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3.
Sheng Wu Gong Cheng Xue Bao ; 35(12): 2295-2307, 2019 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-31880137

RESUMO

In the 1960s, scientists first raised the idea of curing genetic diseases using gene therapy. This new conceptual strategy aimed to achieve a much longer therapeutic effect by introducing exogenous genetic materials into the patients. After more than five decades of ups and downs, gene therapy has been brought into a new era by those milestone breakthroughs in the 21st century. Here we reviewed and summarized the history and breakthroughs of gene therapy, including some critical clinical trials, approved drugs, and emerging gene editing techniques. We believe that with their unique advantages over traditional therapies, more gene therapies will become practical approaches to genetic diseases and benefit the entire human race.


Assuntos
Sistemas CRISPR-Cas , Terapia Genética , Edição de Genes , Humanos
4.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(11): 1381-1386, 2019 Nov 30.
Artigo em Chinês | MEDLINE | ID: mdl-31852637

RESUMO

The CRISPR/Cas9 technology has developed rapidly in recent years with fast, simple and accurate editing functions to allow gene knockout, knock in, activation and interference. It has become a powerful genetic screening tool and been widely used in various models including cell lines, mice and zebrafish. The application of CRISPR system in constructing genome library for high-throughput screening is the main strategy for target gene research of diseases, especially neoplasms. Here we summarize the rationales and recent development of CRISPR/Cas9 library screening technology, the strategies for improving the off-target effects, the basic workflow of library screening and the application of this technology in tumor research.


Assuntos
Sistemas CRISPR-Cas , Animais , Detecção Precoce de Câncer , Técnicas de Inativação de Genes , Biblioteca Gênica , Camundongos
5.
J Law Med ; 26(4): 866-873, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31682364

RESUMO

The discovery of CRISPR systems has been one of the most exciting developments in the field of genetics in the past decade. The recent proliferation of intellectual property rights for CRISPR genome editing technology carries the risk of potential bottlenecks for further basic biological research and development of commercial products. To make CRISPR-based technology widely available, the reliance by the industry on efficient methods of collective management of intellectual property rights through patent pools seems inevitable. A packager of patent pools could be used as a mechanism to facilitate transactions in the market for technology and allow interested parties to deal with a single entity. This article argues that, while a global licensing platform could be effectively achieved in non-therapeutic applications of genome editing, it is questionable whether patent pooling would provide the ideal balance of incentive and reward for CRISPR genome editing technologies for human gene therapy.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Sistemas CRISPR-Cas , Humanos
7.
Biochemistry (Mosc) ; 84(9): 1074-1084, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31693467

RESUMO

The CRISPR/Cas technology has a great potential in the treatment of many hereditary diseases. One of the prospective models for the CRISPR/Cas-mediated therapy is spinal muscular atrophy (SMA), a disease caused by deletion of the SMN1 gene that encodes the SMN protein required for the survival of motor neurons. SMA patients' genomes contain either single or several copies of SMN2 gene, which is a paralog of SMN1. Exon 7 of SMN2 has the single-nucleotide substitution c.840C>T leading to the defective splicing and decrease in the amounts of the full-length SMN. The objective of this study was to create and test gene-editing systems for correction of the single-nucleotide substitution c.840C>T in exon 7 of the SMN2 gene in fibroblasts, induced pluripotent stem cells, and motor neuron progenitors derived from a SMA patient. For this purpose, we used plasmid vectors expressing CRISPR/Cas9 and CRISPR/Cpf1, plasmid donor, and 90-nt single-stranded oligonucleotide templates that were delivered to the target cells by electroporation. Although sgRNA_T2 and sgRNA_T3 guiding RNAs were more efficient than sgRNA_T1 in fibroblasts (p < 0.05), no significant differences in the editing efficiency of sgRNA_T1, sgRNA_T2, and sgRNA_T3 was observed in patient-specific induced pluripotent stem cells and motor neuron progenitors. The highest editing efficiency in induced pluripotent stem cells and motor neuron progenitors was demonstrated by the sgRNA_T1 and 90-nt single-stranded oligonucleotide donors.


