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GENETICS

A tool to edit DNA

A system copied from bacteria, CRISPR-Cas9 may catalyze discoveries in biology and medicine, but it raises ethical concerns

038-041_Crispr_240-AA system that allows bacteria to recognize and combat viral invasions promises significant innovation in genetics.  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, 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.

“It’s a great equalizer, until we manage to do it,” jokes Dr. José 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.  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). Such procedures are within reach of most genetics laboratories, and this gives autonomy to researchers.

It all began in Xavier Neto’s laboratory with Ângela Saito, who at the time was studying for her doctorate at the University of Campinas (Unicamp) under the supervision of biologist Jörg 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’s 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 São 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.

Albinism mutation: to produce a homogeneous effect, injection in the early embryonic stage is necessary

Carolina Clemente/LNBio Albinism mutation: to produce a homogeneous effect, injection in the early embryonic stage is necessaryCarolina Clemente/LNBio

Fighting diseases
The 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, which causes Chagas disease, and who can make a contribution to the development of alternative therapies.  Lander is currently doing a postdoctoral internship at Unicamp’s School of Medical Sciences (FCM – Unicamp) in collaboration with biochemists Aníbal 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. She broke down the genes linked to the parasite’s flagellum—a tail-like structure that allows it to move. “It’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,” 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. “Calcium levels change a lot when the parasite infects the host,” she says. “If we can move these proteins, which differ between the parasite and the vertebrate host, it could be the pathway to an alternative therapy.”

The fight against dengue transmission is the goal of biologist Jayme de Souza Neto, at the Botucatu campus of São Paulo State University (Unesp). In the Aedes aegypti mosquito, he compared the transcribed RNAs of infected mosquitoes resistant to the virus in wild populations in Botucatu, São Paulo State, and Neópolis, in the northeastern state of Sergipe, and identified genes that may be linked to resistance. “We are beginning to mutate the genes of mosquitoes,” 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 Pesquisa FAPESP issue nº 230). Souza Neto spent three months in the laboratory of Julien Pelletier, who was in Botucatu for four months. “In April he must return to start the injections into the mosquito embryos,” he says.

Biologist Natália Gonçalves 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 Pesquisa FAPESP issue nº 237).  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 Ambrósio, Faculty of Animal Science and Food Engineering, University of São 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,” 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.

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 Pesquisa FAPESP issue nº 209), to study cleft lip and palate malformations. “We 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,” 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.

Lygia da Veiga Pereira, a geneticist at IB-USP, is also beginning to directly alter human cells. Her master’s student, Juliana Sant’Ana, 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 Pesquisa FAPESP issue nº 153). “I want to produce heart cells and bone cells with the mutation,” 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. “Science will begin when we can compare these cells.”

Crispr_bebesFundoCAETOProtein structure
Bueno 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.

German-Chilean biochemist Martin Würtele, at the Federal University of São 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’s Max Planck Institute for Infection Biology in 2012. “About 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,” he says. “But 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.” 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. “The Csm2 protein is completely different from those described in other complexes,” says Würtele. 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. “There is a great interest in using bacteriophages as potential substitutes for antibiotics.”

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.  “The harm in overriding evolution can be much greater than the benefits,” he warns. “The possibility of off-target effects can make us shoot at what we see and hit what we don’t see, producing ‘programmed’ 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.

Projects
1. 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 (nº 2015/10166-9); Grant Mechanism Scholarships in Brazil – Postdoctoral; Principal Investigator José Xavier Neto (CNPEM); Grant Recipient Ângela Saito (CNPEM); Investment R$ 169,558.00.
2. Calcium signaling in trypanosomes (nº 2013/50624-0); Grant Mechanism Research Grant – SPEC program; Principal Investigator Roberto Docampo (Unicamp); Investment R$ 1,955,088.00.
3. Gene editing by CRISPR-Cas9 in the correction of Duchenne Muscular Dystrophy in a canine model (GRMD) from induced pluripotent cells (nº 2015/09575-1); Grant Mechanism Scholarships in Brazil – Postdoctoral; Principal Investigator Carlos Eduardo Ambrósio (USP); Grant Recipient Natalia Juliana Nardelli Gonçalves (USP); Investment R$ 169,558.00.
4. Characterization of microbiota-mediated anti-dengue mechanisms of action in wild Aedes aegypti populations (nº 2013/11343-6) Grant Mechanism Research Grant – Young Investigators; Principal Investigator Jayme Augusto de Souza-Neto (Unesp); Investment R$ 2,209,619.50.
5. Generation of FBN1 gene mutations in Induced Pluripotent Stem Cells (iPSCs) using the CRISPR-Cas9 system (nº 2015/01339-7); Grant Mechanism Scholarships in Brazil – Master’s/Capes; Principal Investigator Lygia da Veiga Pereira Carramaschi (USP); Grant Recipient Juliana Borsoi Sant’Ana (USP); Investment R$ 38,823.80.
6. A genomic analysis to comprehend the etiological genetic mechanisms of cleft lip and palate in the Brazilian population (nº 2011/23416-2); Grant Mechanism Scholarships in Brazil – Doctoral; Principal Investigator Maria Rita dos Santos e Passos Bueno (USP); Grant Recipient Luciano Abreu Brito (USP); Investment R$ 146,770.80.
7. Structural biology of protein processors of nucleic acids in bacteria with high biomedical relevance (nº 2011/50963-4); Grant Mechanism Regular Research Grant; Principal Investigator Martin Rodrigo Alejandro Würtele Alfonso (Unifesp); Investment R$ 496,766.00.

Scientific articles
JIANG, F. et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science. Online. January 14, 2016.
LANDER, N. et al. CRISPR-Cas9-induced disruption of paraflagellar rod protein 1 and 2 genes in Trypanosoma cruzi reveals their role in flagellar attachment. mBio. V. 6, No. 4, pp. e01012-15. July-August, 2015
GALLO, G. et al. Structural basis for dimer formation of the CRISPR-associated protein Csm2 of Thermotoga maritima. FEBS Journal. Online. December 10, 2015.
GALLO, G. et al. Purification, crystallization, crystallographic analysis and phasing of the CRISPR-associated protein Csm2 from Thermotoga maritima. Structural Biology Communications. F71, pp. 1223-27. October 2015.