Design and Engineering of Light‐Induced Base Editors Facilitating Genome Editing with Enhanced Fidelity

Abstract Base editors, which enable targeted locus nucleotide conversion in genomic DNA without double‐stranded breaks, have been engineered as powerful tools for biotechnological and clinical applications. However, the application of base editors is limited by their off‐target effects. Continuously expressed deaminases used for gene editing may lead to unwanted base alterations at unpredictable genomic locations. In the present study, blue‐light‐activated base editors (BLBEs) are engineered based on the distinct photoswitches magnets that can switch from a monomer to dimerization state in response to blue light. By fusing the N‐ and C‐termini of split DNA deaminases with photoswitches Magnets, efficient A‐to‐G and C‐to‐T base editing is achieved in response to blue light in prokaryotic and eukaryotic cells. Furthermore, the results showed that BLBEs can realize precise blue light‐induced gene editing across broad genomic loci with low off‐target activity at the DNA‐ and RNA‐level. Collectively, these findings suggest that the optogenetic utilization of base editing and optical base editors may provide powerful tools to promote the development of optogenetic genome engineering.


Table of Contents
Supporting Tables Table S1.Target sgRNA-protospacer sequence for E. coli DH10B and HEK293T in this study.
Table S2.Sequences of protospacers and primers for sgRNA-independent and dependent off-target sites for E. coli DH10B and HEK293T in this study.

Supporting Sequences
Sequence S1.Amino acid sequences used for E. coli DH10B in this study.
Sequence S2.Amino acid sequences used for HEK293T in this study.

Figure S1
. The construction and functional verification of blue light-activated adenine base editor.a) The number of clones with rifampicin resistance for all chimeric sfGFP-TadA-8e proteins.Counts of four independent replicates (n = 4) for each protein variant are displayed.b) Photos of clones for E. coli DH10B containing rifampicin resistance.Intact TadA-8e servers as the positive control.The photos from three independent experiments.c) Schematic diagram of split sfGFP for analyzing the spontaneous dimerization of split TadA-8e variants.The dimerization of split-TadA-8e (N-TadA-8e and C-TadA-8e) induces the polymerization of sfGFP 1-10 and sfGPF 11 to recover the function of fluorescence.Introduce a DNA sequence within the sfGFP coding gene to split sfGFP, including a stop codon (TAA), ribosome binding site (RBS), and an initiation codon (ATG).d) DNA sequencing chromatograms of different BLABE systems with different split sites in the presence and absence of blue light.The arrow indicates that the red base is the potential editing site.The target site ABES4 is treated with different light intensities (2.5 mW cm -2 ; 5 mW cm - 2 ; 10 mW cm -2 ) for 300 min.b) Bar plots showing on-target DNA base editing efficiency of BLCBE under various blue light intensities.The target site CBES3 is treated with different light intensities (dark; 2.5 mW cm -2 ; 5 mW cm -2 ; 10 mW cm -2 ) for 300 min.The editing efficiency of ABES4 and CBES3 are calculated from three independent replicates (n = 3).

Figure S8
. Total RNA mutation types in the transcriptome.a-c) The frequencies of total RNA mutation types in the transcriptome for E. coli DH10B (a, negative control), adenine base editor (b), and cytosine base editor (c).The E. coli DH10B is not treated as a negative control.Left, the donut chart shows the proportion of various types of RNA mutation in the total transcriptomic single nucleotide polymorphism (SNP) mutation.Right, the mean values of RNA mutation frequencies across all mutation types of the transcriptome.The RNA mutation frequencies of three independent replicates are displayed (n = 3).Supporting Tables Table S1.Target sgRNA-protospacer sequence for E. coli DH10B and HEK293T in this study.

Figure S1 .
Figure S1.The construction and functional verification of blue light-activated adenine base editor.

Figure S2 .
Figure S2.Split deaminases strategy for the construction of BLCBE Figure S3.The optimization of linker length of BLABE and BLCBE.

Figure S4 .
Figure S4.The performance of BLABE and BLCBE for base editing in E. coli DH10B.

Figure S6 .
Figure S6.Allele frequencies in the entire amplicon of DNA on-target and sgRNA-dependent offtarget editing at diverse loci in E. coli DH10B.

Figure S7 .
Figure S7.Allele frequencies in the amplicon of DNA on-target and sgRNA-independent off-target editing at diverse genomic loci in E. coli DH10B.

Figure S8 .
Figure S8.Total RNA mutation types in the transcriptome.

Figure S9 .
Figure S9.The optimization of expression strategies and plasmid ratio for HEK293T cells transfection of BLCBE.

Figure S10 .
Figure S10.Allele frequencies in the amplicon of sgRNA-dependent off-target editing at genomic loci for ABE, BLABE, CBE, and BLCBE systems.

Figure S11 .
Figure S11.Allele frequencies in the amplicon of sgRNA-independent off-target editing at diverse genomic loci for ABE and BLABE systems.

Figure S12 .
Figure S12.Allele frequencies in the amplicon of sgRNA-independent off-target editing at diverse genomic loci for CBE and BLCBE systems.

Figure S13 .
Figure S13.Off-target editing of base editor systems on the transcriptome in HEK293T cells.

Figure S14 .
Figure S14.RNA off-target editing induced by ABE and BLABE at all chromosome locations.

Figure S15 .
Figure S15.RNA off-target editing induced by CBE and BLCBE at all chromosome locations.

