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What's All This Stuff About CRISPR??

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What's All This Stuff About CRISPR?? What’s all this stuff about CRISPR?? CLUSTERED RANDOMLY INTERSPERSED SHORT PALINDROMIC REPEATS Scientists Find Form of Crispr Gene Editing With New Capabilities Gene Editing Spurs Hope for Transplanting Pig Organs Into A common bacterium contains molecules that target RNA, not DNA. If it can be harnessed for use in humans, the process may Humans lead to new forms of bioengineering. Geneticists have created piglets free of retroviruses, an important step toward creating a new supply of organs for transplant patients. Scientists Can Design ‘Better’ Babies. Should They? Advances in reproductive technology have put genetic choices within reach of prospective parents. But critics warn of ethical peril. 1. SCIENCE Scientists Find Form of Crispr Gene Editing With New Capabilities A common bacterium contains molecules that target RNA, not DNA. If it can be harnessed for use in humans, the process may lead to new forms of bioengineering. As D.I.Y. Gene Editing Gains Popularity, ‘Someone Is Going to Get Hurt’ June 3, 2016 After a virus was created from mail-order DNA, scientists are sounding the alarm about the genetic tinkering carried out in garages and living rooms. U.S. Scientists Can Design ‘Better’ Babies. Should They? Advances in reproductive technology have put genetic choices within reach of prospective parents. But critics warn of ethical peril. June 10 The CRISPR-CAS 9 System First, a primer on DNA What’s the takeaway from this?? Base pairing in DNA is unique: Adenine always bonds to Thymine Guanine always bonds to Cytosine Genes are sequences of DNA. The code in a gene lies in the sequence of base pairs. So, if you know the sequence in a gene or even a part of the sequence, you can design a “probe” with complementary bases which will seek out that gene in the nucleus. This is the basis of PCR and other molecular techniques such as ancestry tracing. It is also the basis of CRISPR. Viruses and Bacterial Cells Viruses that attack bacterial cells are called bacteriophages. They act by inserting their nuclear (genetic) material (DNA or RNA) into the cell. The viral genetic material takes over the protein and nucleic acid synthetic machinery of the cell to make more viruses. Eventually the new viruses erupt from the cell, destroying it, and go on to infect other cells. CRISPR is based on the bacterial cell’s “immune” or defensive system to protect against viruses (or allospecific plasmids). Overview of the CRISPR/Cas mechanism of action. Philippe Horvath, and Rodolphe Barrangou, Science 327, 167-170 (2010) What do we mean by “palindromic sequence”? Note that in RNA A pairs with U, not T as in DNA Formation of complex Role of the PAM Cas9 will not cleave the protospacer sequence unless there is an adjacent PAM sequence. The spacer in the bacterial CRISPR loci will not contain a PAM sequence (because that’s where the Cas attached to the original viral DNA and cut downstream of that to make the protospacer) , and will thus not be cut by the nuclease. But the protospacer in the invading virus or plasmid will contain the PAM sequence, and will thus be cleaved by the Cas9 nuclease. The canonical PAM is the sequence 5'-NGG-3' where "N" is any nucleobase followed by two guanine ("G") nucleobases. Taking advantage of CRISPR system: DNA surgeon. With just a guide RNA and a protein called Cas9, researchers first showed that the CRISPR system can home in on and cut specific DNA, knocking out a gene or enabling part of it to be replaced by substitute DNA. More recently, Cas9 modifications have made possible the repression (lower left) or activation (lower right) of specific genes. Jennifer Doudna explains CRISPR (16 min) Published by AAAS Elizabeth Pennisi Science 2013;341:833-836 Some Applications of CRISPR in genetic engineering Top 9 CRISPR Startup Companies Changing the Future of Biotech and Medicine1 Inscripta Therapeutics Plantedit Biotech startup companies using CRISPR- Beam Therapeutics Cas9 technology as a main component eGenesis of their strategy have existed almost since the discovery that CRISPR could be Ligandal ntrans reprogrammed to target essentially any region of any genome. Several CRISPR Synthetic technology companies, such as CRISPR Genomics Therapeutics, Editas Medicine, and Mammoth Biosciences Intellia Therapeutics, have already outgrown startup status and are now publicly traded companies. 1The Official Synthego Blog where we explore the exciting, rapidly emerging field of CRISPR genome engineering. Some companies and applications using CRISPR After https://labiotech.eu/policy-legal- finance/doudna-charpentier-crispr-patent- europe/ Ethical Implications Somatic Gene Therapy Gene replacement therapy Insert “good” gene to replace “bad” one Gene augmentation therapy Insert a functioning gene to provide the protein not produced by a mutated gene. Gene inhibition therapy Inhibits an undesirable gene such as a cancer-causing gene Killing of specific cells Cause cells expressing a certain form of a gene to commit suicide Germline Gene Therapy Therapy applied to germ cells and passed on to all subsequent offspring Elimination of inherited diseases • As of March 2015 at least four labs in the US, labs in China and the UK had announced plans for ongoing research to apply CRISPR to human embryos. • 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,. The experiments resulted in changing only some genes, and had off-target effects on other genes. (The study had been rejected by both Nature and Science in part because of ethical concerns. ) • In December, following the Chinese studies, Doudna and others urged a worldwide moratorium on applying CRISPR to the human germline, especially for clinical use. “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". • 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 a gene to make the embryo HIV resistant. • On Jan 21, 2018, The Wall Street Journal reported that 86 people in China have had their genes edited using CRISPR. CRISPR enhances cancer immunotherapy Gene editing expands reach of therapeutic T cells, in mice Date: March 6, 2018 Source: Washington University School of Medicine Summary: The FDA recently approved the first cellular immunotherapies to treat certain blood cancers. But so far, these T cell immunotherapies can't be used if the T cells themselves are cancerous. Such 'CAR-T' cells kill each other because they resemble one another so closely. Scientists now have used the gene-editing technology CRISPR to engineer human T cells that can attack human T cell cancers without succumbing to friendly fire. Science News Patent Battle Doudna and Charpentier (UC) published the mechanism of the CRISPR immune system in prokaryotes. (Science 337 (6096), pp 816-821,17 Aug 2012) Fang Zhang (Broad Institute) shortly filed a patent for CRISPR claiming that he had adapted the prokaryote system to eukaryote cells. UC sued in 2013, claiming that the extension of the Doudna/Charpentier work to eukaryotes was “obvious”. In 2017 the U.S. Patent Trial and Appeals Board (PTAB) sided with Broad. UC brought the case to the U.S. Court of Appeals, where as of June 1st of this year it still resides. Implications of the Patent Fight Whoever wins (including a draw) will get licensing fees from all companies using CRISPR. Licensing fees charged to pharmaceutical companies will be passed on to consumers. CRISPR as a gene-based therapy means that unlike other medicines, it is applied only once to a patient so all costs must be recouped then. Any company currently paying license fees to the losing competitor (UC or Broad) would likely lose venture capital support, delaying further development. To complicate matters the European Patent Office has upheld the UC patent and rejected the Broad’s. If the U.S. rules differently this will greatly complicate international use and development of CRISPR..
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