Assuntos
Sistemas CRISPR-Cas , Éxons/genética , Polimorfismo de Nucleotídeo Único/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Humanos , Proteína 2 de Sobrevivência do Neurônio Motor/genética
8.
Rev. bioét. derecho ; (47): 17-31, nov. 2019.
Artigo em Espanhol | IBECS | ID: ibc-184863

RESUMO

La tecnología de la edición genética por CRISPR ha revolucionado tanto la investigación en biotecnología como en biomedicina. Esta técnica tan poderosa y versátil permite editar los genes de cualquier especie a la carta. A pesar de su potencia y efectividad, quedan muchas cuestiones para resolver y controlar su resultado final, particularmente en las aplicaciones sobre el genoma de los seres humanos. En este artículo se plantean tanto los puntos fuertes y puntos débiles de la técnica, como otras cuestiones abiertas sobre la edición génica, sobre si debe dirigirse a la terapia o a la mejora, si la modificación debe constreñirse a células somáticas o también a editar a embriones, modificando el genoma de los seres humanos del futuro


La tecnologia de l'edició gènica mitjançant CRISPR ha revolucionat tant la recerca en biotecnologia com en biomedicina. Aquesta tècnica tan poderosa i versàtil permet editar els gens de qualsevol espècie a la carta. Malgrat la seva potència i efectivitat, queden moltes qüestions per resoldre i controlar el seu resultat final, particularment en les aplicacions sobre el genoma dels éssers humans. En aquest article es plantegen els punts forts i els punts febles de la tècnica, així com altres qüestions obertes sobre l'edició gènica, si ha de dirigir-se a la teràpia o a la millora, si la modificació ha de constrènyer-se a cèl•lules somàtiques o també es poden editar en embrions, modificant el genoma dels éssers humans del futur


Gene using CRISPR has completely revolutionized the research in biotechnology and biomedicine. This powerful and versatile technique enables the precise edition of genes from any organism. Even though the technique is so effective and amenable, many questions remain to be solved before the final genetic outcome can be fully controlled, particularly in its uses on the human genome. In this article, I discuss the current strengths and weaknesses of the technique. I also pose other open questions on gene editing, such as whether it should be used for either therapy or genetic enhancement, and whether it should be used only on somatic cells or also for embryo gene editing, the latter resulting in the modification of future human beings


Assuntos
Humanos , Edição de Genes/tendências , Sistemas CRISPR-Cas , Terapia Genética/legislação & jurisprudência , Tecnologia de Impulso Genético/legislação & jurisprudência , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células Germinativas
9.
Rev. bioét. derecho ; (47): 33-41, nov. 2019.
Artigo em Espanhol | IBECS | ID: ibc-184864

RESUMO

La mejora genética en la especie humana ha suscitado desde siempre un amplio debate por lo que respecta a los aspectos éticos asociados a ella. Dicho debate había quedado circunscrito al ámbito meramente especulativo hasta la aparición de una nueva tecnología de edición genómica (CRISPR/Cas9) la cual lo ha reabierto al vislumbrarse una aplicabilidad a medio plazo que nos obliga a retomarlo y a reflexionar sobre la conveniencia o no de iniciar un camino excepcional en la historia de la evolución humana. En este artículo se detallan algunos de los argumentos esgrimidos a favor y en contra referentes a esta tecnología


La millora genètica en l'espècie humana ha suscitat des de sempre un ampli debat pel que respecta als aspectes ètics associats a ella. Aquest debat havia quedat circumscrit a l'àmbit merament especulatiu fins l'aparició d'una nova tecnologia d'edició genòmica (CRISPR/Cas9) la qual l'ha reobert en albirar-se una aplicabilitat a mitjà termini que ens obliga a reflexionar sobre la conveniència o no d'iniciar un camí excepcional en la història de l'evolució humana. En aquest article es detallen alguns dels arguments esgrimits a favor i en contra referents a aquesta tecnologia


