Alternative of this AS event have yet to

Alternative gene splicing
(AS) is one of the major contributors to proteome diversity, playing an
essential role in normal development of humans. Contrastingly,
aberrant AS is recognized as a driver of cancer, though the
specific factors and mechanisms that produce oncogenic splice variants are
poorly understood. One example
of oncogenic AS is the AML1-ETO9a splice variant in acute myeloid
leukemia (AML) patients with the t(8;21) chromosomal translocation. AML is caused by abnormal and rapid proliferation
of myeloid progenitor cells. It is the most common cause of acute leukemia in
adults.  In t(8;21) AML patients, the
chimeric protein, AML1-ETO, is formed. This protein retains the DNA-binding
specificity of AML1 and the ability to recruit ETO-associated co-repressors to support
self-renewal of hematopoietic progenitor cells. While AML1-ETO expression can
induce myeloproliferative disorders in mice, its expression alone is not
sufficient for leukemogenesis. In contrast, a
splice variant of AML1-ETO, including exon 9a, is sufficient to induce leukemogenesis. Regulators of this AS event have yet to be

To identify these modulators, it is pivotal to employ a method
that will target the pre-spliced messenger RNA (mRNA) of AML1-ETO along with
the upstream splicing factors. Recently, the clustered regularly interspaced short
palindromic repeat (CRISPR) family member Cas9, was shown to target
specific endogenous RNA molecules, with the help of a guide RNA (gRNA) sequence
and an additional oligonucleotide probe. Based on this finding, we
propose to develop a novel CRISPR-assisted proteomic method to identify RNA
binding proteins (RBPs) that interact with the AML1-ETO pre-mRNA in t(8;21) AML

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Objectives: Aim 1: Prepare CRISPR components for AML-ETO pre-mRNA enrichment. CRISPR components were assembled as follows: dCas9 (a nuclease-dead Cas9 mutant) was expressed in Escherichia Coli BL21
DE3 and purified with a HisTrap Column. dCas9 was then tagged with a biotin
labeling reagent (ThermoScientific). For gRNA, different sequences were
designed to target AML1-ETO pre-mRNA and ordered as DNA oligos (Mobix). Oligos
were then transcribed using a transcription kit (NEB). DNA probes for Cas9 targeting were designed to form
a short DNA/RNA double stranded region upstream of the gRNA targets to enable
dCas9 binding. Probes were ordered from Mobix.

Aim 2: Determine RBPs associated with
AML1-ETO pre-mRNA in t(8;21) AML cells. Briefly,
t(8;21) AML cells will be irradiated with UV light to cross-link RNA molecules
with their RBPs. These cells will be lysed and incubated with the biotin-dCas9,
gRNA, and DNA probes. The sample will then be incubated with immobilized
streptavidin beads. The enriched RBPs will be released by RNase digestion of
the AML-ETO pre-mRNA and followed with the subsequent mass spectrometry
workflow for protein identification.

Aim 3: Validate the RNA-protein
interactions with AML-ETO pre-mRNA in t(8;21) AML cells. We
plan to perform Aim 2 methods along with western blots to confirm the
RNA-protein interactions discovered in Aim 2. We will further confirm these
interactions reciprocally, by isolating the identified RBPs and detecting the
AML1-ETO pre-mRNA with real-time PCR.

This study will identify key AS regulators that direct expression of the
leukemogenic isoform of AML1-ETO. This method could also be applied to other
splice isoforms. Thus providing a tool for specific endogenous RBP
identification, which has not been shown before.