Loop engineering of AtCas9 for effective and broad genome editing

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Efficient genome editing in mammalian cells is crucial for developing CRISPR-based therapeutics.
• Current Cas9 enhancement strategies primarily focus on point mutations, leaving loop engineering underexplored.

Purpose of the Study:

• To investigate loop engineering as a method to enhance Cas9 nuclease and base editing efficiency.
• To develop improved Cas9 variants for therapeutic applications and expanded genome editing capabilities.

Main Methods:

• Engineered surface-exposed loops of thermophilic AtCas9 by substituting with counterparts from mesophilic Nme1Cas9 to create the AtCas9-Z7 variant.
• Utilized biochemical assays to assess RNP-DNA interactions and binding affinity under magnesium-limiting conditions.
• Employed molecular dynamics simulations to analyze the conformational stability of engineered Cas9 variants.
• Combined loop engineering with structure-guided point mutations to further enhance Cas9 activity.

Main Results:

• The AtCas9-Z7 variant demonstrated significantly improved nuclease and base editing efficiency.
• Z7 maintained high Cas9 binding affinity under magnesium-limiting conditions, overcoming a common constraint in mammalian cells.
• The Z7-E78-ABE variant showed a 5.76-fold increase in editing efficiency, expanded PAM recognition, and enabled editing in primary human T cells.
• Loop transplantation into GeoCas9 and ThermoCas9 resulted in median efficiency increases of 14.50-fold and 7.37-fold, respectively, at non-canonical PAMs.

Conclusions:

• Loop engineering is an effective and streamlined strategy for enhancing Cas9 performance in genome editing.
• This approach can be combined with other optimization methods to further boost Cas9 activity and expand its utility.
• Loop engineering presents a rational and modular strategy for Cas9 optimization with significant therapeutic potential for CRISPR-based applications.