Prime editing for precise and highly versatile genome manipulation

Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

Prime editing: advances and therapeutic applications.

Engineered CRISPR prime editors with compact, untethered reverse transcriptases

CRISPR technologies for genome, epigenome and transcriptome editing.

Prime editor (PE), which is developed by combining Cas9 nickase and an engineered reverse transcriptase, can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here, we develop spegRNA by introducing same-sense mutations at proper positions in the reverse-transcription template of pegRNA to increase PE's base-editing efficiency up-to 4,976-fold (on-average 353-fold). We also develop apegRNA by altering the pegRNA secondary structure to increase PE's indel-editing efficiency up-to 10.6-fold (on-average 2.77-fold). The spegRNA and apegRNA can be combined to further enhance editing efficiency. When spegRNA and apegRNA are used in PE3 and PE5 systems, the efficiencies of sPE3, aPE3, sPE5 and aPE5 systems are all enhanced significantly. The strategies developed in this study realize highly efficient prime editing at certain previously uneditable sites. 

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Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure

Reactivating silenced γ-globin expression through the disruption of repressive regulatory domains offers a therapeutic strategy for treating β-hemoglobinopathies. Here, we used transformer base editor (tBE), a recently developed cytosine base editor with no detectable off-target mutations, to disrupt transcription-factor-binding motifs in hematopoietic stem cells. By performing functional screening of six motifs with tBE, we found that directly disrupting the BCL11A-binding motif in HBG1/2 promoters triggered the highest γ-globin expression. Via a side-by-side comparison with other clinical and preclinical strategies using Cas9 nuclease or conventional BEs (ABE8e and hA3A-BE3), we found that tBE-mediated disruption of the BCL11A-binding motif at the HBG1/2 promoters triggered the highest fetal hemoglobin in healthy and β-thalassemia patient hematopoietic stem/progenitor cells while exhibiting no detectable DNA or RNA off-target mutations. Durable therapeutic editing by tBE persisted in repopulating hematopoietic stem cells, demonstrating that tBE-mediated editing in HBG1/2 promoters is a safe and effective strategy for treating β-hemoglobinopathies. 

Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs.

Design and application of the transformer base editor in mammalian cells and mice.


 Activating the silent γ-globin expression is a universal therapeutic strategy for treating β-hemoglobinopathies including β-thalassemia and sickle cell disease. Several therapeutic target sites have been identified and the editing at these sites can reactivate γ-globin expression. In this study, we used a recently developed cytosine base editor, transformer base editor (tBE), which triggers no observable off-target mutations, to edit the therapeutic target sites for triggering γ-globin expression. Through editing six therapeutic sites, we found that the editing at BCL11A binding motif in the HBG1/2 promoters induced the highest γ-globin expression level. By comparing to other approaches such as Cas9 nuclease or other base editors, tBE-meditated editing at BCL11A binding motif induced higher fetal hemoglobin (HbF) expression and meanwhile did not trigger any detected DNA or RNA off-target mutations. Of note, tBE-mediated editing at the HBG1/2 promoters induced more robust HbF expression in the erythroid progeny from β 0/β 0 thalassemia patient-derived hematopoietic stem and progenitor cells (HSPCs) than CRISPR/Cas9 approach. Moreover, persistent base editing by tBE was found in long-term repopulating hematopoietic stem cells. Taken together, this study demonstrates that tBE-mediated therapeutic base editing at BCL11A binding motif in HBG1/2 promoters is a safe and highly effective strategy for treating β-hemoglobinopathies.

Innovative Transformer Base Editor (tBE)-Mediated Editing at BCL11A Binding Motif Triggers High Level of γ-Globin Expression with No Observable Off-Target Mutations

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Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure

Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs.

Design and application of the transformer base editor in mammalian cells and mice.

Innovative Transformer Base Editor (tBE)-Mediated Editing at BCL11A Binding Motif Triggers High Level of γ-Globin Expression with No Observable Off-Target Mutations