CRISPR Approaches for the Diagnosis of Human Diseases.

CRISPR/Cas is a prokaryotic self-defense system, widely known for its use as a gene-editing tool. Because of their high specificity to detect DNA and RNA sequences, different CRISPR systems have been adapted for nucleic acid detection. CRISPR detection technologies differ highly among them, since they are based on four of the six major subtypes of CRISPR systems. In just 5 years, the CRISPR diagnostic field has rapidly expanded, growing from a set of specific molecular biology discoveries to multiple FDA-authorized COVID-19 tests and the establishment of several companies. CRISPR-based detection methods are coupled with pre-existing preamplification and readout technologies, achieving sensitivity and reproducibility comparable to the current gold standard nucleic acid detection methods. Moreover, they are very versatile, can be easily implemented to detect emerging pathogens and new clinically relevant mutations, and offer multiplexing capability. The advantages of the CRISPR-based diagnostic approaches are a short sample-to-answer time and no requirement of laboratory settings; they are also much more affordable than current nucleic acid detection procedures. In this review, we summarize the applications and development trends of the CRISPR/Cas13 system in the identification of particular pathogens and mutations and discuss the challenges and future prospects of CRISPR-based diagnostic platforms in biomedicine.

Detection of chromosome instability by interphase FISH in mouse and human tissues.

Chromosomal instability (CIN), a type of genomic instability, favors changes in chromosome number and structure and it is associated with the progression and initiation of multiple diseases, including cancer. Therefore, CIN identification and analysis represents a useful tool for cancer diagnosis and treatment. Here, we report an optimized molecular cytogenetic protocol to detect CIN in formalin-fixed, paraffin-embedded mouse and human tissues, using fluorescent in situ hybridization to visualize and quantify chromosomal alterations such as amplifications, deletions, and translocations. For complete information on the generation and use of this protocol, please refer to Brandt et al. (2018).

Functional characterization of two enhancers located downstream FOXP2.

BACKGROUND: Mutations in the coding region of FOXP2 are known to cause speech and language impairment. However, it is not clear how dysregulation of the gene contributes to language deficit. Interestingly, microdeletions of the region downstream the gene have been associated with cognitive deficits. METHODS: Here, we investigate changes in FOXP2 expression in the SK-N-MC neuroblastoma human cell line after deletion by CRISPR-Cas9 of two enhancers located downstream of the gene. RESULTS: Deletion of any of these two functional enhancers downregulates FOXP2, but also upregulates the closest 3' gene MDFIC. Because this effect is not statistically significant in a HEK 293 cell line, derived from the human kidney, both enhancers might confer a tissue specific regulation to both genes. We have also found that the deletion of any of these enhancers downregulates six well-known FOXP2 target genes in the SK-N-MC cell line. CONCLUSIONS: We expect these findings contribute to a deeper understanding of how FOXP2 and MDFIC are regulated to pace neuronal development supporting cognition, speech and language.

CRISPR/Cas9 for Cancer Therapy: Hopes and Challenges.

Cancer is the second leading cause of death globally and remains a major economic and social burden. Although our understanding of cancer at the molecular level continues to improve, more effort is needed to develop new therapeutic tools and approaches exploiting these advances. Because of its high efficiency and accuracy, the CRISPR-Cas9 genome editing technique has recently emerged as a potentially powerful tool in the arsenal of cancer therapy. Among its many applications, CRISPR-Cas9 has shown an unprecedented clinical potential to discover novel targets for cancer therapy and to dissect chemical-genetic interactions, providing insight into how tumours respond to drug treatment. Moreover, CRISPR-Cas9 can be employed to rapidly engineer immune cells and oncolytic viruses for cancer immunotherapeutic applications. Perhaps more importantly, the ability of CRISPR-Cas9 to accurately edit genes, not only in cell culture models and model organisms but also in humans, allows its use in therapeutic explorations. In this review, we discuss important considerations for the use of CRISPR/Cas9 in therapeutic settings and major challenges that will need to be addressed prior to its clinical translation for a complex and polygenic disease such as cancer.

CRISPR/Cas9 Technology: Applications and Human Disease Modeling.

The CRISPR/Cas9 system development has revolutionized the field of genome engineering through the efficient creation of targeted breaks in the DNA of almost any organism and cell type, opening an avenue for a wide range of applications in biomedical research and medicine. Apart from gene edition through knock-in or knock-out approaches, CRISPR/Cas9 technology has been used for many other purposes, including regulation of endogenous gene expression, epigenome editing, live-cell imaging of chromosomal loci, edition of RNA and high-throughput screening. With all those technological improvements, CRISPR/Cas9 system has broadened the number of alternatives for studying gene function and the generation of more accurate disease models. Although many mechanistic questions remain to be answered and several challenges have yet to be addressed, the use of CRISPR/Cas9-based genome engineering technologies will increase our knowledge of disease processes and their treatment in the near future.

Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9.

Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma.