Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

CALR frameshift mutations in MPN patient-derived iPSCs accelerate maturation of megakaryocytes

“…To obtain isogenic CALR -repaired clones for CALR ins5 and CALR del52-mutated iPSCs, we used CRISPR/Cas9 technology to correct the mutations. Until now, the repair of CALR ins5 mutations has been reported only as a conference abstract ( Wang et al., 2018 ), and a CRISPR/Cas9 approach has been used to model CALR mutations in murine cell lines ( Abdelfattah and Mullally, 2018 ). To our knowledge, the repair of CALR del52 mutations in MPN-specific iPSCs has not been reported.…”

CALR frameshift mutations in MPN patient-derived iPS cells accelerate maturation of megakaryocytes

“…To obtain isogenic CALR -repaired clones for CALR ins5 and del52-mutated iPS cells, CRISPR/Cas9 technology was used to correct the mutations. Until now, the repair of CALR ins5 mutations was reported at a conference abstract (Wang et al, 2018), and a CRISPR/Cas9 approach was used to model CALR mutations in murine cell lines (Abdelfattah and Mullally, 2018). To our knowledge, the repair of CALR del52 mutations in MPN-specific iPS cells was not reported, yet.…”

Diallyl disulfide down‐regulates calreticulin and promotes C/EBPα expression in differentiation of human leukaemia cells

“…CRT is a multiprocess calcium buffering chaperone of the endoplasmic reticulum that is essential for numerous cellular functions . Some studies have identified increased expression of CRT in many tumour tissues or cells .…”