{"id":219809,"date":"2016-06-29T17:00:01","date_gmt":"2016-06-29T20:00:01","guid":{"rendered":"http:\/\/revistapesquisa.fapesp.br\/en\/?p=219809"},"modified":"2016-06-29T18:23:39","modified_gmt":"2016-06-29T21:23:39","slug":"a-tool-to-edit-dna","status":"publish","type":"post","link":"https:\/\/revistapesquisa.fapesp.br\/en\/a-tool-to-edit-dna\/","title":{"rendered":"A tool to edit DNA"},"content":{"rendered":"

\"038-041_Crispr_240-A\"<\/a>A system that allows bacteria to recognize and combat viral invasions promises significant innovation in genetics.\u00a0 It concerns a protein guided by an RNA molecule that cuts DNA strands at specific points and activates repair pathways. A number of researchers in Brazil are preparing to incorporate the technique, created in 2012<\/a>, into their lines of research. It is a story that is only beginning to be told and for now has yielded few tangible results. The system bears watching, both for what it promises and its potential to alter human genes and produce designer babies, which raises concerns to the point of discussing a moratorium on its use.<\/p>\n

\u201cIt’s a great equalizer, until we manage to do it,\u201d jokes Dr. Jos\u00e9 Xavier Neto, of the Brazilian Biosciences National Laboratory (LNBio) in Campinas, about the system that became known as CRISPR-Cas9. The acronym stands for Clustered Regularly Interspaced Short Palindromic Repeats, which works with an associated protein, Cas.\u00a0 CRISPR-Cas9 can be inserted into cells using viruses or through DNA injections in the early stages of an embryo. A specially synthesized RNA molecule serves as a guide to reaching the gene that is to be altered (see infographic<\/a>).<\/em> Such procedures are within reach of most genetics laboratories, and this gives autonomy to researchers.<\/p>\n

It all began in Xavier Neto\u2019s laboratory with \u00c2ngela Saito, who at the time was studying for her doctorate at the University of Campinas (Unicamp) under the supervision of biologist J\u00f6rg Kobarg; she had to produce a rodent with a deficiency in the production of a particular protein (knockout or KO) to study its role in leukemia. In the laboratory\u2019s databanks, she began with the tedious traditional process, where it is necessary to generate and track genetic manipulations in hundreds of embryonic stem cell clones. To learn how to do this task on an almost industrial scale, she went, during her doctorate, to the MD Anderson Cancer Center at the University of Texas, where she ultimately learned the new technique with the American geneticist Richard Behringer. She returned to the S\u00e3o Paulo laboratory bringing in her luggage the vectors that would be injected into mouse embryos to produce the knockouts she needed. It worked: Saito taught her colleagues and, in just over a year, the laboratory has produced knockouts for four different genes.<\/p>\n

\"Albinism

Carolina Clemente\/LNBio <\/span>Albinism mutation: to produce a homogeneous effect, injection in the early embryonic stage is necessaryCarolina Clemente\/LNBio <\/span><\/p><\/div>\n

Fighting diseases<\/strong>
\nThe potential of CRISPR-Cas9 in research on disease-causing agents also drew the attention of Venezuelan parasitologists Noelia Lander and Miguel Chiurillo, who are interested in studying the parasite Trypanosoma cruzi,<\/em> which causes Chagas disease, and who can make a contribution to the development of alternative therapies.\u00a0 Lander is currently doing a postdoctoral internship at Unicamp\u2019s School of Medical Sciences (FCM – Unicamp) in collaboration with biochemists An\u00edbal Vercesi and Roberto Docampo, who is from Argentina and is a professor at the University of Georgia as well as a visiting professor at the Unicamp. She has shown that she was able to alter genes in a 2015 article published in the journal mBio.<\/em> She broke down the genes linked to the parasite\u2019s flagellum\u2014a tail-like structure that allows it to move. \u201cIt’s a very easy phenotype to see, because the flagellum is separated from the cell body and the parasite is deposited at the bottom of the bottle,\u201d she says. The proof of concept is a victory because trypanosomes have been very effective in resisting any attempt at genetic manipulation. Now, in studies of genes involved in cell signaling by calcium, an element comes along that can help fight this disease that lacks effective treatment in its chronic phase. \u201cCalcium levels change a lot when the parasite infects the host,\u201d she says. \u201cIf we can move these proteins, which differ between the parasite and the vertebrate host, it could be the pathway to an alternative therapy.\u201d<\/p>\n