Human genetic enhancement has always risen a wide debate regarding the ethical aspects associated with it. This debate had remained into a merely speculative realm until the appearance of a new genome editing technology (CRISPR / Cas9) which has reopened it when a mid-term applicability can be envisaged. This new situation forces us to resume it and ponder the convenience or not of initiating an exceptional path in the history of human evolution. This article details some of the arguments put forward in favour and against this technology


Assuntos
Humanos , Genética Humana/ética , Sistemas CRISPR-Cas , Proteína 9 Associada à CRISPR , Edição de Genes , Melhoramento Genético/ética
10.
Rev. bioét. derecho ; (47): 77-92, nov. 2019.
Artigo em Espanhol | IBECS | ID: ibc-184867

RESUMO

La reciente aceleración de las investigaciones en ingeniería genética ha situado a Europa en una aporía de difícil solución. Pues bien, no queremos renunciar a determinados valores, aun cuando suponga no poder seguir el ritmo de China o de Estados Unidos; pero si no lo hacemos, corremos el riesgo de ser objeto de una forma de colonialismo muy diferente a la que hemos observado en los siglos precedentes


La recent acceleració de les recerques en enginyeria genètica ha situat a Europa en una aporia de difícil solució. D'una banda, es troba enmig de dos blocs contendents, la Xina i els Estats Units, en una reedició de la Guerra Freda, només que amb la biotecnologia com a mecanisme de dominació; d'un altre, Europa es troba institucionalment fraccionada en tres entitats superposades, el Consell d'Europa, la Unió Europea i l'Oficina Europea de Patents, amb diferències notables quant a legitimitat democràtica i a eficàcia normativa. Doncs bé, no volem renunciar a determinats valors, tot i que suposi no poder seguir el ritme de la Xina o dels Estats Units; però si no ho fem, correm el risc de ser objecte d'una forma de colonialisme molt diferent a la que hem observat en els segles precedents


The recent acceleration of research in genetic engineering has placed Europe in an aporia of difficult solution. On the one hand, it is in the midst of two contending blocs, China and the United States, which in their struggle for world primacy have reissued the Cold War, only with biotechnology as a mechanism of domination. On the other, Europe is institutionally divided into three overlapping entities, the Council of Europe, the European Union and the European Patent Office, with notable differences in terms of democratic legitimacy and normative effectiveness. So, we do not want to give up certain values, even if it means not being able to keep pace with China or the United States; but if we do not, we run the risk of being the object of a form of colonialism very different from that we have observed in previous centuries


Assuntos
Humanos , Biotecnologia/ética , Patentes como Assunto/ética , Edição de Genes , Sistemas CRISPR-Cas , Bioética , Europa (Continente) , Patentes como Assunto/legislação & jurisprudência
11.
Rev. bioét. derecho ; (47): 141-157, nov. 2019.
Artigo em Espanhol | IBECS | ID: ibc-184871

RESUMO

La clonación y transgénesis animal son prácticas biotecnológicas en auge, para nada exentas de problemáticas éticas en lo que respecta al uso que hacen de los animales no humanos. En este artículo se examinan los diversos ámbitos de aplicación de la clonación animal (médico-farmacéutico, industria alimentaria, recreación de especies extintas, clonación de animales de compañía e industria artística y deportiva) y se revisan los principales argumentos éticos que cuestionan la clonación y la transgénesis animal desde una perspectiva antiespecista. Esta perspectiva sostiene que los animales no humanos son merecedores de consideración moral como sujetos de vidas significativas, y no únicamente como medios para la realización de fines humanos


