TY - JOUR

T1 - Engineering of the High-Precision Cas12m Dual Base Editor-Loaded Virus-like Particles for A-to-G and C-to-T Conversions

AU - Aliev, Timur

AU - Imatdinov, Almaz

AU - Prudnikova, Elena

AU - Imatdinov, Ilnaz

N1 - Aliev, T.; Imatdinov, A.; Prudnikova, E.; Imatdinov, I. Engineering of the High-Precision Cas12m Dual Base Editor-Loaded Virus-like Particles for A-to-G and C-to-T Conversions. Genes 2026, 17, 1247. https://doi.org/10.3390/genes17101247 This research was funded by the Ministry of Science and Higher Education of the Russian Federation (The Federal Scientific-Technical Program for Genetic Technologies Development for 2019–2030, agreement № 075-15-2025-526).

PY - 2026/10/9

Y1 - 2026/10/9

N2 - Background/Objectives: CRISPR/Cas-dependent base editing enables the precise correction of genomic sequences by direct modification of DNA bases without creating potentially mutagenic DNA double-strand breaks. Base editing is considered a therapeutic approach for rare monogenic disorders or viral infections. However, many variants of base editors suffer from limited specificity because they induce bystander edits within a broad editing window. However, nucleotide substitution is sufficient to correct a mutation or to achieve a therapeutic effect. Methods: PCR, cloning by restriction digest, and ligation were used to create genetic constructs. The editing efficiency was evaluated using NGS. Virus-like particles were produced in HEK293T cells using transient expression. VLPs were visualized by electron microscopy. Results: Using dual base editors that induce C-to-T and A-to-G substitutions, we show that high editing precision can be achieved by engineering the linker between the MmCas12m domain and the deaminase domain of the editor. By testing different linker sequences, we have created high-precision dual base editors with narrowed editing windows that can edit a single nucleotide at a target position with high accuracy. Conclusions: We found that shortening the linker to an acceptable length provides a single-nucleotide substitution in vitro with minimal bystander activity (less than 0.8%), while the high-precision MmCas12m dual base editor retains up to 90% of the editing efficiency of the original base editor. These high-precision base editors will be useful in cases where high precision is required, and editing of adjacent nucleotides is undesirable.

AB - Background/Objectives: CRISPR/Cas-dependent base editing enables the precise correction of genomic sequences by direct modification of DNA bases without creating potentially mutagenic DNA double-strand breaks. Base editing is considered a therapeutic approach for rare monogenic disorders or viral infections. However, many variants of base editors suffer from limited specificity because they induce bystander edits within a broad editing window. However, nucleotide substitution is sufficient to correct a mutation or to achieve a therapeutic effect. Methods: PCR, cloning by restriction digest, and ligation were used to create genetic constructs. The editing efficiency was evaluated using NGS. Virus-like particles were produced in HEK293T cells using transient expression. VLPs were visualized by electron microscopy. Results: Using dual base editors that induce C-to-T and A-to-G substitutions, we show that high editing precision can be achieved by engineering the linker between the MmCas12m domain and the deaminase domain of the editor. By testing different linker sequences, we have created high-precision dual base editors with narrowed editing windows that can edit a single nucleotide at a target position with high accuracy. Conclusions: We found that shortening the linker to an acceptable length provides a single-nucleotide substitution in vitro with minimal bystander activity (less than 0.8%), while the high-precision MmCas12m dual base editor retains up to 90% of the editing efficiency of the original base editor. These high-precision base editors will be useful in cases where high precision is required, and editing of adjacent nucleotides is undesirable.

KW - базовое редактирование

KW - CRISPR/Cas

KW - линкер

KW - Cas12m

KW - Cas9

KW - TadDE

KW - VLP

KW - base editing

KW - CRISPR/Cas

KW - linker

KW - Cas12m

KW - Cas9

KW - TadDE

KW - VLP

UR - https://www.mendeley.com/catalogue/ad747114-4ddd-3c2f-996e-a2ae3905b1a2/

UR - https://www.scopus.com/pages/publications/105054775671

U2 - 10.3390/genes17101247

DO - 10.3390/genes17101247

M3 - Article

C2 - 42938363

VL - 17

JO - Genes

JF - Genes

SN - 2073-4425

IS - 10

M1 - 1247

ER -