The fight against dengue transmission is the goal of biologist Jayme de Souza Neto, at the Botucatu campus of S\u00e3o Paulo State University (Unesp). In the Aedes aegypti <\/em>mosquito, he compared the transcribed RNAs of infected mosquitoes resistant to the virus in wild populations in Botucatu, S\u00e3o Paulo State, and Ne\u00f3polis, in the northeastern state of Sergipe, and identified genes that may be linked to resistance. \u201cWe are beginning to mutate the genes of mosquitoes,\u201d he reports. By July 2016, he intends to have populations in the laboratory in which he will be able to determine if their susceptibility to the virus has been altered. Still far away on the horizon, the idea is to produce resistant mosquitoes, which, because they are not infected, do not transmit the disease to humans. The project has advanced under the collaboration framework established between FAPESP, Unesp and the University of Keele in the United Kingdom (see <\/em>Pesquisa FAPESP\u00a0issue n\u00ba 230<\/em><\/a>). Souza Neto spent three months in the laboratory of Julien Pelletier, who was in Botucatu for four months. \u201cIn April he must return to start the injections into the mosquito embryos,\u201d he says.<\/p>\n

Biologist Nat\u00e1lia Gon\u00e7alves is dealing with larger subjects: Golden Retrievers used as a model for studies of Duchenne muscular dystrophy, a degenerative disease in which patients ultimately cannot walk or eat (see <\/em>Pesquisa FAPESP\u00a0issue n\u00ba 237<\/em><\/a>).\u00a0 For work on her doctorate she established reprogrammed cell lines (induced pluripotent stem cells or iPSCs) from the skin cells of dogs. Now, for her postdoctoral internship under the supervision of Carlos Eduardo Ambr\u00f3sio, Faculty of Animal Science and Food Engineering, University of S\u00e3o Paulo (FZEA-USP), she plans to establish a line with cells from dystrophic dogs and correct the defective gene that produces the dystrophin protein, in partnership with French geneticist Jean-Paul Concordet, of the National Museum of Natural History in Paris. “We already know which region of the gene is missing, then the idea is to produce this small piece and insert it,\u201d she says. She has a lot of preliminary work to do: while the technique for inactivating genes with CRISPR-Cas9 is already fairly well mastered, her success rate in inserting specific stretches is still low.<\/p>\n

Geneticist Maria Rita Passos Bueno, of the USP Biosciences Institute (IB-USP), is also focusing on DNA editing to study human diseases with the help of researcher Erika Kague, who learned the technique at the end of her postdoctoral internship at the University of Pennsylvania in the United States. Doctoral student Luciano Abreu Brito established a zebrafish line (see <\/em>Pesquisa FAPESP\u00a0issue n\u00ba 209<\/em><\/a>), to study cleft lip and palate malformations. \u201cWe found the mutation by sequencing in patients, which we can now insert into the fish to test if it is even relevant to the disease,\u201d he says. In isolated human cells, doctoral student Danielle Moreira inserted mutations linked to autism. In the future, she plans to use iPSCs that can give rise to neurons, to determine if the genetic changes identified in patients alter the functioning of nerve cells.<\/p>\n

Lygia da Veiga Pereira, a geneticist at IB-USP, is also beginning to directly alter human cells. Her master’s student, Juliana Sant\u2019Ana, is in contact with geneticist Chad Cowan of Harvard University in the United States to learn how to use CRISPR-Cas9. The idea is to provoke in the fibrillin protein gene the mutation typical of Marfan syndrome. Once successful in easy-to-grow cells, Pereira intends to pass the BR-1 to the stem cell line developed in her laboratory (see <\/em>Pesquisa FAPESP\u00a0issue n\u00ba 153<\/em><\/a>). \u201cI want to produce heart cells and bone cells with the mutation,\u201d she says. The ease of working with CRISPR-Cas9 allows this stage to be completed relatively quickly and to reach the goal: the study of how the disease behaves in different tissues. \u201cScience will begin when we can compare these cells.\u201d<\/p>\n

