Applications of CRISPR-Cas9 Technology to Genome Editing in Glioblastoma Multiforme

Abstract

Glioblastoma multiforme (GBM) is an aggressive malignancy of the brain and spinal cord with a poor life expectancy. The low survivability of GBM patients can be attributed, in part, to its heterogeneity and the presence of multiple genetic alterations causing rapid tumor growth and resistance to conventional therapy. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) nuclease 9 (CRISPR-Cas9) system is a cost-effective and reliable gene editing technology, which is widely used in cancer research. It leads to novel discoveries of various oncogenes that regulate autophagy, angiogenesis, and invasion and play important role in pathogenesis of various malignancies, including GBM. In this review article, we first describe the principle and methods of delivery of CRISPR-Cas9 genome editing. Second, we summarize the current knowledge and major applications of CRISPR-Cas9 to identifying and modifying the genetic regulators of the hallmark of GBM. Lastly, we elucidate the major limitations of current CRISPR-Cas9 technology in the GBM field and the future perspectives. CRISPR-Cas9 genome editing aids in identifying novel coding and non-coding transcriptional regulators of the hallmarks of GBM particularly in vitro, while work using in vivo systems requires further investigation.

Keywords: CRISPR-Cas9 genome editing; angiogenesis; apoptosis; autophagy; cell invasion and migration; glioblastoma multiforme (GBM); proliferation.

Figure 1

Principle of CRISPR-Cas9 genome editing and sources of its major elements for application to GBM research. CRISPR-Cas9 genome editing involves three major steps: Step 1, guide RNA binds to complementary sequence in the gene of interest; Step 2, Cas9 performs double-strand DNA break; Step 3, activation of non-homologous end joining (NHEJ) mechanism repairs DNA by directly ligating double-strand DNA break ends after DNA sequences are inserted or deleted, or activation of homology-directed repair (HDR) mechanism inserts the targeted DNA sequence in the presence of donor DNA template with homology regions to the cut-ends resulting in more precise gene insertion. Majority of CRISPR-Cas9 genome editing applications were performed in GBM cell lines or GSCs. The sources of guide RNA used in GBM research were variable such as plasmid, viral, and synthetic. Similarly, the sources of Cas9 were varying including plasmid Cas9, viral Cas9, and Cas9 protein.

Figure 2

Application of CRISPR-Cas9 genome editing to identifying genes that correlate with different hallmarks of GBM. CRISPR-Cas9 mediated gene knockdown, knockout, and overexpression have been used for identifying several genes that have unique and overlapping roles in GBM development, progression, and recurrence. The listed genes with their references are the ones already edited using CRISPR-Cas9 genome editing technology in GBM research.