Poster Presentation Australasian RNA Biology and Biotechnology Association 2024 Conference

Using CRISPR-cas13 to identify functional lncRNAs in breast cancer (#112)

Maina Bitar 1 , Lu Wang 1 , Xue Lu 1 , Stacey Edwards 1 , Juliet French 1
  1. QIMR Berghofer, Brisbane / Herston, QUEENSLAND, Australia

The number of known long noncoding RNAs (lncRNAs) has surpassed that of protein-coding genes in humans. However, determining the biological roles of novel lncRNAs remains a difficult task, typically performed on a gene-by-gene basis. Moreover, lncRNAs work at different levels - as a DNA element, through their transcription and/or as functional RNA transcripts - making it difficult to pinpoint the relevant functional agent. Recently, CRISPR technologies have emerged as a powerful platform for high-throughput identification of genes involved in specific phenotypes. The CRISPR-cas13 system is an RNA-cleaving variation that can be leveraged to silence lncRNA transcripts. We were pioneers in using CRISPR-cas13 for large-scale screens, targeting over 800 novel lncRNAs identified from breast cancer genome-wide association studies and assessing their impact on cell proliferation. This world-first screen led to the characterisation of KILR, a ~7kb novel lncRNA whose overexpression induces breast cancer cell apoptosis. Follow-up functional studies revealed KILR inhibits DNA replication by sequestering the RPA1 protein, thereby affecting replication fork speed and stability. CRISPR-cas13 can also reveal lncRNAs involved in phenotypes beyond cell proliferation. We have discovered thousands of novel lncRNAs that respond to irradiation in breast cancer cells. In a subsequent large-scale screen, we targeted nearly 3,000 lncRNAs to identify those capable of synergising with the PARP inhibitor drug Olaparib, which is currently used in the treatment of breast cancer. Among the identified lncRNAs we have selected a novel isoform of POL2RJ4, which we have shown to affect the expression of BRCA1. These examples clearly illustrate how CRISPR-based technologies are advancing the field of lncRNA functional characterisation, enabling the parallel assessment of thousands of candidates to identify those with therapeutic potential for cancer research.