Identification of early flowering mutant gene in Phalaenopsis amabilis (L) Blume for sgRNA construction in CRISPR/Cas9 genome editing system / Identificação do gene mutante de floração precoce em Phalaenopsis amabilis (L) Blume para construção de sgRNA no sistema de edição de genoma CRISPR/Cas9

Phalaenopsis amabilis (L.) Blume commonly called Moth Orchid (Orchidaceae) is a natural orchid species designated as the National Flower of Indonesia for its beautiful flower shape and long-lasting flowering period. Basically, P. amabilis has a long vegetative phase that cause late flowering, about 2 to 3 years for flowering, hence a method to shorten vegetative period is desired. The latest technological approach that can be used to accelerate flowering of P. amabilis is the CRISPR/Cas9 genome editing method to inactivate the GAI (Gibberellic Acid Insensitive) gene as a mutant gene that can accelerate the regulation of FLOWERING TIME (FT) genes flowering biosynthesis pathway. The approach that needs to be taken is to silence the GAI gene with a knockout system which begins with identifying and characterizing the GAI target gene in the P. amabilis which will be used as a single guide RNA. CRISPR/Cas9 mediated knockout efficiency is highly dependent on the properties of the sgRNA used. SgRNA consists of a target sequence, determining its specificity performance. We executed phylogenetic clustering for the PaGAI protein with closely related orchid species such as Dendrobium capra, Dendrobium cultivars and Cymbidium sinensis. SWISS-Model as tool webserver for protein structure homology modeling. Results show that P. amabilis has a specific domain with the occurrence of point mutations in the two conservative domains. Therefore, a single guide RNA reconstruction needs to be implemented.

Phalaenopsis amabilis (L.) Blume, comumente chamada de orquídea mariposa (Orchidaceae), é uma espécie natural de orquídea designada como a flor nacional da Indonésia por seu belo formato de flor e período de floração duradouro. Basicamente, P. amabilis tem uma longa fase vegetativa que causa floração tardia, cerca de 2 a 3 anos para a floração, portanto, um método para encurtar o período vegetativo é desejado. A mais recente abordagem tecnológica que pode ser utilizada para acelerar a floração de P. amabilis é o método de edição do genoma CRISPR/Cas9 para inativar o GAI (Gibberellic Acid Insensitive) que pode ser usado como um gene mutante para acelerar a regulação da floração dos genes FLOWERING TIME (FT), via de biossíntese. Para isto, a melhor abordagem é silenciar o GAI gene com um sistema knockout que deve ser iniciado com a identificação e caracterização do gene alvo GAI no P. amabilis, e que, posteriormente, será utilizado como um único RNA guia. A eficiência de nocaute mediada por CRISPR/Cas9 é altamente dependente das propriedades do sgRNA usados. O SgRNA consiste em uma sequência alvo, determinando seu desempenho de especificidade. Executamos agrupamento filogenético para a proteína PaGAI com espécies de orquídeas intimamente relacionadas, como Dendrobium capra, Dendrobium cultivars e Cymbidium sinensis. SWISS-Model foi utilizado como ferramenta webserver para modelagem de homologia de estruturas de proteínas. Os resultados mostram que P. amabilis possui um domínio específico com ocorrência de mutações pontuais nos dois domínios conservativos. Portanto, uma única reconstrução de RNA guia precisa ser implementada.

Towards Understanding Long COVID: SARS-CoV-2 Strikes the Host Cell Nucleus.

Despite what its name suggests, the effects of the COVID-19 pandemic causative agent "Severe Acute Respiratory Syndrome Coronavirus-2" (SARS-CoV-2) were not always confined, neither temporarily (being long-term rather than acute, referred to as Long COVID) nor spatially (affecting several body systems). Moreover, the in-depth study of this ss(+) RNA virus is defying the established scheme according to which it just had a lytic cycle taking place confined to cell membranes and the cytoplasm, leaving the nucleus basically "untouched". Cumulative evidence shows that SARS-CoV-2 components disturb the transport of certain proteins through the nuclear pores. Some SARS-CoV-2 structural proteins such as Spike (S) and Nucleocapsid (N), most non-structural proteins (remarkably, Nsp1 and Nsp3), as well as some accessory proteins (ORF3d, ORF6, ORF9a) can reach the nucleoplasm either due to their nuclear localization signals (NLS) or taking a shuttle with other proteins. A percentage of SARS-CoV-2 RNA can also reach the nucleoplasm. Remarkably, controversy has recently been raised by proving that-at least under certain conditions-, SARS-CoV-2 sequences can be retrotranscribed and inserted as DNA in the host genome, giving rise to chimeric genes. In turn, the expression of viral-host chimeric proteins could potentially create neo-antigens, activate autoimmunity and promote a chronic pro-inflammatory state.

