Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips

Cheulhee Jung; John A. Hawkins; Stephen K. Jones; Yibei Xiao; James R. Rybarski; Kaylee E. Dillard; Jeffrey Hussmann; Fatema A. Saifuddin; Cagri A. Savran; Andrew D. Ellington; Ailong Ke; William H. Press; Ilya J. Finkelstein

doi:10.1016/j.cell.2017.05.044

Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips

Cheulhee Jung, John A. Hawkins, Stephen K. Jones, Yibei Xiao, James R. Rybarski, Kaylee E. Dillard, Jeffrey Hussmann, Fatema A. Saifuddin, Cagri A. Savran, Andrew D. Ellington, Ailong Ke, William H. Press, Ilya J. Finkelstein

Research output: Contribution to journal › Article › peer-review

73 Citations (Scopus)

Abstract

CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and ∼10⁷ unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA.

Original language	English
Pages (from-to)	35-47.e13
Journal	Cell
Volume	170
Issue number	1
DOIs	https://doi.org/10.1016/j.cell.2017.05.044
Publication status	Published - 2017 Jun 29
Externally published	Yes

Bibliographical note

Publisher Copyright:
© 2017 Elsevier Inc.

Keywords

CRISPR
Cas3
Cascade
biophysics
fluorescence microscopy
next generation sequencing

ASJC Scopus subject areas

General Biochemistry,Genetics and Molecular Biology

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10.1016/j.cell.2017.05.044

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@article{d33b041a6e544878bbfe72b2cf1c5245,

title = "Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips",

abstract = "CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and ∼107 unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA.",

keywords = "CRISPR, Cas3, Cascade, biophysics, fluorescence microscopy, next generation sequencing",

author = "Cheulhee Jung and Hawkins, {John A.} and Jones, {Stephen K.} and Yibei Xiao and Rybarski, {James R.} and Dillard, {Kaylee E.} and Jeffrey Hussmann and Saifuddin, {Fatema A.} and Savran, {Cagri A.} and Ellington, {Andrew D.} and Ailong Ke and Press, {William H.} and Finkelstein, {Ilya J.}",

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year = "2017",

month = jun,

day = "29",

doi = "10.1016/j.cell.2017.05.044",

language = "English",

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AU - Jung, Cheulhee

AU - Hawkins, John A.

AU - Jones, Stephen K.

AU - Xiao, Yibei

AU - Rybarski, James R.

AU - Dillard, Kaylee E.

AU - Hussmann, Jeffrey

AU - Saifuddin, Fatema A.

AU - Savran, Cagri A.

AU - Ellington, Andrew D.

AU - Ke, Ailong

AU - Press, William H.

AU - Finkelstein, Ilya J.

PY - 2017/6/29

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N2 - CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and ∼107 unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA.

AB - CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and ∼107 unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA.

KW - CRISPR

KW - Cas3

KW - Cascade

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KW - fluorescence microscopy

KW - next generation sequencing

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Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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