Fig. 1: Tumoricidal oligonucleotide libraries created by response-directed in vitro evolution.

a Consistent enrichment of the binding capacity of a random library during the initial stage of the process, implemented on HCT116, a colorectal cancer cell line. Each point represents an independent binding assay (only rounds 0, 2, 4, 7 were sampled for enrichment analysis). b A representative binding assay showing the success of the initial stage (round 7 [final] vs round 0 of the process) on HCT116 cells. c Representative sorting plot during the functional stage of the process. Bead-clustered library (x-axis) is tagged by Cy5; Cas-3/7 is a green fluorescent reporter. Red gate within the upper right quadrant includes cas-3/7⁺ (cas⁺) cells bound to an oligo cluster (clust⁺). These events are sorted and carried forward to the next round. d Consistent enrichment of the ability to induce cas-3/7 activation in HCT116 cells by the oligo library. Inset shows flow cytometric analysis of cas-3/7 activity in HCT116 cells (black, cells treated with round 1; red, cells treated with round 8). e–g Representative runs of response-directed in vitro evolution, resulting in oligo libraries with cas-3/7 activity-inducing capacity (only rounds 1, 3, 5, 8 were sampled for enrichment analysis); e patient-derived xenograft (PDX)-derived triple negative breast cancer (TNBC) cells, termed TNBC9 (only rounds 1, 4, 6, 7, 8 were sampled); f human acute lymphoblastic leukemia cell line (CCRF-CEM; only rounds 1, 6, 7 were sampled, additional intermediate data points are missing due to insufficient material); g patient-derived acute myeloblastic leukemia (AML), termed AML1. h The selectivity of tumoricidal oligo library towards AML1 compared with primary peripheral blood mononuclear cells (PBMC) from a healthy donor. Shown is a representative analysis of cas-3/7 activity in both targets induced by the same library. The response observed in primary PBMC is statistically zero. i The exclusivity of a library evolved against AML1 target cells, to AML cells from other patients (AML4, AML5) and an AML cell line, kasumi-1. Shown is a flow cytometric analysis of cas-3/7 activity (black, round 1; red, round 6 [final] of the process).