Differential Divalent Metal Binding by SpyCas9's RuvC Active Site Contributes to Nonspecific DNA Cleavage

Area of Science:

• Molecular Biology
• Biochemistry
• Biotechnology

Background:

• Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated (CRISPR-Cas) systems provide adaptive immunity in bacteria and archaea against mobile genetic elements (MGEs).
• CRISPR-Cas systems utilize CRISPR RNAs (crRNAs) and Cas nucleases for sequence-specific DNA cleavage, a mechanism adapted for gene editing applications.
• Safety concerns for gene editing include off-target cleavage and guide RNA (gRNA)-independent DNA cleavage by nucleases like Streptococcus pyogenes Cas9 (SpyCas9).

Purpose of the Study:

• To engineer a variant of SpyCas9 with reduced or eliminated gRNA-free DNA cleavage activity.
• To enhance the specificity and safety of SpyCas9 for gene editing applications.
• To investigate the role of cation cofactors in gRNA-free DNA cleavage by SpyCas9.

Main Methods:

• Site-directed mutagenesis was used to create a SpyCas9 variant with an H982A substitution in the RuvC active site.
• In vitro assays were performed to measure gRNA-free DNA cleavage activity in the presence of different divalent metal cations (Mn2+ and Mg2+).
• Mechanistic molecular dynamics simulations were employed to analyze the structural basis of cation-dependent DNA cleavage.

Main Results:

• The SpyCas9 H982A variant exhibited significantly reduced gRNA-free DNA cleavage activity, approximately 167-fold less than wild-type SpyCas9 in the presence of Mn2+.
• Molecular dynamics simulations revealed that Mn2+ facilitates a gRNA-free DNA cleavage competent state in wild-type SpyCas9, which is disrupted by the H982A substitution.
• The H982A substitution specifically targets Mn2+-dependent gRNA-free DNA cleavage, while Mg2+-dependent cleavage activity remains largely unaffected.

Conclusions:

• The H982A substitution effectively eliminates Mn2+-dependent gRNA-free DNA cleavage, leading to a safer and more selective SpyCas9 variant for gene editing.
• Modulating cation:protein interactions offers a viable strategy for engineering improved and safer CRISPR-Cas gene editing tools.
• This study provides a mechanistic understanding of cation-dependent DNA cleavage and a pathway for developing next-generation gene editing technologies.