor interested genome regions but not other regions. Theoretically, this bacterial originated DNA editing system is specific because it usually uses 20 nucleotides, the length of which guarantees the required specificity in human genome, to recognize genome DNA. However, off-target effect of genome editing is still necessary. The Science paper did not resolve this concern.

        An exciting thing about this bacteria originated gene editing tool is there is more space to develop this technology. Both DNA scaffold and degradation proteins can be engineered to improve their efficacy and specificity.

Luck control

        People fail to realize how lucky they are except when some tragedies hit someone else. Every day, by watching TVs, surfing internets all of which report so many accidents, we got the idea that the world is full of risks. China is occupied by hazardous air; United States is discussing gun control; North Korean is testing nuke weapons; Solomon islands are experiencing earthquakes. Thus, fortune is not the arrival of good luck in the future, but the escape of bad luck in the past. Chinese cherish this kind of fortune by coining a word “houpa” that means the fear after the event. Western culture has also a similar expression: no news is good news.

        It makes more sense that we did not realize how lucky we are when it is about cancer. Everyone will develop cancer very early if their bodies fail to repair a great deal of DNA mutations caused by environments. Majority of people have no idea how many mechanisms in our body work together to protect us from cancer. For example, when DNA in a cell was hurt by UV, radiation or other stress, a protein called p53 will be activated to induce a cascade of events that lead to the death of the cell. So many similar proteins exist that DNA mutations will be eliminated under different circumstances. Every day, our bodies have fixed a great volume of mistakes in DNA without our realization.

        Among all hiding safeguard mechanisms preventing us from cancer, immune system is fundamental and vital. Immune reactions are tamed to help to reject cancer. Research of how to tame immune system for cancer treatment is called cancer immunotherapy. Historically, cancer is controlled by surgery, radiation therapy, chemotherapy, and latest targeted therapy. All these methods kill cancer cells using foreign materials without considering if these materials will also do harm to our body. Different from these methods, cancer immunotherapy focuses on provoking our immune system to work better to kill cancer cells. As a result, the side effects of cancer immunotherapy are less and the efficacy of it is better.

        Everyone has immune system so we are all lucky. But under some circumstances like cancer, luck is not enough. We need to control luck. Luckily, we are on the way.

Cancer: task impossible? Depending on what measurements we use

It is better to admit failing to win the war against cancer than fail to admit it.

        The Wall Street Journal published an article by Bill Gates a couple of days ago. The heading is so striking that I cannot help reading the contents. The title is “my plan to fix the world’s biggest problems”. However, after I went through the paper, I was a little bit disappointed because what Mr. Gates had declared as great plan for resolving biggest problems is actually very basic. Mr. Gates described the process of solving problems as simple 4 stages “setting clear goals, choosing an approach, measuring results, and then using those measurements to continually refine our approach” and emphasized that measurements are extremely important and the key to fix the biggest problems in today’s world: poverty, disease, lack of education and so on. Although it sounds simple, what Mr. Gates had said is indeed instructive. Setting goals is easy. People who tend to make New Year’s resolution will be able to know how easy to set goals. Selecting an appropriate approach is harder and requires smart thinking. But they are not in the same street with measuring and subsequent refining in term of the measuring. Measuring enables us to know how close we are from the goal.

        Where are we on the road map of cancer research in the light of current measurements we adopted?

       The answer, according to today's starndard, is probably we stay where we were decades ago in cancer research. About forty years ago, the president Richard Nixon set the goal to eradicate cancer by signing the National Cancer Act in 1971. The war on cancer campaign also fosters mounting cancer research. However, cancer incidence and death rates, which are two main standards to measure cancer research, keep unchanged during the past 40 years. The measurements have told us that cancer remains a big burden for Unites States. Moreover, some cancers show increasing incidence and death rates in United States and other countries. 
        It may be smart for us to accept the fact that we failed to win the war on cancer based on today's measurements. Only by accepting this fact, we can make some adjustment to better control cancer.

          Once we admit the fact that we failed the war on cancer, we will be able to figure out what we should do next. The direction for cancer control should be averted to cancer diagnostics and cancer prevention both of which entail broad and deep understanding of cancer genetic factors and environmental factors.      

