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Tiny New CRISPR Protein Could Make Human Gene-Hacking Less Risky

February 11, 2019

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Double Strand DNA Breaks Introduced by CRISPR-Cas9 Allows Further Genetic Manipulation By Exploiting Endogenous DNA Repair Mechanisms.
Graphic credit: By Guido4 - Own work, CC BY-SA 4.0,
https://commons.wikimedia.org/w/index.php?curid=63789049

Editor's Note: We just saw the CNN Documentary “Three Identical Strangers.“ The story relates a bizarre experiment begun around 1960, where by design, identical twins and triplets were separated at birth, and adopted as singlets by unsuspecting parents. The ostensible goal of the study was to differentiate “nature from nurture“ on development and personality. While this type of unethical experiment could never occur now without an ethics committee approval, informed consent and clear transparency, we must all be aware, no matter how noble the intention, although in this case perhaps not so noble, there is the law of unintended consequences, when we interfere with basic biology and human development. In our era of genetic engineering, this documented story serves as a cautionary tale. See the documentary and you will understand.  

When people talk about the gene-editing tool CRISPR, they usually mean CRISPR-Cas9. But Cas9 is just one of several CRISPR-associated proteins. A few others are Cas12a, CasY, and CasX. These proteins act as the “scissors“ in the CRISPR system, which acts as a natural defense against viruses for some bacteria, similarly to the immune system in humans.

A dead Cas9 protein coupled with epigenetic modifiers which are used to repress certain genome sequences rather than cutting it all together.
Graphic credit: Mariuswalter - Own work, CC BY-SA 4.0,
https://commons.wikimedia.org/w/index.php?curid=62766589

Using “dead“ versions of Cas9 (dCas9) eliminates CRISPR's DNA-cutting ability, while preserving its ability to target desirable sequences. Multiple groups added various regulatory factors to dCas9s, enabling them to turn almost any 1) ___ on or off or adjust its level of activity. Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. The targeted site is methylated, epigenetically modifying the gene. This modification inhibits transcription. These precisely placed modifications may then be used to regulate the effects on gene expressions and DNA dynamics after the inhibition of certain genome sequences within DNA. Within the past few years, epigenetic marks in different human 2) ___ have been closely researched and certain patterns within the marks have been found to correlate with everything ranging from tumor growth to brain activity. Conversely, CRISPR-mediated activation (CRISPRa) promotes gene transcription. Cas9 is an effective way of targeting and silencing specific genes at the DNA level. In bacteria, the presence of Cas9 alone is enough to block transcription. For mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA sequences called promoters that immediately precede the target gene. Cas9 was used to carry synthetic transcription factors that activated specific 3) ___ genes. The technique achieved a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter.

In 2016, researchers demonstrated that CRISPR from an ordinary mouth bacterium could be used to edit RNA. The researchers searched databases containing hundreds of millions of genetic sequences for those that resembled Crispr genes. They considered the fusobacteria Leptotrichia shahii. It had a group of genes that resembled CRISPR genes, but with important differences. When the researchers equipped other bacteria with these genes, which they called C2c2, they found that the organisms gained a novel defense. Many viruses encode their genetic information in RNA rather than DNA that they repurpose to make new 4) ___. HIV and poliovirus are such viruses. Bacteria with C2c2 make molecules that can dismember RNA, destroying the virus. Tailoring these genes opened any RNA molecule to editing. CRISPR-Cas systems can also be employed for editing of micro-RNA and long-noncoding RNA genes in plants. CRISPR simplifies creation of animals for research that mimic disease or show what happens when a gene is knocked down or mutated. CRISPR may be used at the germline level to create animals where the gene is changed everywhere, or it may be targeted at non-germline cells.

CRISPR can be utilized to create human cellular models of disease. For instance, applied to human pluripotent stem cells, CRISPR when introduced, targeted mutations in genes relevant to polycystic kidney disease (PKD) and focal segmental glomerulosclerosis (FSGS). These CRISPR-modified pluripotent 5) ___ cells were subsequently grown into human kidney organoids that exhibited disease-specific phenotypes. Kidney organoids from stem cells with PKD mutations formed large, translucent cyst structures from kidney tubules. The cysts were capable of reaching macroscopic dimensions, up to one centimeter in diameter. Kidney organoids with mutations in a gene linked to FSGS developed junctional defects between podocytes, the filtering cells affected in that disease. This was traced to the inability of podocytes to form microvilli between adjacent cells. Importantly, these disease phenotypes were absent in control organoids of identical genetic background, but lacking the CRISPR modifications. A similar approach was taken to model long QT syndrome in cardiomyocytes derived from pluripotent stem cells. These CRISPR-generated cellular models, with isogenic controls, provide a new way to study human disease and test drugs.

