화학공학소재연구정보센터
학회 한국공업화학회
학술대회 2018년 봄 (05/02 ~ 05/04, 대구 엑스코(EXCO))
권호 22권 1호
발표분야 (생물공학) 생물공학 신기술 최신 동향
제목 Massively parallel biophysical analysis of a CRISPR-Cas complex on repurposed next generation sequencing chips
초록 Bacterial CRISPR-Cas systems recognize foreign DNA via complementary base pairing with an RNA-guided nucleoprotein complex. Although off-target DNA binding has been reported, a quantitative description of sequence-dependent DNA binding and nuclease activation remains elusive. Here, we describe a chip-hybridized affinity mapping platform (CHAMP) that repurposes next-generation sequencing chips to directly measure the interactions between proteins and ~10^7 unique DNA sequences. Using CHAMP, we interrogate DNA recognition by a Type I-E CRISPR/Cas (Cascade) complex and the Cas3 nuclease. These results show that Cascade is sensitive to an extended protospacer adjacent motif (PAM). Analysis of mutated target sequences reveals a three-nucleotide periodicity in Cascade-DNA interactions. The PAM-proximal nucleotides control Cas3 recruitment, providing an additional DNA-guided proofreading mechanism. These findings provide a framework for high-throughput analysis of protein-DNA interactions.
저자 정철희
소속 고려대
키워드 NGS; high-throughput; CHAMP; CRISPR-Cas
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