CRISPR-Cas9 bends and twists DNA to read its sequence.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• The CRISPR-Cas9 system is vital for bacterial immunity and genome editing.
• Cas9 protein identifies target DNA sequences through complementarity with guide RNA and a protospacer-adjacent motif (PAM).
• The mechanism by which Cas9 unwinds DNA for target interrogation is not fully understood.

Purpose of the Study:

• To elucidate the ATP-independent mechanism of DNA unwinding and target interrogation by Cas9.
• To reveal the structural dynamics of Cas9-RNA-DNA complexes during target search.
• To understand how DNA geometry influences Cas9's search efficiency.

Main Methods:

• Cryogenic-electron microscopy (cryo-EM) to determine structures of Cas9-RNA-DNA complexes.
• Solution conformational probing to analyze DNA dynamics.
• Biophysical analysis of DNA bending and base flipping.

Main Results:

• Cas9 binding to the PAM induces significant DNA bending and undertwisting.
• This DNA distortion results in the flipping of nucleotides out of the duplex.
• Structural analysis revealed global protein rearrangements and an unstacked DNA hinge during target interrogation.

Conclusions:

• Cas9 utilizes DNA bending and base flipping, driven by PAM recognition, to interrogate DNA sequences.
• This mechanism facilitates efficient identification of target DNA within a vast genomic landscape.
• The findings provide insights into the physical basis of DNA search and its implications for genome editing efficiency.