AlPaCas: allele-specific CRISPR gene editing through a protospacer-adjacent-motif (PAM) approach

Gene therapy of dominantly inherited genetic diseases requires either the selective disruption of the mutant allele or the editing of the specific mutation. The CRISPR-Cas system holds great potential for the genetic correction of single nucleotide variants (SNVs), including dominant mutations. However, distinguishing between single-nucleotide variations in a pathogenic genomic context remains challenging. The presence of a PAM in the disease-causing allele can guide its precise targeting, preserving the functionality of the wild-type allele. The AlPaCas (Aligning Patients to Cas) webserver is an automated pipeline for sequence-based identification and structural analysis of SNV-derived PAMs that satisfy this demand. When provided with a gene/SNV input, AlPaCas can: (i) identify SNV-derived PAMs; (ii) provide a list of available Cas enzymes recognizing the SNV (s); (iii) propose mutational Cas-engineering to enhance the selectivity towards the SNV-derived PAM. With its ability to identify allele-specific genetic variants that can be targeted using already available or engineered Cas enzymes, AlPaCas is at the forefront of advancements in genome editing. AlPaCas is open to all users without a login requirement and is freely available at https://schubert.bio.uniroma1.it/alpacas

Coevolving Residues and the Expansion of Substrate Permissibility in LAGLIDADG Homing Endonucleases

Genome-editing (GE) is a form of genetic engineering that permits the deliberate manipulation of genetic material for the study of biological processes, agricultural and industrial biotechnologies, and developing targeted therapies to cure human disease. While the potential application of GE is wide-ranging, the efficacy of most strategies is dependent upon the ability to accurately introduce a double-stranded break at the genomic location where alterations are desired. LAGLIDADG homing endonucleases (LHEs) are a class of mobile genetic element that recognize and cleave 22-bp sequences of DNA. Given this high degree of specificity, LHEs are powerful GE reagents, but re-engineering their recognition sites has been hindered by a limited understanding of structural constraints within the family, and how cleavage specificity is regulated in the central target site region. In the present studies, a covariation analysis of the LHE family recognized a set of coevolving residues within the enzyme active site. These positions were found to modulate catalytic efficiency, and are thought to create a barrier to active site evolution and re-engineering by constraining the LHE fitness landscape towards a set of functionally permissive combinations. Interestingly, mutation of these positions led to the identification of a catalytic residue variant that demonstrates cleavage activity against a greater number of central target site substrates than wild-type enzymes. To facilitate these investigations, high-throughput and unbiased methods were developed to functionally screen large mutagenic libraries and simultaneously profile cleavage specificity against 256 different substrates. Lastly, structural studies aimed at increasing our understanding of the LHE coevolving network led to the discovery of direct protein-DNA contacts in the central target site region. Significantly, these findings increase our understanding of functionally important structural constraints within the LHE family and have the potential to increase the sequence targeting capacity of LHE scaffolds. More broadly, the methodologies described in this thesis can assist large-scale structure-function studies and facilitate investigations of substrate specificity for most DNA-binding proteins. Finally, the thorough biochemical validation I provide for computational predictions of coevolution showcases a strategy to infer protein function-structure from genetic information and emphasizes the need to expand these studies to other protein families

Aplicación de CRISPR en la edición de células T para el tratamiento de pacientes con cáncer

Treball Final de Grau en Medicina. Codi: MD1158. Curs acadèmic: 2020/2021La tecnología CRISPR-Cas es una potente herramienta molecular que permite editar el genoma de forma simple y específica. Su aplicación en el ámbito de la inmunoterapia ha permitido optimizar la función de las células T humanas para combatir el cáncer. Esta revisión sistemática planteó como principal objetivo comprobar la seguridad, viabilidad y eficacia de la terapia con células T genéticamente modificadas mediante CRISPR-Cas, en pacientes con cáncer refractario. Para ello, analizamos los dos primeros ensayos clínicos llevados a cabo con esta técnica, ambos en fase I. Los artículos fueron extraídos de la base de datos Pubmed y la metodología empleada sigue las pautas establecidas por el manual Cochrane 5.1.0. Los efectos adversos reportados en ambos estudios fueron escasos y reversibles. Por otro lado, la eficiencia de edición genética se demostró mediante una baja frecuencia de eventos “off-target”, término utilizado para designar la aparición de mutaciones no deseadas. Por último, la persistencia in vivo de las células T editadas, fue prolongada. En cuanto a la eficacia antitumoral, cabe señalar que se logró la estabilidad de la enfermedad en algunos pacientes durante un período limitado de tiempo, pero ninguno de ellos mostró una regresión tumoral significativa. Concluimos que la aplicación de células T editadas con CRISPR es segura y viable. Sin embargo, se necesitarán próximos estudios para determinar su eficacia.CRISPR-Cas technology is a powerful molecular tool providing a simple and promising way of genome editing. Its application in immunotherapy field enables to optimize the function of human T cells to fight cancer. The primary aim of this systematic review was to check safety, feasibility and efficacy of CRISPRCas edited T cells therapy in patients with refractory cancer. Therefore, the first-in-human phase I clinical trials using this technology have been evaluated. Articles were extracted from Pubmed database, and the methodology employed follows Cochrane Handbook 5.1.0 guidelines. The adverse events reported in both studies were rare and reversible. On the other hand, editing efficiency was shown by the low frequency of “off-target” events, term used to designate the occurrence of unwanted mutations. Finally, in vivo persistence of edited T cells was stable in the long-term. Regarding antitumor efficacy, it should be noted that some patients achieved disease stability during a limited period of time, but none of them showed a significant tumor regression. We conclude that CRISPR-edited T cells technique is safe and feasible. Nevertheless, further studies would be needed to establish its efficac