Engineering a transposon-associated TnpB-ωRNA system for efficient gene editing and phenotypic correction of a tyrosinaemia mouse model

Abstract

Transposon-associated ribonucleoprotein TnpB is known to be the ancestry endonuclease of diverse Cas12 effector proteins from type-V CRISPR system. Given its small size (408 aa), it is of interest to examine whether engineered TnpB could be used for efficient mammalian genome editing. Here, we showed that the gene editing activity of native TnpB from Deinococcus radiodurans (ISDra2 TnpB) in mouse embryos was already higher than previously identified small-sized Cas12f1. Further stepwise engineering of noncoding RNA (ωRNA or reRNA) component of TnpB significantly elevated the nuclease activity of TnpB. Notably, an optimized TnpB-ωRNA system could be efficiently delivered in vivo with single adeno-associated virus (AAV) and corrected the disease phenotype in a tyrosinaemia mouse model. Thus, the engineered miniature TnpB system represents a new addition to the current genome editing toolbox, with the unique feature of the smallest effector size that facilitate efficient AAV delivery for editing of cells and tissues.

Fig. 1. Mouse embryo injection of TnpB-ωRNA induced efficient gene editing.

a Characteristics of SpCas9, IscB, LbCas12a, Un1Cas12f1 and ISDra2 TnpB nucleases. b Comparison of editing efficiency between TnpB and Cas12f1 on Tyr gene in mice. c Coat color phenotype of Tyr gene-edited mice by Un1Cas12f1 and TnpB. d TnpB-mediated gene editing efficiency for Dmd gene. e Dystrophin and laminin-2 immunostaining for TA, DI, and heart muscle tissues in wild-type and Dmd-edited mice by TnpB. f Western blotting of dystrophin and vinculin protein for three muscle tissues in wild-type and Dmd-edited mice by TnpB. Data are represented as means ± SEM. A dot represents a biological replicate (n = 3 or more). Unpaired two-tailed Student’s t tests. Significant differences between conditions are indicated by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS non-significant). Scale bars, 100 μm. Source data are provided as a Source Data file.

Fig. 2. Stepwise engineering of TnpB-associated ωRNA improved gene editing efficiency.

a Reporter assay schematics of detecting cleavage activity of TnpB-ωRNA. b Predicted secondary structure of cognate ωRNA (231 nt). Cognate ωRNA was divided into 6 segments, named from S1 to S6. c Reporter assay results using engineered ωRNA by one-by-one truncation of S1 to S6. d Reporter assay results with engineered ωRNA by different combined truncations of S1 to S5. e Predicted secondary structure of a ωRNA-v1 variant with simultaneous truncation of S1, S2, and S3. f Reporter assay results for ωRNA variants with different SL deletion and modifications. g Predicted secondary structure of final optimized ωRNA-v2 (ωRNA*) variant. Data are represented as means ± SEM. A dot represents a biological replicate (n = 3). Source data are provided as a Source Data file.

Fig. 3. Characterization of endogenous gene editing activity and off-target effect with optimized TnpB-ωRNA system.

a The experimental workflow for detecting editing efficiency of original and optimized TnpB-ωRNA in HEK293T cells. b Gene efficiency comparison results for 14 human endogenous gene loci targeted by wild-type and optimized TnpB-ωRNA in HEK293T. c Gene editing activity comparison among TnpB-ωRNA, TnpB-ωRNA*, SaCas9 and SpCas9. All data points with median at center line and 25th and 75th quartile lines. Detailed statistical results in Source Data file. d Off-target analysis for top predicted off-target genomic loci via Cas-OFFinder. e Genome-wide off-target analysis with PEM-seq for the engineered TnpB-ωRNA. Data are represented as means ± SEM. A dot represents a biological replicate (n = 3 or more). Unpaired two-tailed Student’s t tests. Significant differences between conditions are indicated by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS non-significant.). Source data are provided as a Source Data file.

Fig. 4. Correction of fatal liver disease with in vivo delivery of TnpB-ωRNA via single AAV.

a Diagram of AAV-TnpB-ωRNA/ωRNA* vector and gene therapy schematics in Fah^−/− mouse model of type I hereditary tyrosinaemia. b Survival curve for disease mice treated with AAV-TnpB-ωRNA/ωRNA* or AAV-TnpB without ωRNA (TnpB only group). c Body weight change during the observation period for disease mice in different treatment groups. d Body weight ratio for TnpB-ωRNA or TnpB only versus TnpB-ωRNA*-treated mice in 19-day after NTBC withdrawal. e Histology analysis with H&E and Sirius red staining for mouse liver from different treatment groups. f Western blot for HPD protein from untreated and TnpB-ωRNA-treated HT1 mice. Data are represented as means ± SEM. A dot represents a biological replicate (n = 3 or more). Unpaired two-tailed Student’s t tests. Significant differences between conditions are indicated by an asterisk (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS non-significant.). Scale bars, 200 μm. Source data are provided as a Source Data file.