Precise and efficient silencing of mutant Kras^G12D by CRISPR-CasRx controls pancreatic cancer progression

Abstract

Rationale: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with few therapeutic targets and rare effective treatments. Over 90% of PDAC tumors bear a Kras mutation, and the single-site mutation G12D (Kras G12D ) is most prevalent. Methods: Here, we applied the CRISPR-CasRx system to silence the mutant Kras G12D transcript in PDAC cells. We also used a capsid-optimized adenovirus-associated virus 8 vector (AAV8) to deliver the CRISPR-CasRx system into PDAC orthotopic tumors and patient-derived tumor xenografts (PDX). Results: Our data showed that guided by a KrasG12D-specific gRNA, CasRx is able to precisely and efficiently silence the mutant KrasG12D expression in PDAC cells. The knockdown of mutant KrasG12D by CasRx abolishes the aberrant activation of downstream signaling induced by mutant KrasG12D and subsequently suppresses the tumor growth and improves the sensitivity of gemcitabine in PDAC. Additionally, delivering CasRx-gRNA via AAV8 into the orthotopic KrasG12D PDAC tumors substantially improves the survival of mice without obvious toxicity. Furthermore, targeting KrasG12D through CasRx suppresses the growth of PDAC PDXs. In conclusion, our study provides a proof-of-concept that CRISPR-CasRx can be utilized to target and silence mutant KrasG12D transcripts and therefore inhibit PDAC malignancy.

Comparative genomic analysis of head and body/tail of pancreatic ductal adenocarcinoma at early and late stages

“…Furthermore, KRAS driver mutations lead to the activation of Wnt and MAPK pathways, which controls tumour cell proliferation, motility, metabolism and survival 13 . Silencing of mutant KRAS by CRISPR‐CasRx system suppresses PDAC progression 14 . The results were consistent with a large cohort study including 9,952 patients with metastasized PDAC from the Netherlands Cancer Registry (2005‐2015), which demonstrated that pancreatic tail cancers had more metastatic sites and worse survival 15 .…”

“…13 Silencing of mutant KRAS by CRISPR-CasRx system suppresses PDAC progression. 14 There were strengths and limitations in this study. This was the first study including both early-and late-stage PDACs and comparing the genomic profiles between pancreatic head and body/tail cancers.…”

Drugging multiple same-allele driver mutations in cancer

“…K-Ras A146T is a weak driver. It is mostly in colorectal and hematopoietic cancers, but not in pancreatic cancer [24] where K-Ras G12D is extremely highly populated [25]. K-Ras A146T acts by promoting nucleotide exchange rather than blocking GTP hydrolysis, which is the common mechanism for strong K-Ras mutations, such as K-Ras G12D .…”

CRISPR-CasRx Targeting LncRNA LINC00341 Inhibits Tumor Cell Growth in vitro and in vivo

“…Although traditional methods including siRNA/shRNA also offers a possibility for inhibiting LINC00341, the unexpected off-target effects significantly limit their applications. As a newly identified Cas system from RNA-targeting CRISPR enzymes, CRISPR-CasRx exhibits high efficiency and specificity for RNA cleavage ( Jiang et al, 2020 ; Zhou et al, 2020 ). Inhibition of cell proliferation, escalated the rate of apoptosis, and reduced the motility rate in LINC00341 sgRNA-transfected T24 as well as 5,637 bladder cancer cell lines.…”