\"Crispr_bebesFundo\"CAETO<\/span>Protein structure<\/strong>
\nBueno notes that knowledge of the CRISPR-Cas9 system is advancing rapidly in the search for ever greater accuracy in editing. One of the fronts now being explored by Jennifer Doudna, at the University of California at Berkeley, one of the protagonists in developing the technique, is to figure out how the protein’s structure allows it to fit into the DNA and the cut at a specific point, as shown in an article published in the January 2016 issue of the journal Science.<\/em><\/p>\n

German-Chilean biochemist Martin W\u00fcrtele, at the Federal University of S\u00e3o Paulo (Unifesp), was already engaged in unraveling the three-dimensional structure of these proteins even before the discovery by Doudna and her French colleague Emmanuelle Charpentier, at Germany\u2019s Max Planck Institute for Infection Biology in 2012. \u201cAbout five years ago we started working with several CRISPR-Cas proteins for their contribution to the protection of bacteria against their main natural enemies, the bacteriophages, and the possibility of editing DNA,\u201d he says. \u201cBut since then they discovered Cas9, which, unlike the CRISPR-Cas systems we work with, does pretty much the whole process with a single protein and is a serious candidate for the Nobel Prize.\u201d He says that a protein called Csm2, withdrawn from bacteria, consists of a long chain of amino acids in a helix, surrounded by three shorter helices. \u201cThe Csm2 protein is completely different from those described in other complexes,\u201d says W\u00fcrtele. He believes this protein is part of a major defense of bacteria and knowledge of how it works might be used against the bacteria themselves. \u201cThere is a great interest in using bacteriophages as potential substitutes for antibiotics.\u201d<\/p>\n

The applications are so numerous that the ability to edit human genes has generated enormous fear of the consequences. For the time being research continuity is guaranteed, with the proposal to prohibit the implantation of altered human embryos. Many researchers are concerned, but Souza Neto does not believe there is a real risk.\u00a0 \u201cThe harm in overriding evolution can be much greater than the benefits,\u201d he warns. \u201cThe possibility of off-target effects can make us shoot at what we see and hit what we don\u2019t see, producing \u2018programmed’ babies for appearance or performance, but with leukemia or worse problems.” There are control mechanisms to guard against this, such as ethics committees and, in Brazil, the Biosafety Law, which prohibits genetic engineering on human embryos. The United Kingdom also made a decision: on February 1, 2016, it authorized gene editing in human cells for purposes of scientific research.<\/p>\n

