Structure-Guided Engineering of a Cas12i Nuclease Unlocks Near-PAMless Genome Editing

Area of Science:

• Molecular Biology
• Gene Editing Technologies

Background:

• CRISPR-Cas nucleases are powerful genome editing tools but are limited by strict Protospacer Adjacent Motif (PAM) recognition sequences.
• This PAM specificity restricts the accessible sites within the genome for therapeutic and research applications.

Purpose of the Study:

• To engineer a compact Cas12i nuclease (SF01) with relaxed PAM specificity.
• To expand the targeting range of CRISPR-Cas genome editing tools.

Main Methods:

• Structure-guided engineering of the SF01 nuclease using AlphaFold-predicted models.
• Systematic mutagenesis of 38 residues at the PAM-interacting interface.
• Construction and testing of adenine base editors (ABEs) with engineered variants.

Main Results:

• Developed three superior SF01 variants (KR, IKRR, STKRR) with relaxed PAM specificity, enabling editing at 5'-NNTN-3' sites.
• Achieved a four-fold increase in targetable genome portion (over 25%) with near-PAMless activity.
• Engineered ABEs demonstrated high-efficiency editing (~80%) at endogenous loci with expanded targeting scope.
• Off-target analysis confirmed no loss of specificity despite enhanced on-target activity.

Conclusions:

• Engineered SF01 variants significantly expand the genome editing toolkit by overcoming PAM constraints.
• These variants enable previously inaccessible genome editing applications.
• The enhanced specificity and broad targeting scope offer a powerful new tool for molecular biology and gene therapy.