טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentNir Zvi Inbal
SubjectUsing CRISPR/Cas9 to Study the Mechanisms of Action of
Semaphorins and Lysyl Oxidases.
DepartmentDepartment of Medicine
Supervisor PROFESSOR EMERITUS Gera Neufeld
Full Thesis textFull thesis text - English Version


Abstract

The CRISPR/Cas9 knockout process causes random insertion/deletion (indel) mutations, occurring within the coding region of the gene of interest resulting in gene knockout. CRISPR/Cas9 can therefore be used in order to fully, completely and permanently silence targeted genes at the DNA level in cultured cells. Furthermore, it does not require the introduction of any selection markers, thus enabling easy rescue experiments.


Class-3 semaphorin guidance factors bind to receptor complexes containing neuropilin and plexin receptors. A semaphorin may bind to several receptor complexes resulting in diverse effects regarding cell migration. U87MG glioblastoma cells express both neuropilins and the four class-A plexins. Here, we demonstrate these cells respond to Sema3A or Sema3B by cytoskeletal collapse and cell contraction, but fail to contract in response to Sema3C, Sema3D, Sema3G or Sema3E, even in the case of over-expressed class-A plexins in the cells. However, following plexinD1 expression in the cells, these four semaphorins were able to induce cell contraction indicating that they require plexinD1 to enable signal transduction. Surprisingly, unlike Sema3D and Sema3G Sema3C induced the contraction and repulsion of plexin-D1- expressing U87MG cells in which we have knocked-out the genes encoding both neuropilins using CRISPR/Cas9. In addition,  we found that neuropilins function as enhancers of plexin-D1-mediated Sema3C signaling. Interestingly, in the absence of neuropilins, plexin-A4 and was required in addition to plexin-D1 to enable Sema3C induced signal transduction.


Lysyl oxidase (LOX) and its paralogs, lysyl oxidase like (LOXL1-4), form a family of five secreted enzymes that act as copper dependent amine oxidases. They catalyze the cross-linking of collagen and elastin in the extracellular matrix (ECM). In order to compare the properties of the individual lysyl-oxidases, and in order to identify differences between them, we have generated two cell lines, derived from MDA-MB-231 and LM2-4 breast cancer derived cells, in which we have knocked-out the five lysyl-oxidase family genes  using CRISPR/Cas9. These cells displayed significant inhibition of invasiveness and were unable to form colonies in soft agar. However, re-expression of LOX and LOXL2 failed to rescue the tumorigenic phenotype both in-vitro and in-vivo. Deep sequencing and mass spectrometry analysis of cells over-expressing LOX cDNA revealed several genes, whose expression was specifically regulated by LOX. For example, we found that LOX up-regulates the expression of the STOX2 transcription factor, which had been previously identified as a gene contributing to oral squamous cell carcinoma progression. Further research is required in order to determine the role of such identified lysyl-oxidase regulated genes, in the complex biological functions affected by the different lysyl-oxidases.

Applying the CRISPR/Cas9 method to explore class-3 semaphorins signaling dependence on neuropilins was successful perhaps because class-3 semaphorin signaling requires a direct contact between the semaphorins and their receptors. In contrast, the influence of the lysyl oxidases on complex biological process is frequantly indirect, and involves many participants. At this point, further understanding and improvement of the CRISPR/Cas9 method is required in order to apply it effectively for the investigation of cancer biology.