Development of a CRISPR-Cas9(D10A) targetable, high-complexity, single-cell barcoding approach for capture of treatment resistant subclones from heterogeneous cancers

Cancers 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 capturing 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 by constructing a Cas9D10A and paired-gRNA targetable unique reporter (CAPTURE) barcoding library with up to 36 million unique barcodes. This approach enabled us to uniquely barcode > 1 million cells, track barcode distribution following treatment and then isolate the resistant subpopulation using the subpopulation-specific barcode. Proof of principle studies showed that specific barcoding cells, even at as low as 0.1%, could be efficiently isolated using barcode targeting and cell sorting. Applied our barcoding approach, we found that A375 cell acquired BRAF inhibitor resistance both from pre-exiting and late-emerging mechanisms. Colony formation experiments showed that the isolated subpopulations were indeed resistant clones. Whole exome, transcriptome and methylome analysis were applied to study the captured subpopulations. Our CAPTURE barcoding approach will enable the identification of the both pre-exiting and late-emerging genetic or epigenetic changes driving treatment resistance. Citation Format: Ze-yan Zhang, Ravesanker Ezhilarasan, Yingwen Ding, Qianghu Wang, Jie Yang, Lihong Long, Roel G. Verhaak, Erik P. Sulman. Development of a CRISPR-Cas9D10A targetable, high-complexity, single-cell barcoding approach for capture of treatment resistant subclones from heterogeneous cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4698.