CRISPR/Cas9 system and fluorescence-enhanced BA-LIFT bioprinting technique as a toolbox for the study of the Immune System

• Autores: Miguel Gómez Fontela
• Directores de la Tesis: Pilar Lauzurica Gómez (dir. tes.), Carlos Molpeceres Alvarez (dir. tes.), Núria Gironés Pujol (tut. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2020
• Idioma: inglés
• Número de páginas: 184
• Tribunal Calificador de la Tesis: Manuel Fresno Escudero (presid.), Pablo Gómez del Arco (secret.), Sara Lauzurica Santiago (voc.), Elena Fernández Ruiz (voc.), Miguel Marcilla Goldaracena (voc.)
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Materias:
  • Ciencias de la vida
    • Inmunología
    • Biología molecular
  • Ciencias tecnológicas
    • Tecnología electrónica
      • Dispositivos láser
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • In this work, the CRISPR/Cas system genome editing toolbox and the fluorescence enhanced BA-LIFT laser bioprinting tools have been used to study the immune system cells behavior. CD69 is tightly regulated at the transcription level by the CNS2 regulatory region, whose epigenetic marks define as a bivalent regulatory element that match with the previously definition of CD69 as a bivalent gene. By CRISPR/Cas9 transcriptional assays and genome editing, two major areas within the CNS2 enhancer with antagonistic but complementary regulatory activities in vivo were defined in T cells: a Core E region of ~60 bp with a dual repressor and activator function; and a 5’ region adjacent to the Core E (5’C) of ~160 bp where the major activation transcriptional machinery is associated. Deletion of the Core E led to a CD69 overexpression both at resting and after stimulation, supporting its repressive role at steady state and avoiding CD69 overexpression upon stimulation. The Core E is enriched in the Oct1 and Chd4 TFs, whose bivalent function have been previously defined. RNA-seq analysis of ΔCore E T cells showed that CD69 overexpression is associated with an increase of the chemokine receptors CCR1, CCR2 and CCR5 at steady state, and of its ligands CCL3L3, CCL4 and CCL4L1 after stimulation, highlighting the role of CD69 in the regulation of chemokines and its receptors, as has been previously observed in mouse models.

    The role of the human ERAP2 aminopeptidase in the antigen presentation was assessed by generating a functional ERAP2 KO in B cells. Comparison of the HLA-B*27:05 ligandomes of ERAP+/+ and ERAP-/- cells determine that ERAP2 destroy ligands with N-terminal basic residues and peptides with minority anchor motifs at P2 (K and Q). A compensation effect in other peptide positions was observed, since the overall stability of the peptides remained constant.

    A newly developed fluorescence enhanced BA-LIFT laser bioprinting technique has been integrated. A laser-based bioprinting device for accurate identification, selection and printing of single or grouped cells within a complex population was developed and validated. FE BA-LIFT has proven to be an efficient and precise tool with high viability and resolution when depositing biological material in hydrogels or scaffolds with different characteristics. An IFN-γ human NK cell reporter has been generated by CRISPR/Cas9 to be used for the validation of the FE BA-LIFT bioprinter