CRISPR/Cas technology has been proposed as a treatment for multiple human diseases, especially those with a genetic cause. Its ability to modify specific DNA sequences makes it a tool with potential to fix disease-causing mutations. Early research in animal models suggest that therapies based on CRISPR technology have potential to treat a wide range of diseases, including cancer, beta-thalassemia, sickle cell disease, hemophilia, cystic fibrosis, Duchenne's muscular dystrophy, Huntington's, and heart disease. CRISPR/Cas-based “RNA-guided nucleases“ can be used to target virulence factors, genes encoding antibiotic resistance and other medically relevant sequences of interest. This technology thus represents a novel form of antimicrobial therapy and a strategy by which to manipulate bacterial populations. Recent studies suggested a correlation between the interfering of the CRISPR/Cas locus and acquisition of antibiotic resistance. This system provides protection of bacteria against invading foreign DNA, such as transposons, bacteriophages and plasmids. This system was shown to be a strong selective pressure for the acquisition of antibiotic resistance and virulence factor in bacterial pathogens. Some of the affected genes are tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and fetal hemoglobin.

Research suggests that CRISPR is an effective way to limit replication of multiple herpesviruses. It was able to eradicate viral DNA in the case of Epstein-6) ___ virus (EBV). Anti-herpesvirus CRISPRs have promising applications such as removing cancer-causing EBV from tumor cells, helping rid donated organs for immunocompromised patients of viral invaders, or preventing cold sore outbreaks and recurrent eye infections by blocking HSV-1 reactivation. As of August 2016, these were awaiting testing. CRISPR is being applied to develop tissue-based treatments for cancer and other diseases. CRISPR may revive the concept of transplanting animal organs into people. Retroviruses present in animal genomes could harm transplant recipients. In 2015, a team eliminated 62 copies of a retrovirus's DNA from the pig genome in a kidney epithelial cell. Researchers recently demonstrated the ability to birth live pig specimens after removing these retroviruses from their genome using 7) ___ for the first time. CRISPR may have applications in tissue engineering and regenerative medicine, such as creating human blood vessels that lack expression of MHC class II proteins, which often cause transplant rejection. As of 2016 CRISPR had been studied in animal models and cancer cell lines, to learn if it can be used to repair or thwart mutated genes that cause cancer.

The first clinical trial involving CRISPR started in 2016. It involved removing immune cells from people with lung cancer, using CRISPR to edit out the gene expressed PD-1, then administrating the altered cells back to the same person. 20 other trials were under way or nearly ready, mostly in China, as of 2017. In 2016, US FDA approved a clinical trial in which CRISPR would be used to alter T cells extracted from people with different kinds of cancer and then administer those engineered T cells back to the same people. In May 2018, the company CRISPR Therapeutics received approval to start a 8) ___ trial with a CRISPR-based treatment for the blood disorder beta-thalassemia, which was scheduled to start in late 2018. In 2015, multiple studies attempted to systematically disable each individual human gene, in an attempt to identify which genes were essential to human biology. Between 1,600 and 1,800 genes passed this test - of the 20,000 or so known human genes. Such genes are more strongly activated, and unlikely to carry disabling mutations. They are more likely to have indispensable counterparts in other species. They build proteins that unite to form larger collaborative complexes. The studies also cataloged the essential genes in four cancer-cell lines and identified genes that are expendable in healthy cells, but crucial in specific tumor types and drugs that could target these rogue genes. The specific functions of some 18% of the essential genes are unidentified. In one 2015 targeting experiment, disabling individual genes in groups of cells attempted to identify those involved in resistance to a melanoma drug. Each such gene manipulation is itself a separate “drug“, potentially opening the entire genome to CRISPR-based regulation.