        We might also need to adjust the standard we used to measure cancer control. Incidence rate and death rate of cancer have been used as golden standard for measuring cancer situation but may mask what we have achieved in cancer research. New measurements should be adopted in evaluating cancer control. Early detection rate, for example, may be used as a standard to measure cancer control and incorporated in cancer statistics.    

        Due to the aging population and deteriorating environment, the world will suffer tougher cancer burden. However, if we follow the direction of cancer diagnostic and prevention and adopt new measurements for cancer control, we might witness a tremendous progression in cancer control.

Discovery of Richard III's skeleton: DNA meets archaeology

Analysis of DNA in ancient remains enables us to reevaluate history.

        A 500-year-old remains found beneath parking lot in Greyfriars, Leicester was confirmed to belong to King Richard III, the last Plantagenet king of England, who is the subject of many works of literature including Shakespeare’s play Richard III.

        How was the skeleton regarded to be that of Richard III? On the one hand, ancient maps and records support that the parking lot which used to be a friary is actually the place where Richard III resided in. On the other hand, DNA test provided the decisive proof. DNA test was performed by comparing DNA from the remains discovered in Greyfriars and that from Canadian-born Michael Ibsen who is a direct descendent of Anne of York, Richard’s elder sister. Researchers responsible for this project have alleged: “It is the academic conclusion of the University of Leicester that beyond reasonable doubt, the individual exhumed at Greyfriars in September 2012 is indeed Richard III, the last Plantagenet king of England.”

        How can they be so sure that skeleton belongs to Richard III? Researchers did not disclose many details. But what they have announced is not convincing.

        The DNA test University of Leicester researchers have done must be like paternity testing which can tell if two individuals have a biological parent-child relationship by analyzing specific DNA information between two individuals that will be inherited from parent to child. In a standard DNA paternity test, usually a child, alleged father, and the mother are involved. The mother’s participation in the paternity test helps to exclude half of the child’s DNA, leaving the other half for comparison with the alleged father’s DNA. The DNA test about the identity of Richard III must be based on a method similar to paternity testing: comparing DNA information between the skeleton and Michael Ibsen. But at least two controls (controls with known information are used in biological experimental designs for ruling out or ruling in a candidate) should be set: first, an irrelevant random DNA should be incorporated as a negative control, and second, DNA sample from another descendent of Richard III should be incorporated as a positive control. These two controls are useful to rule out systematic errors.      

        DNA has played a role in identifying one of great names in history. In the future, DNA may be useful to disclose the health conditions of those great names in history that may shape the characters of those people or influence key events in history. In this way, the involvement of DNA in archaeology will change the landscape of archaeology.

The future of cancer genome sequencing

Three trends in cancer genome sequencing

        No doubt that genome sequencing will dominate future cancer research and clinic. A great number of papers published in high impact journals have adopted genome sequencing to resolve questions in cancer research that were once unrealistic to answer. Many oncologists hope to acquire the genome information of patients to guide their clinical administration. A few growing biotechnology companies have launched genome sequencing based products for cancer diagnostics. Media also pay much attention to genome sequencing. The essential reason of this trend is the genomic origin of cancers. Like what famous cancer geneticist Robert A. Weinberg has proposed in one of his most broadly cited papers, cancer actually originates from genome instability and relevant mutations. Another important reason for the blossom of genome sequencing is tumor heterogeneity which means there are no two tumors alike. Only by genome sequencing can we know exactly how special a tumor is and what therapy is best for it. Thus, the capacity of detecting genome information using sequencing tool has been favored by both Ph.Ds and M.Ds.

        The genome sequencing is in adolescence for basic research and in babyhood in clinic. How will it grow up? There might be three kinds of trends.

        Single cancer cell sequencing

        There are no two pieces of leaves alike. So are tumor cells. A tumor cell’s special identity can be recognized by expressed proteins, RNAs in the cell. But the fundamental way to distinguish a tumor cell is genome sequencing. Single cancer cell sequencing is thus important.

