Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding.

Abstract:

:The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has led to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable nonspecific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of nuclease-dead Cas12a (dCas12a) from Francisella novicida as it inspects and binds to its DNA target. Our results reveal concealed thermodynamic factors affecting dCas12a DNA binding, which should guide the design and optimization of crRNA that limits off-target effects, including the crucial role of an extended protospacer adjacent motif (PAM) sequence and the impact of the specific base composition of crRNA-DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.

authors

Specht DA,Xu Y,Lambert G

doi

10.1073/pnas.1918685117

subject

Has Abstract

pub_date

2020-05-26 00:00:00

pages

11274-11282

issue

21

eissn

0027-8424

issn

1091-6490

pii

1918685117

journal_volume

117

pub_type

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