DRES-04. DEVELOPMENT OF A CRISPR-CAS9D10A TARGETABLE HIGH-COMPLEXITY SINGLE-CELL BARCODING APPROACH FOR ISOLATION OF TREATMENT RESISTANT SUBCLONES FROM HETEROGENOUS MALIGNANT GLIOMAS

摘要

Malignant gliomas are composed of heterogeneous cell populations and the clonal evolution of these cells is one of the key reasons for treatment resistance and tumor recurrence. A fundamental challenge in studying clonal evolution in these tumors is the difficulty in isolating the phenotype-associated (e.g. treatment resistant) sub-populations from the heterogeneous population. We developed an approach to individually barcode and isolate specific cell subpopulations using a CRISPR (clustered, regularly interspaced, short palindromic repeat)-Cas9D10A targetable, single-cell barcoding library with a complexity of 36 million unique barcodes. This approach enabled us to uniquely barcode > 1 million cells, identify enriching barcoded sub-populations following treatment and then isolate the resistant subpopulation using the subpopulation-specific barcode. Each barcode sequence carrying two guide RNAs so that CRISPR-Cas9D10A targeting can lead to “switching” (i.e. frameshift) of an EGFP (Enhanced Green Fluorescent Protein) reporter. Thus, targeted cells lose their EGFP signal and can be specifically isolated by cell sorting. Proof of principle studies showed that specific barcoding cells could be efficiently targeted to turn off EGFP and subpopulations isolated. Subsequent large-scale implementation of this approach has been performed to isolate resistant subpopulations following radiation and temozolomide. Our approach will lead to identification of the both pre-existing and acquired specific somatic genetic or epigenetic changes driving treatment resistance in patients with malignant gliomas.