La clonació i transgènesi animal són pràctiques biotecnològiques creixents i no exemptes de problemàtiques ètiques pel que fa a l'ús que fan dels animals no humans. En aquest article s'examinen els diversos àmbits d'aplicació de la clonació animal (metge-farmacèutic, indústria alimentària, recreació d'espècies extintes, clonació d'animals de companyia i indústria artística i esportiva) i es revisen els principals arguments ètics que qüestionen la clonació i la transgènesi animal des d'una perspectiva antiespecista. Aquesta perspectiva sosté que els animals no humans són mereixedors de consideració moral com a subjectes de vides significatives, i no únicament com a mitjans per a la realització de finalitats humanes


Animal cloning and animal transgenesis are growing biotechnological practices, not at all exempt from ethical problems regarding the use they make of non-human animals. This article examines the different areas of application of animal cloning (medical-pharmaceutical, food industry, recreating of extinct species, cloning of companion animals and the art and sport industries) and reviews the main ethical arguments that question cloning and animal transgenesis from an antispeciesist perspective. This perspective argues that non-human animals deserve moral consideration as subjects of meaningful lives, and not only as means for the achievement of human ends


Assuntos
Animais , Clonagem de Organismos/ética , Clonagem de Organismos/veterinária , Biotecnologia/ética , Direitos dos Animais/legislação & jurisprudência , Sistemas CRISPR-Cas , Proteína 9 Associada à CRISPR , Pesquisa Biomédica/ética , Animais de Laboratório
12.
Yi Chuan ; 41(10): 939-949, 2019 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-31624056

RESUMO

Mutations in Hypoxanthine-guanine Phosphoribosyltransferase1 (HPRT1) gene can lead to metabolic disorder of hypoxanthine and guanine metabolism, and other severe symptoms such as hypophrenia, gout, and kidney stones, called the Lesch-Nyhan disease (LND). Although the mutations are widely distributed throughout the HPRT1 gene, there are some isolated hot spots. In this study, we aim to introduce two previously reported hot spots, c.508 C>T and c.151 C>T, which could lead to premature translational termination in HPRT1 gene. Through CRISPR/Cas9 mediated homology-directed repair (HDR) by using single-stranded oligo-deoxyribonucleotides (ssODN) as donor template, we obtained cell clones containing these two mutations in HEK293T or HeLa cells. Targeted mutation of c.508 C>T and c.151 C>T reached to 16.3% and 10%, respectively. We further detect HPRT1 protein levels with Western blot and enzyme activity with 6-TG in 5 different cell clones. HPRT1 protein and its enzymatic activity both was hardly detected in homozygous mutant cells, while reduced HPRT1 protein expression and enzymatic activity was detected in heterozygous mutant cells. Our study will be beneficial to those who working on generation of cell or animal models of HRPT1 mutations, and provides a basis for further investigations on the genetic mechanism of Lesch-Nyhan disease.


Assuntos
Sistemas CRISPR-Cas , Hipoxantina Fosforribosiltransferase/genética , Mutação Puntual , Células HEK293 , Células HeLa , Humanos , Síndrome de Lesch-Nyhan/genética
13.
Medicina (B Aires) ; 79 Suppl 3: 77-81, 2019.
Artigo em Espanhol | MEDLINE | ID: mdl-31603849

RESUMO

Duchenne muscular dystrophy is a genetically determined disease, linked to the X chromosome, c haracterized clinically by producing progressive muscle weakness, with an incidence of 1 per 3500-6000 males born. It is caused by the mutation of the DMD gene, which encodes dystrophin, a sub-sarcolemmal protein essential for structural muscle stability. The genetic defects in the DMD gene are divided into: deletions (65%) duplications (5.10%) and point mutations (10-15%). At present there is no curative treatment, the only drug that has been shown to modify the natural history of the disease (independently of the genetic mutation) are corticosteroids, currently indicated in early stages of the disease. In relation to clinical trials, in the last ten years, has experienced great advances in the field of therapeutic options, divided into two major therapeutic targets: 1) the area of gene therapies and 2) trying to reverse or block the pathophysiological processes of the disease, such as inflammation, fibrosis, muscle regeneration, etc. It is likely that an effective treatment for Duchenne muscular dystrophy requires combinations of therapies that address both the primary defect and its secondary pathophysiological consequences.