Projects<\/strong>
\n1.<\/strong> Generation of a knockout mouse for the orphan nuclear receptor Coup-TFII: Investigation of the molecular mechanisms underlying the atrial-specific expression of the promoter of the SMyHC3 gene (n\u00ba 2015\/10166-9<\/a>); Grant Mechanism<\/strong>\u00a0Scholarships in Brazil \u2013 Postdoctoral; Principal Investigator<\/strong>\u00a0Jos\u00e9 Xavier Neto (CNPEM); Grant Recipient<\/strong>\u00a0\u00c2ngela Saito (CNPEM); Investment<\/strong>\u00a0R$ 169,558.00.
\n2.<\/strong> Calcium signaling in trypanosomes (n\u00ba 2013\/50624-0<\/a>); Grant Mechanism<\/strong>\u00a0Research Grant \u2013 SPEC program; Principal Investigator<\/strong>\u00a0Roberto Docampo (Unicamp); Investment<\/strong>\u00a0R$ 1,955,088.00.
\n3.<\/strong> Gene editing by CRISPR-Cas9 in the correction of Duchenne Muscular Dystrophy in a canine model (GRMD) from induced pluripotent cells (n\u00ba 2015\/09575-1<\/a>); Grant Mechanism<\/strong>\u00a0Scholarships in Brazil \u2013 Postdoctoral; Principal Investigator<\/strong>\u00a0Carlos Eduardo Ambr\u00f3sio (USP); Grant Recipient<\/strong>\u00a0Natalia Juliana Nardelli Gon\u00e7alves (USP); Investment<\/strong>\u00a0R$ 169,558.00.
\n4.<\/strong> Characterization of microbiota-mediated anti-dengue mechanisms of action in wild Aedes aegypti populations (n\u00ba 2013\/11343-6<\/a>) Grant Mechanism<\/strong>\u00a0Research Grant – Young Investigators; Principal Investigator<\/strong>\u00a0Jayme Augusto de Souza-Neto (Unesp); Investment<\/strong>\u00a0R$ 2,209,619.50.
\n5.<\/strong> Generation of FBN1 gene mutations in Induced Pluripotent Stem Cells (iPSCs) using the CRISPR-Cas9 system (n\u00ba 2015\/01339-7<\/a>); Grant Mechanism<\/strong>\u00a0Scholarships in Brazil \u2013 Master\u2019s\/Capes; Principal Investigator<\/strong>\u00a0Lygia da Veiga Pereira Carramaschi (USP); Grant Recipient<\/strong>\u00a0Juliana Borsoi Sant\u2019Ana (USP); Investment<\/strong>\u00a0R$ 38,823.80.
\n6.<\/strong> A genomic analysis to comprehend the etiological genetic mechanisms of cleft lip and palate in the Brazilian population (n\u00ba 2011\/23416-2<\/a>); Grant Mechanism<\/strong>\u00a0Scholarships in Brazil \u2013 Doctoral; Principal Investigator<\/strong>\u00a0Maria Rita dos Santos e Passos Bueno (USP); Grant Recipient<\/strong>\u00a0Luciano Abreu Brito (USP); Investment<\/strong>\u00a0R$ 146,770.80.
\n7.<\/strong> Structural biology of protein processors of nucleic acids in bacteria with high biomedical relevance (n\u00ba 2011\/50963-4<\/a>); Grant Mechanism<\/strong>\u00a0Regular Research Grant; Principal Investigator <\/strong>Martin Rodrigo Alejandro W\u00fcrtele Alfonso (Unifesp); Investment<\/strong>\u00a0R$ 496,766.00.<\/p>\n

Scientific articles<\/em>
\nJIANG, F. et al<\/em>. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage<\/a>. Science<\/strong>. Online. January 14, 2016.
\nLANDER, N. et al.<\/em> CRISPR-Cas9-induced disruption of paraflagellar rod protein 1 and 2 genes in Trypanosoma cruzi reveals their role in flagellar attachment<\/a>. mBio<\/strong>. V. 6, No. 4, pp. e01012-15. July-August, 2015
\nGALLO, G. et al<\/em>. Structural basis for dimer formation of the CRISPR-associated protein Csm2 of Thermotoga maritima<\/a>. FEBS Journal<\/strong>. Online. December 10, 2015.
\nGALLO, G. et al<\/em>. Purification, crystallization, crystallographic analysis and phasing of the CRISPR-associated protein Csm2 from Thermotoga maritima.<\/a> Structural Biology Communications<\/strong>. F71, pp. 1223-27. October 2015.<\/p>\n","protected":false},"excerpt":{"rendered":"A system to edit DNA raises ethical concerns","protected":false},"author":3,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_exactmetrics_skip_tracking":false,"_exactmetrics_sitenote_active":false,"_exactmetrics_sitenote_note":"","_exactmetrics_sitenote_category":0,"footnotes":""},"categories":[159],"tags":[209,210,230,237,247,250],"coauthors":[1601],"class_list":["post-219809","post","type-post","status-publish","format-standard","hentry","category-science","tag-biology","tag-cellular-biology","tag-ethics","tag-genetics","tag-medicine","tag-neuroscience"],"acf":[],"_links":{"self":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts\/219809","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/comments?post=219809"}],"version-history":[{"count":0,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts\/219809\/revisions"}],"wp:attachment":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/media?parent=219809"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/categories?post=219809"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/tags?post=219809"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/coauthors?post=219809"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}