        Sequencing itself requires a certain amount of DNA that usually cannot be acquired from a single cell. Amplification of DNA extracted from a single cell is necessary to obtain enough DNA for sequencing. Latest state-of-the-art sequencing technology is viable for single cell sequencing.

        Single cell sequencing may not be suitable for clinic because analyzing a tumor instead of a single tumor cell is more appropriate for patient’s benefit. However, it is indispensible for studying fundamental questions in cancer research like cancer origin, cancer stem cell and so on.

        Dynamic sequencing

        No man ever steps in the same river twice. Tumors change their genome quickly, especially when they undergo anti-cancer drug exposure. Genome-changed tumors may not be sensitive any more to the drug that works formerly. Finding out the changed genome information is necessary before a new therapy will be applied. This implies that the genome sequencing should be performed periodically to monitor genome dynamic so that optimized therapy can be administrated. This dynamic genome sequencing will be especially important when tumors have improved drug resistance to a drug by producing new mutations. 

        Noninvasive sequencing

        Current genome sequencing requires pathology tissues. However, sometimes acquiring pathology tissues are painful and even dangerous for patients. Melanoma represents such an example. This situation calls for noninvasive test. At least under one condition noninvasive test becomes possible. It is when tumor cells shed from the primary tumor into bloodstream and circulate there. This phenomenon is christened as circulating tumor cells. By analyzing genomes obtained from circulating tumor cells, the cancer mutation signature can be captured and appropriate therapy options can be suggested. 

        These three trends are not mutual exclusive and combining these methods will enable us to better understand cancer.

Cancer genome sequencing: a routine examination in the near future?

        Bill Gates is not necessarily right in every investment. However, as a shrewd businessman, he will not invest without a solid reason.  Recently, Foundation Medicine, a new biotechnology company launched in 2010, said they received an investment of 13.5 millions from Bill Gates. Its first product, FoundationOne, is an integrated service of cancer genomic test which will suggest the best therapy for a cancer patient based on his genome information. What made Gates send money to this company? The answer is simple: this company is promising. It was rated as one of the 15 fierce biotechnology companies in 2012. It has won investments not only from Gates but also from big capitals such as the Third Rock and Google Ventures.

        In fact, I would rather say that Bill Gates and other venture capitals invested in the future of cancer genome sequencing than the company. Cancer genome sequencing and relevant analysis hold the promise for cancer control in the future.

        Currently four types of cancer treatment are available in clinic: surgery, radiation therapy, chemotherapy, and targeted therapy. These four treatments have all been adopted as routine therapy in clinic practice. Targeted therapy, in which the drug is designed against specific cancer mutations, becomes increasingly popular for its specificity and low toxicity. It is likely to surpass radiation and chemical therapy to become the primary option for cancer patients in the future. Since cancerous mutations are tremendously diverse among individuals, cell types, and clinical stages, the success of targeted therapy is highly dependent on how accurately the mutations can be measured. FoundationOne, as the company has described, can probe the whole genome information of a patient using a piece of solid tumor tissue. Moreover, FoundationOne service can be updated regularly as more mutations are indentified and more targeted therapies become available. FoundationOne represents one of available clinical practices translated from the so-called personalized medicine, a concept favored by today’s oncologists.  

        How good is FoundationOne?

        It is not as good as it sounds to be. There are some reasons for the imperfection of personalized cancer treatment. First of all, targeted therapy itself is not perfect. The biggest problem of targeted therapy is drug resistance. All known anti-tumor drugs targeting mutations result in drug resistance in 6 to 18 months after usage. Secondly, targeted therapy is more expensive than the traditional therapies. For example, Erlotinib, a drug targeting mutations in lung cancer, extends life by an average of 3.3 month at a cost of $95000. Thirdly, not all cancerous mutations are suitable for drug targets. For example, there is currently no drug against Ras mutation, a frequently mutated gene in cancer. Fouthly, tumor genome, which is different from normal genome, changes rapidly, thus requires periodical examination.       