Unenriched sequencing libraries often have abundant undesired sequences. Cas9 can specifically deplete the undesired sequences with double strand breakage with up to 99% efficiency and without significant off-target effects as seen with restriction enzymes. Treatment with Cas9 can deplete abundant rRNA while increasing pathogen sensitivity in RNA-seq libraries. CRISPR/Cas-9 can be used to edit the DNA of organisms in vivo and entire chromosomes can be eliminated from an organism at any point in its development. Chromosomes that have been deleted in vivo are the Y chromosomes and X chromosomes of adult lab mice and human chromosomes 14 and 21, in embryonic stem cell lines and aneuploid mice respectively. This method might be useful for treating genetic aneuploid diseases such as Down 9) ___. Successful in vivo genome editing using CRISPR/Cas9 has been shown in several model organisms, such as Escherichia coli, Saccharomyces cerevisiae, Candida albicans, Caenorhadbitis elegans, Arabidopsis, Danio rerio, Mus musculus. Successes have been achieved in the study of basic biology, in the creation of disease models, and in the experimental treatment of disease models. Concerns have been raised that off-target effects (editing of genes besides the ones intended) may obscure the results of a CRISPR gene editing experiment (the observed phenotypic change may not be due to modifying the target gene, but some other gene). Modifications to CRISPR have been made to minimize the possibility of off-target effects. In addition, orthogonal CRISPR experiments are recommended to confirm the results of a gene editing experiment.

As of December 2014, patent rights to CRISPR were contested. Several companies formed to develop related drugs and research tools. As companies ramp up financing, doubts as to whether CRISPR can be quickly monetized were raised. In February 2017 the US Patent Office ruled on a patent interference case brought by University of California with respect to patents issued to the Broad Institute, and found that the Broad patents, with claims covering the application of CRISPR/cas9 in eukaryotic cells, were distinct from the inventions claimed by University of California. Shortly after, University of California filed an appeal of this ruling. As of November 2013, SAGE Labs (part of Horizon Discovery group) had exclusive rights from one of those companies to produce and sell genetically engineered rats and non-exclusive rights for mouse and rabbit models. By 2015, Thermo Fisher Scientific had licensed intellectual property from ToolGen to develop CRISPR reagent kits. In March 2017, the European Patent Office (EPO) announced its intention to allow broad claims for editing all kinds of cells to Max-Planck Institute in Berlin, University of California, and University of Vienna, and in August 2017, the EPO announced its intention to allow CRISPR claims in a patent application that MilliporeSigma had filed. As of August 2017 the patent situation in Europe was complex, with MilliporeSigma, ToolGen, Vilnius University, and Harvard contending for claims, along with University of California and Broad. As of November 2018, all the CRISPR patent holders and institutes associated with them have set up companies to commercialize the patents by sub-licensing them for therapeutic, agriculture and many other application areas to biotech firms, pharmaceuticals, agri-businesses etc. Caribou Biosciences, ERS Genomics, Editas Medicine, Intellia Therapeutics, and CRISPR Therapeutics are the spin offs associated with CRISPR landscape. Not many large scale commercial assignees have actively participated in the early phases of the CRISPR-Cas patent landscape. The only large establishments making it to the top ten are Dow AgroSciences and DuPont Nutrition Science (now merged as DowDuPont), together holding 20 inventions in CRISPR-Cas9 applications in agriculture and animal biotechnology.

As of March 2015, multiple groups had announced ongoing research to learn how they one day might apply CRISPR to human embryos, including labs in the US, China, and the UK, as well as US biotechnology company OvaScience. Scientists, including a CRISPR co-discoverer, urged a worldwide moratorium on applying CRISPR to the human germline, especially for clinical use. These groups said that “scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans“ until the full implications “are discussed among scientific and governmental organizations“. These scientists support further low-level research on CRISPR and do not see CRISPR as developed enough for any clinical use in making heritable changes to humans. In April 2015, Chinese scientists reported results of an attempt to alter the DNA of non-viable human embryos using CRISPR to correct a mutation that causes beta thalassemia, a lethal heritable disorder. The study had previously been rejected by both Nature and Science in part because of ethical concerns. The experiments resulted in successfully changing only some of the intended genes, and had off-target effects on other genes. The researchers stated that CRISPR is not ready for clinical application in reproductive medicine. In April 2016, Chinese scientists were reported to have made a second unsuccessful attempt to alter the DNA of non-viable human embryos using CRISPR - this time to alter the CCR5 gene to make the embryo HIV resistant. In December 2015, an International Summit on Human Gene Editing took place in Washington under the guidance of David Baltimore. Members of national scientific academies of America, Britain and China discussed the ethics of germline modification. They agreed to support basic and clinical research under certain legal and ethical guidelines. A specific distinction was made between somatic cells, where the effects of edits are limited to a single individual, versus germline cells, where genome changes could be inherited by descendants. Heritable modifications could have unintended and far-reaching consequences for human evolution, genetically (e.g. gene/environment interactions) and culturally (e.g. Social Darwinism). Altering of gametocytes and embryos to generate inheritable changes in humans was defined to be irresponsible. The group agreed to initiate an international forum to address such concerns and harmonize regulations across countries. In November 2018, Jiankui He announced that he had edited two human embryos, to attempt to disable the gene for CCR5, which codes for a receptor that HIV uses to enter cells. He said that twin girls, Lulu and Nana, had been born a few weeks earlier. He said that the girls still carried functional copies of CCR5 along with disabled CCR5 (mosaicism) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature.