Assuntos
Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Animais , Sistemas CRISPR-Cas , Distrofina/genética , Genótipo , Humanos , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/genética , Fenótipo
14.
Zhonghua Gan Zang Bing Za Zhi ; 27(8): 610-614, 2019 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-31594078

RESUMO

Objective: To determine whether single-stranded guided RNA (gRNA) and Cas9 protein exist in the exosome secreted by cells transfected with CRISPR/Cas9 expression plasmid. Furthermore, to explore whether CRISPR/Cas9 system can edit target genes of peripheral cells through the intercellular transmission of exosomes. Methods: (1) The CRISPR/Cas9 expression plasmid was transfected into HuH7 cells. The supernatant was collected and the exosomes were concentrated and purified by differential centrifugation. The morphology and particle size of exosomes were determined by electronic microscopy and Malvern laser scatter granulometry. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the levels of gRNA and Cas9 protein. (2) HuH7 cells transfected with pBB4.5 1.2×HBV and HBV specific CRISPR/Cas9 expression plasmids were co-cultured. After 2 days, HBV DNA was extracted and sequenced by PCR. Results: There were not only full-length gRNA and Cas9 protein in the exosomes of Huh7 cells transfected with CRISPR / Cas9 expression plasmid. In addition, gene-editing functions were delivered between the cells to achieve the destruction of HBV genome of surrounding cells. Conclusion: The CRISPR-Cas9 gene-editing system has the potential to deliver exosomes between cells via carrying functional gRNA and Cas9 proteins. This phenomenon hints that we are in the process of using the CRISPR/Cas9 system for gene therapy. Therefore, it is necessary to consider the potential effects of exosomes-carried gRNA and Cas9 proteins on the surrounding cells of the distal tubules.


Assuntos
Sistemas CRISPR-Cas , Exossomos , Vírus da Hepatite B/genética , Linhagem Celular , Genoma Viral , Humanos , Transfecção
15.
Cytogenet Genome Res ; 159(1): 48-53, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31610539

RESUMO

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.


Assuntos
Amaryllidaceae/genética , Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Replicação do DNA/genética , Zea mays/genética , Cromatina/metabolismo , Cromossomos/genética , DNA de Plantas/genética , Endonucleases/genética , Hibridização in Situ Fluorescente/métodos , RNA Guia/genética , Telômero/genética
16.
Microb Cell Fact ; 18(1): 162, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581942

RESUMO

BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.


Assuntos
Edição de Genes/métodos , Genoma Bacteriano , Zymomonas/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Francisella/enzimologia , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Zymomonas/metabolismo
18.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(5): 1682-1690, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31607332

RESUMO

OBJECTIVE: To knockout ADRB2 gene rapidly and efficiently in human primary T cells by using CRISPR/Cas9 technology and multiple sgRNAs strategy. METHODS: Six paired-sgRNAs, which were designed to target the 5' constitutive coding exons of ADRB2 gene, were cloned into pGL3-U6-sgRNA-PGK-Puro vector separately. The expre-ssion vectors containing the single sgRNAs were constructed and transiently co-transfected into HEK-293T cell line with Cas9 expression vector. The sgRNA-mediated cleavage efficiency was tested by T7EN I digestion assay. Concatenating four highly efficient paired sgRNAs were cloned into pGL3-U6-sgRNA-ccdB-EF1α-Puro expression vector. The reco-mbinant plasmid allows the cells to express 4 sgRNAs, which target different sites on the ADRB2 genomic locus. The cleavage efficiency and mutation model were tested by T7EN I digest assay and T-A cloning technique. Multiple sgRNAs plasmid and Cas9 plasmid was transiently transferred into human primary T cells by electroporation. Flow cytometry (FCM) was used to detect the knockout efficiency of ß2 adrenergic receptor (ß2-AR). RESULTS: The results of T7EN I digestion and TA cloning sequencing showed that the multiple sgRNAs strategy could obtain more abundant mutation types and higher gene editing efficiency than single sgRNA. In addition to the deletion and insertion of bases, large fragment DNA deletions and inversions could be observed. All of the random 10 TA clones for detection were genetically modified, thus the mutation efficiency was as high as 100%. FCM assay showed that 43.09% of the cells in the control T cells were ß2-AR positive, but the proportion of ß2-AR positive cells in the multiple sgRNAs electrotransformed T cells decreased to 25.61%. CONCLUSION: A method, which is simple and operable, for knocking out ß2-AR in human primary T cells has been established preliminarily. The results are helpful for the further study of the role of ß2-AR in human T cells.