NFigure S2 .Figure S3 .Figure S4 .
Figure S2.Split deaminase strategy for the construction of BLCBE.a) Schematic of potential split sites on the APOBEC3A (A3A) amino acid sequence.Four candidate sites for splitting are located in the loop area, marked with the red triangle, and split positioned between two amino acids.The secondary structure of A3A is highlighted in different colors (α helix, dark blue; β sheet, light blue).b) Cartoon representation of A3A protein.The potential sfGFP insertion sites for A3A are behind the red-labeled amino acids.c-f) Base editing efficiency of various BLCBE systems, including N42 (c), D85 (d), T118 (e), and G147 (f).Base editing efficiency is calculated by EditR (N.D. no detected; n = 3 independent replicates).g) DNA sequencing chromatograms of selection of photoswitches for different BLCBE systems.The Magnets are classified into three levels, and the reverse sgRNA sequences are shown.All possible editing sites are marked with black arrows.

Figure S5 .
Figure S5.Fluorescence image showing sfGFP variants expression.a, b) The E. coli DH10B with plasmids expressing the sfGFP mutants and BLABE (a) or BLCBE (b) are cultured for 540 min and at 240 min treated with blue light.The fluorescence images are obtained at 60 and 540 min under light and dark conditions.Scar bar, 20 μm.Intact ABE and CBE serve as positive controls.

Figure S6 .
Figure S6.Allele frequencies in the entire amplicon of DNA on-target and sgRNA-dependent offtarget editing at diverse loci.a) Allele nucleotide percentages of DNA on-target and off-target base editing of BLABE targeting two sites, ABES4 and ABES19.ABE serves as the positive control, and the target editing efficiency of BLABE is tested in the presence and absence of blue light.b) Allele frequencies of DNA on-target editing within target sites and off-target allele efficiency at diverse genomic loci in the presence and absence of blue light for BLCBE.The possible editing sites within the protospacer sequence are marked by red arrows and the unexpected editing are indicated by black arrows.The base substitutions and deletions are represented with bold letters and short dashes, respectively.The editing window is indicated by red double-ended dotted lines.PAM sequences for SpCas9 (NGG) and SaCas9 (NNGRRT) are marked by short blue lines.The values on the right of the graph represent frequencies and mutation alleles' reads (n = 3).

Figure S7 .
Figure S7.Allele frequencies in the amplicon of DNA on-target and sgRNA-independent off-target editing at diverse genomic loci.a,b) Allele frequencies of on-target editing and sgRNA-independent off-target for BLABE (a) and BLCBE (b) in the presence and absence of blue light.The allele frequencies of off-target within the R-loop region are calculated by amplicon sequencing, and the protospacer sequence is pointed by the purple font.The possible editing sites within the protospacer sequence are marked by red arrows and the unexpected editing are indicated by black arrows.The base substitutions and deletions are represented with bold letters and short dashes, respectively.The editing window is indicated by red double-ended dotted lines.PAM sequences for SpCas9 (NGG) and SaCas9 (NNGRRT) are marked by short blue lines.The values on the right of the graph represent frequencies and mutation alleles' reads (n = 3).

Figure S9 .
Figure S9.The optimization of expression strategies and plasmid ratio for HEK293T cells transfection of BLCBE.a) Target cytosine base editing efficiency for various strategies of expression for BLCBE, HEK293T cells were transfected using BLCBE with various protein expression strategies, where the BLCBE systems were expressed using P2A, IRES, and cleavage assays.The base editing efficiency of HEK2 is shown by the bar chart (n = 3, N.D. no detected).b) Target cytosine base editing efficiency for transfection of HEK293T using dual plasmids system.The numbers of 0.3 ~ 3 in the legend mean the ratios of transfected plasmids (pCMV-A3AN:pCMV-pMag-A3AC).The bar chart shows the efficiency of HEK2 editing efficiency using different ratios of the plasmids for the expression of BLCBE.(n = 3, N.D. no detected).

Figure S10 .Figure S11 .Figure S12 .Figure S13 .Figure S14 .Figure S15 .
Figure S10.Allele frequencies in the amplicon of sgRNA-dependent off-target editing at genomic loci for ABE, BLABE, CBE, and BLCBE systems.a) Allele frequencies of sgRNA-dependent offtarget for ABE and BLABE with sgRNA targeting HEK2.The off-target site was selected by Cas-OFFinder and named HEK2-OT1.The BLABE system was treated under blue light and darkness respectively.b) Allele frequencies of sgRNA-dependent off-target for CBE and BLCBE with sgRNA targeting HEK2.The off-target site was selected by Cas-OFFinder and named HEK2-OT1.The BLCBE system was treated under blue light and darkness respectively.The grey rectangle indicates the sequence of protospacer within sgRNA.The allele frequencies of off-target within the R-loop region are calculated by amplicon sequencing.The values on the right of the graph represent frequencies and mutation alleles' reads.The possible editing sites within the protospacer sequence are marked by red arrows and the unexpected editing are indicated by black arrows.The base substitutions and deletions are represented with bold letters and short dashes, respectively.The editing window is indicated by red double-ended dotted lines.PAM sequences for SpCas9 (NGG) and SaCas9 (NNGRRT) are marked by short blue lines.The values on the right of the graph represent frequencies and mutation alleles' reads (n = 3).