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47. CRISPR Cas9-mediated targeted sequencing on nanopore: a powerful technology to map complex structural chromosome abnormalities

      Complex structural chromosome abnormalities are common in cancer cells. Besides the well-known mechanism of gradual accumulation of mutations to prefer cell duplication/survival in cancer cells, complex and massive chromosomal and genomic alterations can also be generated by a chromoanagenesis event, which is characterized by the simultaneous occurrence of multiple structural alterations through a single catastrophic cellular event. Despite improvements in genomics technology, the detection of complex structural chromosome abnormalities from short-read sequencing still poses challenges. The nanopore can sequence stretches of DNA of up to hundreds of kilobases in length and clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 is a novel gene-editing technique, which can efficiently induce targeted genetic modifications. Here we analyzed a cancer genome of a patient using the CRISPR Cas9-mediated targeted sequencing on nanopore. The patient suffered from acute myeloid leukemia resulting from a complex karyotype: 44,XY,add(1)(p12),t(1;4)(p13;q31.1), add(2)(p13),-3,-5,add(6)(q25),der(7)?hsr(7)(q11.2)?hsr(7)(q?22)t(3;7)(p12;q?31),-16, der(16)t(16;17)(q12.1;q11.2),-17,add(17)(p13),del(17)(p13),add(20)(q11.2), der(?)t(?;1)(?;p22)[cp18]/46,XY[2]. FISH revealed amplification of chromosome 7 centromere. Illumina genome-wide SNP microarray revealed 9 gains, 20 losses, as well as three chromoanagenesis regions (3q12.2-q13.31, 3q13.32-q21.3, and 7p12.2-q11.2). CRISPR Cas9 probes for breakpoints of these abnormalities were designed. Protein nanopores were used to measure DNA sequence directly and in real time. The long reads from the nanopore not only determined simple structural abnormalities, but also enabled us to resolve the long-range structure of the complex chromoanagenesis. In summary, sequencing cancer genome of our patient using CRISPR Cas9-mediated targeted sequencing on nanopore detected breakpoints of complex structural chromosome abnormalities, at high sensitivity. We demonstrated the value of this approach in mapping complex structural chromosome abnormalities for both clinical and research applications. Characterization of these complex structural chromosome abnormalities will not only help understand the molecular mechanisms responsible for a process of chromoanagenesis, but also may identify specific molecular targets and their impact on therapy and overall survival.
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