The CRISPR‐Cas13a Gene‐Editing System Induces Collateral Cleavage of RNA in Glioma Cells

Abstract

RNA is rarely used as a therapeutic target due to its flexible structure and instability. CRISPR‐Cas13a is a powerful tool for RNA knockdown, and the potential application of CRISPR‐Cas13a in cancer cells should be further studied. In this study, overexpression of LwCas13a by lentivirus in glioma cells reveals that crRNA‐EGFP induces a “collateral effect” after knocking down the target gene in EGFP‐expressing cells. EGFRvIII is a unique EGFR mutant subtype in glioma, and the CRISPR‐Cas13a system induces death in EGFRvIII‐overexpressing glioma cells. Bulk and single‐cell RNA sequencing analysis in U87‐Cas13a‐EGFRvIII cells confirm the collateral effect of the CRISPR‐Cas13a system. Furthermore, CRISPR‐Cas13a inhibits the formation of glioma intracranial tumors in mice. The results demonstrate the collateral effect of the CRISPR‐Cas13a system in cancer cells and the powerful tumor‐eliminating potential of this system.

Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes

“…In the study, exogenous gene GFP or EGFRVIII were overexpressed and targeted by Cas13a/crRNA. In this context, a partial degradation of ribosomal RNA was observed, and the abundance of non-target transcripts of GAPDH, HOTHAIR and L3MTL1 was reduced as well 25 . Furthermore, the RNA integrity was compared between the LN229 glioma cell line and HEK293T cells after treatment with the Cas13a/crRNA, the LN229 cells tended to be more sensitive to the collateral effect than the HEK293T cells 25 .…”

“…These data have warranted its safety to be used as an effector to target against RNA viruses that infect humans 21 . However, a Cas13a/crRNA associated collateral cleavage was shown recently in human cancerous U87 glioblastoma cells 25 . In the study, exogenous gene GFP or EGFRVIII were overexpressed and targeted by Cas13a/crRNA.…”

“…The Cas13a gene from L. wadei belongs to the class 2 type VI RNA-guided RNA nuclease 8 . It's RNA targeting effect has been demonstrated in eukaryotic and plant cells 8,18,25,28 .…”

“…Interestingly, no collateral effect was observed in three studies of CRISPR-Cas13 using human or plant cell lines 8,23,24 , but Cas13a associated collateral RNA cleavage was reported in human glioma cancer cells 25 . Fascinatingly, Cas13 can effectively silence serval transcripts in parallel 8,18,24,26 .…”

Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes

“…In bacteria, Cas13 HEPN-nuclease is able to cleave not only the target-RNA in cis but also other non-target RNA present in trans [10]. Interestingly, no collateral effect was observed in three studies of CRISPR-Cas13 using human or plant cell lines [11,26,27], but Cas13a associated collateral RNA cleavage was reported in human glioma cancer cells [28]. Notably, Cas13 can effectively silence several transcripts in parallel [11,20,27,29].…”