CRISPR-Cas, el editor de genes / CRISPR, gene editor

El término CRISPR, por su acrónimo en inglés refiere a Clustered Regularly Interspaced Short Palindromic Repeats, es decir, repeticiones palindrómicas cortas, agrupadas y regularmente esparcidas, por sus características en el genoma, pertenece naturalmente al sistema de defensa de bacterias y arqueas. Este ha sido adaptado biotecnológicamente para la edición del ADN de células eucariotas, incluso de células humanas. El sistema CRISPR-Cas para editar genes consta, en forma generalizada, de dos componentes: una proteína nucleasa (Cas) y un ARN guía (sgRNA). La simplicidad del complejo lo hace una herramienta molecular reprogramable capaz de ser dirigida y de editar cualquier sitio en un genoma conocido. Su principal foco son las terapias para enfermedades hereditarias monogénicas y para el cáncer. Sin embargo, además de editor de genes, la tecnología CRISPR se utiliza para edición epigenética, regulación de la expresión génica y método de diagnóstico molecular. Este artículo tiene por objetivo presentar una revisión de las aplicaciones de la herramienta molecular CRISPR-Cas, particularmente en el campo biomédico, posibles tratamientos y diagnósticos, y los avances en investigación clínica, utilizando terapia génica con CRISPR/Cas más relevantes hasta la fecha. (AU)

CRISPR are Clustered Regularly Interspaced Short Palindromic Repeats, which naturally belong to the defense system of bacteria and archaea. It has been biotechnologically adapted for editing the DNA of eukaryotic cells, including human cells. The CRISPR-Cas system for editing genes generally consists of two components, a nuclease protein (Cas) and a guide RNA (sgRNA). The simplicity of the complex makes it a reprogrammable molecular tool capable of being targeted and editing any site in a known genome. Its main focus is therapies for monogenic inherited diseases and cancer. However, in addition to gene editor, CRISPR technology is used for epigenetic editing, regulation of gene expression, and molecular diagnostic methods. This article aims to present a review of the applications of the CRISPR-Cas molecular tool, particularly in the biomedical field, possible treatments and diagnoses, and the advances in clinical research, using the most relevant CRISPR-Cas gene therapy to date. (AU)

CRISPR systems: Novel approaches for detection and combating COVID-19.

Type V and VI CRISPR enzymes are RNA-guided, DNA and RNA-targeting effectors that allow specific gene knockdown. Cas12 and Cas13 are CRISPR proteins that are efficient agents for diagnosis and combating single-stranded RNA (ssRNA) viruses. The programmability of these proteins paves the way for the detection and degradation of RNA viruses by targeting RNAs complementary to its CRISPR RNA (crRNA). Approximately two-thirds of viruses causing diseases contain ssRNA genomes. The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has caused the outbreak of the coronavirus disease 2019 (COVID-19), which has infected more than 88 million people worldwide with near 2 million deaths since December 2019. Thus, accurate and rapid diagnostic and therapeutic tools are essential for early detection and treatment of this widespread infectious disease. For us, the CRISPR based platforms seem to be a plausible new approach for an accurate detection and treatment of SARS-CoV-2. In this review, we talk about Cas12 and Cas13 CRISPR systems and their applications in diagnosis and treatment of RNA virus mediated diseases. In continue, the SARS-CoV-2 pathogenicity, and its conventional diagnostics and antivirals will be discussed. Moreover, we highlight novel CRISPR based diagnostic platforms and therapies for COVID-19. We also discuss the challenges of diagnostic CRISPR based platforms as well as clarifying the proposed solution for high efficient selective in vivo delivery of CRISPR components into SARS-CoV-2-infected cells.

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents.

Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9-guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9-guide RNA-lipid complexes targeting the Tmc1Bth allele into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1Bth/+ mice. These findings suggest that protein-RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.

The therapeutic application of CRISPR/Cas9 technologies for HIV.

INTRODUCTION: The use of antiretroviral therapy has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless, gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) gene editing system. AREAS COVERED: CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease-deficient Cas9 fused to transcription activation domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus. EXPERT OPINION: The diversity of the CRISPR/Cas9 technologies offers great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV.

CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.

CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.