Assuntos
Sistemas CRISPR-Cas , Receptores Adrenérgicos beta 2/genética , Edição de Genes , Técnicas de Inativação de Genes , Humanos , RNA Guia , Linfócitos T
19.
BMC Bioinformatics ; 20(1): 517, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31651233

RESUMO

BACKGROUND: One of the main challenges for the CRISPR-Cas9 system is selecting optimal single-guide RNAs (sgRNAs). Recently, deep learning has enhanced sgRNA prediction in eukaryotes. However, the prokaryotic chromatin structure is different from eukaryotes, so models trained on eukaryotes may not apply to prokaryotes. RESULTS: We designed and implemented a convolutional neural network to predict sgRNA activity in Escherichia coli. The network was trained and tested on the recently-released sgRNA activity dataset. Our convolutional neural network achieved excellent performance, yielding average Spearman correlation coefficients of 0.5817, 0.7105, and 0.3602, respectively for Cas9, eSpCas9 and Cas9 with a recA coding region deletion. We confirmed that the sgRNA prediction models trained on prokaryotes do not apply to eukaryotes and vice versa. We adopted perturbation-based approaches to analyze distinct biological patterns between prokaryotic and eukaryotic editing. Then, we improved the predictive performance of the prokaryotic Cas9 system by transfer learning. Finally, we determined that potential off-target scores accumulated on a genome-wide scale affect on-target activity, which could slightly improve on-target predictive performance. CONCLUSIONS: We developed convolutional neural networks to predict sgRNA activity for wild type and mutant Cas9 in prokaryotes. Our results show that the prediction accuracy of our method is improved over state-of-the-art models.


Assuntos
Bactérias/genética , Aprendizado Profundo , Edição de Genes , RNA Guia/genética , Sistemas CRISPR-Cas , Deleção de Sequência
20.
Nature ; 574(7779): 549-552, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645729

RESUMO

About half of all bacteria carry genes for CRISPR-Cas adaptive immune systems1, which provide immunological memory by inserting short DNA sequences from phage and other parasitic DNA elements into CRISPR loci on the host genome2. Whereas CRISPR loci evolve rapidly in natural environments3,4, bacterial species typically evolve phage resistance by the mutation or loss of phage receptors under laboratory conditions5,6. Here we report how this discrepancy may in part be explained by differences in the biotic complexity of in vitro and natural environments7,8. Specifically, by using the opportunistic pathogen Pseudomonas aeruginosa and its phage DMS3vir, we show that coexistence with other human pathogens amplifies the fitness trade-offs associated with the mutation of phage receptors, and therefore tips the balance in favour of the evolution of CRISPR-based resistance. We also demonstrate that this has important knock-on effects for the virulence of P. aeruginosa, which became attenuated only if the bacteria evolved surface-based resistance. Our data reveal that the biotic complexity of microbial communities in natural environments is an important driver of the evolution of CRISPR-Cas adaptive immunity, with key implications for bacterial fitness and virulence.


Assuntos
Bacteriófagos/genética , Bacteriófagos/imunologia , Biodiversidade , Sistemas CRISPR-Cas/genética , Evolução Molecular , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/virologia , Bacteriófagos/patogenicidade , Sistemas CRISPR-Cas/imunologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Receptores Virais/metabolismo
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