Massively parallel biophysical analysis of a CRISPR-Cas complex on rep…

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  • 2017-01-06


[BK21 Plus Seminar]
                  ▶Subject: Massively parallel biophysical analysis of a CRISPR-Cas complex on repurposed next generation sequencing chips
                  ▶Speaker: Cheulhee Jung, PH.D. (University of Texas at Austin)

                  ▶Date: 2:00 PM/Oct. 17(Mon.)/2016
                  ▶Place: Conference room(#179), Postech Biotech Center
                    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 modern next-generation sequencing chips to directly measure the interactions between proteins and ~107 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 surprising three-nucleotide periodicity in Cascade-DNA interactions. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment, providing an additional DNA-guided proofreading mechanism. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding and provide a framework for high-throughput, quantitative analysis of protein-DNA interactions.

•Chip-hybridized affinity mapping platform (CHAMP) allows massively parallel profiling of protein-nucleic acid interactions on modern next generation sequencing chips.
•CHAMP profiling reveals a promiscuous PAM specificity for a Type I-E CRISPR-Cas system.
•Quantitative profiling of off-target DNA-binding sequences reveals a three-nucleotide periodicity in Cascade-DNA interactions.
•Cas3 recruitment is sensitive to the identity of the PAM and DNA-RNA mismatches within the PAM-proximal region, establishing a novel DNA-guided proofreading mechanism.

                ▶Inquiry: Prof. Sung Ho Ryu (279-2292)
                      * This seminar will be given in English.
                  please refrain from taking photos during seminars. *