        Although FoundationOne is not perfect  at this point, it is  being improved, as stated on the company’s website. As the cost goes down, cancer genome sequencing will hopefully become a routine medical examination in the future, just like blood test and urine test. As new drugs are being designed and drug resistance mechanisms being unraveled, the power of cancer genome sequencing may truly come to play.    

DNA disk

        Animals use urine to mark territory. By using urine, they broadcast the news: I am the boss of this place! Other animals read the information that the place has been owned by someone else from urine. Then the newcomers may choose to leave or stay to fight for the territory. In this case, urine was used to store information. Urine is so cheap that it is practical for animals to claim territory. However, urine cannot hold too much information. For example, it is hard for newcomers to tell how strong the settlers are so that they can decide if they might stay to fight. Urine cannot last too long either so animals have to pee frequently to make a territory claim. To sum up, urine is practical to store simple information for a short while, that can be read by other animals. From this example, we can learn a lesson about how humans store information. When we store information, we usually consider four factors: practicality (how easy the information can be produced), capacity (how much information can be accommodated), maintenance (how long information can be kept), and readability (how easy information can be read).

        Books written in papers are first milestone of information storage. Books are practical because papers are cheap and easy to handle. Books can last many years. People with normal intellect can learn how to read and write without too much difficulty. With the emergency of silicon chips, humans achieved tremendous progression in information storage. Simple symbols, namely 0s and 1s, were used to represent everything. Silicon chips beat books in every aspects: higher capacity, easier to maintain, and easier to read. Smaller hard drives with higher capacity are created continuously.

         However, we still face unmet need for information storage. More and more information was produced especially in biology field. For example, genome sequencing has produced and is producing a vast volume of information. How to store these massive amounts of information poses a huge challenge to humans.

        What should be the next-generation carrier of information deluge after books and silicon chips? DNA may be the qualified candidate since it has been used to store information by nature for millions of years. Scientists figured out how to use DNA to store information.

        Nick Goldman designed a method to store information in DNA. They tried to store in DNA Shakespeare’s sonnets (ASCII text), Waston and Crick’s paper about the identification of DNA double helix (PDF), a colorful photo of European Bioinformatics Institutes (JPEG 2000), Martin Luther King’s “I have a dream” speech (MP3), and Huffman code. These documents were encoded into binary text, namely 0s and 1s. Subsequently, these binary texts were translated into base 3 encoded files (0s, 1s and 2s) that correspond to long DNA sequences. Long DNA sequence is supposed to store information but it is hard to read by sequencing. Thus, short DNA sequences with overlapping segments were designed to represent long DNA.  Moreover, indexing information were added to short DNA sequences so that it is possible to find which DNA corresponds to which document. Overall, the five files listed above were represented by a total of 153,335 strings of DNA, each comprising 117 nucleotides (nt). The DNA was synthesized and lyophilized for shipment in ambient temperature from USA to Germany via UK. In Germany, DNA was resuspended, amplified, purified and sequenced. Then full-length DNA sequence corresponding original files was reconstructed based on sequencing results. For these five files, it turned out to be a 100% accuracy of reconstruction.  Martin Luther King's speech is still clear and heart-stirring. 

        Needless to say that DNA can hold much more information and can be maintained more easily than magnetic tapes. But Is DNA storage practical? Currently the cost of DNA storage is about $12400/MB for information storage and $220/MB for information decoding. This cost is much higher than magnetic tapes. However, information stored in magnetic tapes needs to be copied frequently in case they will not be extracted because magnetic tapes cannot last very long. By contrast, synthesized DNA can last thousands of years under normal maintenance. DNA encoding will also be cheaper if the current trend of DNA manipulating continues. In less than 50 years, DNA storage will be practical. The information writing and reading into DNA is not competitive with current technology but can be accelerated. In summary, DNA storage holds a big promise for massive amounts of information storage.

        It is possible to connect a DNA synthesizing machine and a DNA sequencing machine to a computer. Then we will be able to manipulate information in a DNA based format.  

[1] Towards practical, high-capacity, low-maintenance information storage in synthesized DNA. Nature (2013) doi:10.1038/nature11875 Received 15 May 2012 Accepted 12 December 2012 Published online 23 January 2013