Figure 2.

Cas-SF01 engineering via a 2-step combined strategy

(A) Schematics of the EGxxFP reporter system for screening for high-efficiency Cas12i3 variants carrying combinations of mutations (Cas12i3VC). A short sequence is inserted inside the EGFP to disrupt its fluorescence. Targeted cleavage in the inserted sequence restores EGFP fluorescence due to DNA repair through single-strand annealing (SSA) between 2 flanking repeats. The system also constitutively expresses an mCherry marker. Editing efficiency is assessed by determining the ratio of EGFP⁺ cells over mCherry⁺ cells.

(B and C) The editing efficiencies of Cas12i3 with different mutation combinations for the PAM-interacting region (B) and target DNA strand-interacting region (C), as determined by the EGxxFP reporter. The mutation combinations with the highest editing rate increase in the 2 regions are highlighted in red boxes.

(D) Further combination of mutations identified from the screens results in variants with higher editing efficiency. The variant with highest editing efficiency contains 5 mutations (S7R/D233R/D267R/N369R/S433R) and was called Cas-SF01.

(E) Comparison of indel activity of Cas12i3 and Cas-SF01 at the TTR locus in mammalian cells. Significant difference was determined by Student’s t test.