Policy regulations for the CRISPR/cas9 system vary around the globe. In February 2016, British scientists were given permission by regulators to genetically modify human embryos by using CRISPR-Cas9 and related techniques. However, researchers were forbidden from implanting the embryos and the embryos were to be destroyed after seven days. The US has an elaborate, interdepartmental regulatory system to evaluate new genetically modified foods and crops. For example, the Agriculture Risk Protection Act of 2000 gives the 10) ___the authority to oversee the detection, control, eradication, suppression, prevention, or retardation of the spread of plant pests or noxious weeds to protect the agriculture, environment and economy of the US. The act regulates any genetically modified organism that utilizes the genome of a predefined “plant pest“ or any plant not previously categorized. In 2015, Yinong Yang successfully deactivated 16 specific genes in the white button mushroom, to make them non-browning. Since he had not added any foreign-species (transgenic) DNA to his organism, the mushroom could not be regulated by the USDA under Section 340.2. Yang's white button mushroom was the first organism genetically modified with the Crispr/cas9 protein system to pass US regulation. In 2016, the USDA sponsored a committee to consider future regulatory policy for upcoming genetic modification techniques. With the help of the US National Academies of Sciences, Engineering and Medicine, special interests groups met on April 15 to contemplate the possible advancements in genetic engineering within the next five years and any new regulations that might be needed as a result. The FDA in 2017 proposed a rule that would classify genetic engineering modifications to animals as “animal drugs“, subjecting them to strict regulation if offered for sale, and reducing the ability for individuals and small businesses to make them profitably.

In China, where social conditions sharply contrast with the west, genetic diseases carry a heavy stigma. This leaves China with fewer policy barriers to the use of this technology. In 2012, and 2013, CRISPR was a runner-up in Science Magazine's Breakthrough of the Year award. In 2015, it was the winner of that award. CRISPR was named as one of MIT Technology Review's 10 breakthrough technologies in 2014 and 2016.

On February 9, 2019, the U.S. patent office indicated it will issue a third CRISPR patent to UC. This Patent is involved in interference proceedings and will add to university's gene-editing portfolio. The U.S. Patent and Trademark Office has issued a notice of allowance for a University of California patent application covering systems and methods for using single molecule guide RNAs that, when combined with the Cas9 protein, create more efficient and effective ways for scientists to target and edit genes. U.S. patent application number 13/842,859, which had notably been examined in advance of a prior interference proceeding involving the Broad Institute, specifically focuses on methods and systems for modifying a target DNA molecule in any setting, both in vitro and within live cells, using one or multiple single guide RNAs, across every cell type. The associated patent is expected to issue in the next 6-9 weeks.

This CRISPR-Cas9 DNA-targeting technology, invented by Jennifer Doudna and Martin Jinek of the University of California, Berkeley, along with Emmanuelle Charpentier at Umea University and Krzystof Chylinski at the University of Vienna, is a fundamental molecular tool for editing genes. Together, this patent application and prior U.S. Patent Numbers 10,000,772 and 10,113,167, cover CRISPR-Cas9 methods and compositions useful as gene-editing scissors in any setting, including in vitro, as well as within live plant, animal and human cells.

Sources: Robert Sanders, USC; NIH.gov; Wikipedia

ANSWERS: 1) gene; 2) cells; 3) human; 4) viruses; 5) stem; 6) -Barr; 7) CRISPR; 8) clinical; 9) Syndrome; 10) USDA

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