Inativação do receptor PD1 por CRISPR/Cas9 para melhoramento da imunoterapia com células CAR-T

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) é uma técnica de edição genética capaz de editar regiões do DNA de maneira eficaz e relativamente fácil, sendo considerada uma revolução na engenharia genômica. Suas aplicações na área da saúde são diversas. O nocaute pela técnica de CRISPR pode ser aplicado no melhoramento da imunoterapia, em especial de células CAR-T. Considerando os tumores sólidos, estas células são menos eficazes, devido à expressão de moléculas de checkpoint imunológico em células cancerígenas, que ao se ligarem a receptores de superfície em células T, regulam negativamente a resposta imunológica. Um desses receptores é o PD-1, codificado pelo gene PDCD1. Desta forma, este trabalho sugere a inativação do gene PDCD1, visando demonstrar parte do processo necessário para edição e aplicação da técnica CRISPR/Cas9 para o melhoramento da imunoterapia através da inativação do receptor PD-1 em células T.

In silico performance analysis of web tools for CRISPRa sgRNA design in human genes.

Angiogenic gene overexpression has been the main strategy in numerous vascular regenerative gene therapy projects. However, most have failed in clinical trials. CRISPRa technology enhances gene overexpression levels based on the identification of sgRNAs with maximum efficiency and safety. CRISPick and CHOP CHOP are the most widely used web tools for the prediction of sgRNAs. The objective of our study was to analyze the performance of both platforms for the sgRNA design to angiogenic genes (VEGFA, KDR, EPO, HIF-1A, HGF, FGF, PGF, FGF1) involving different human reference genomes (GRCH 37 and GRCH 38). The top 20 ranked sgRNAs proposed by the two tools were analyzed in different aspects. No significant differences were found on the DNA curvature associated with the sgRNA binding sites but the sgRNA predicted on-target efficiency was significantly greater when CRISPick was used. Moreover, the mean ranking variation was greater for the same platform in EPO, EGF, HIF-1A, PGF and HGF, whereas it did not reach statistical significance in KDR, FGF-1 and VEGFA. The rearrangement analysis of the ranking positions was also different between platforms. CRISPick proved to be more accurate in establishing the best sgRNAs in relation to a more complete genome, whereas CHOP CHOP showed a narrower classification reordering.

Generation of Functional Genetic Study Models in Zebrafish Using CRISPR-Cas9.

CRISPR-Cas9 is a method for genome editing that can be used efficiently for in vivo applications; the basic implementation of this method is used to generate genome site-directed sequence eliminations. Here we describe a protocol for genome editing using CRISPR-Cas9 in zebrafish (Danio rerio) one-cell embryos.

Sistema CRISPR/Cas: Edición genómica de precisión / CRISPR/Cas system: precision genome editing

La función original de los sistemas CRISPR/Cas es destruir el DNA de virus bacterianos. Este sistema ha evolucionado para identificar y cortar secuencias de diferentes DNA de virus de DNA evitando la infección. En la célula, está compuesto de genes Cas que producen nucleasas guiadas por RNA capaces de cortar el DNA. Si el RNA guía encuentra DNA de un virus con el que se puede emparejar, recluta a la nucleasa Cas9 que lo corta. Este sistema es utilizado in vitro para editar genes basándose en la producción de rupturas de doble cadena y su posterior reparación. Actualmente existen varias plataformas para el diseño de RNAs guía, aunque también es posible realizarlo de forma manual. Los componentes del sistema son entregados a la célula mediante un plásmido o una ribonucleoproteína. En esta revisión nos centraremos en la función original de CRISPR/Cas en procariotas y en cómo los investigadores la han modificado para proporcionar nuevas técnicas de edición de genomas. Discutiremos sobre las ventajas de esta nueva técnica, las formas en que podemos utilizarla y algunas de las limitaciones que aún encontramos en su aplicación

The original function of CRISPR/Cas systems is to destroy the DNA of bacterial viruses. This system has evolved to identify sequences of different DNA viruses and cut them in order to avoid infection. In the cell, the system is made up of Cas genes which produce RNA-guided nucleases capable of cutting DNA. If the guide RNA finds viral DNA with which it can pair up, it recruits the Cas9 nuclease to cut it. This system is used in vitro for gene edition, relying on the production of double-strand breaks and their subsequent repair. Currently, there are several platforms for the design of the guide RNA, and it is also possible to design it manually. The components of the system can be delivered to the cell through a plasmid or through a ribonucleoprotein. In this review we will focus on the original function of CRISPR/Cas in prokaryotes, and in how researchers have modified it in order to provide new genome editing techniques. We will discuss the advantages of this new technique, the ways in which it can be used, and some of the limitations found in its application