Base and Prime Editing Technologies for Blood Disorders

Abstract

Nuclease-based genome editing strategies hold great promise for the treatment of blood disorders. However, a major drawback of these approaches is the generation of potentially harmful double strand breaks (DSBs). Base editing is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in the DNA without generating DSBs. Two major classes of base editors have been developed: cytidine base editors or CBEs allowing C>T conversions and adenine base editors or ABEs allowing A>G conversions. The scope of base editing tools has been extensively broadened, allowing higher efficiency, specificity, accessibility to previously inaccessible genetic loci and multiplexing, while maintaining a low rate of Insertions and Deletions (InDels). Base editing is a promising therapeutic strategy for genetic diseases caused by point mutations, such as many blood disorders and might be more effective than approaches based on homology-directed repair, which is moderately efficient in hematopoietic stem cells, the target cell population of many gene therapy approaches. In this review, we describe the development and evolution of the base editing system and its potential to correct blood disorders. We also discuss challenges of base editing approaches-including the delivery of base editors and the off-target events-and the advantages and disadvantages of base editing compared to classical genome editing strategies. Finally, we summarize the recent technologies that have further expanded the potential to correct genetic mutations, such as the novel base editing system allowing base transversions and the more versatile prime editing strategy.

Keywords: CRISPR/Cas9; base editing; blood diseases; genetic disorders; genome editing.

Figure 1

Cytosine and adenine base editors. (A) Cytosine base editors (CBEs), composed of a nickase Cas9 (nCas9) fused to a deaminase and one (in BE3s) or two (in BE4s) UGI (uracil glycosylase inhibitor), convert C:G into T:A base pairs in the editing window (nucleotide 4 to 8 in the protospacer, in green). (B) Adenine base editors (ABEs) are composed of a dead (d) or nickase (n) Cas9 (d/nCas9) fused to two TadA, one evolved to edit adenine in DNA (TadA*) and one wild type (TadA). ABEs convert A:T into G:C base pairs in the editing window (nucleotide 4 to 7 in the protospacer, in purple). Cas9 is guided by the sgRNA to the protospacer [which is followed by the PAM (protospacer adjacent motif)], unwinds the DNA and the deaminase converts the target base. Undesired events (bystander edits, in blue, and unwanted base conversion, in yellow) of CBEs and ABEs are shown in (A,B), respectively. The addition of the second UGI in CBEs (in BE4) and the removal of TadA in ABEs (ABE8) are highlighted with a gray dotted line. The gradient color of the editing window in the upper panels of (A,B) represents the enlarged editing window observed with novel BEs.

Figure 2

Potential ex vivo base editing approaches for genetic blood disorders. Schematic representation of base editing approaches to genetically correct HSCs from SCD and β-thalassemia patients (left) or to generate allogeneic CAR-T cells (right). (Left) Correction of the A>G β-thalassemic mutation (in position-28) and reversion of the SCD A>T mutation can be performed using CBEs and ABEs, respectively. HbF reactivation can be achieved (i) upon generation of HPFH mutations in HBG1/2 promoters by ABEs or CBEs or (ii) upon disruption of the BCL11A erythroid enhancer (located at position +58 kb from BCL11A transcription start site) by CBEs. BEs are delivered to HSCs as mRNA or RNP complexes. In ex vivo gene therapy approaches, HSCs genetically modified by BEs will be transplanted to the patient as a definitive therapy. (Right) Multiplex base editing of loci involved in alloreactivity (e.g., TRAC, B2M, PDC1D, CD7) and lentiviral vectors (LV)-mediated CAR expression to safely generate allogeneic CAR-T cells, which will be infused into patients to kill cancer cells.

Figure 3

Base editing delivery systems and potential off-target activity. BEs are delivered by plasmid chemical transfection (e.g., lipofectamine) or electroporation (yellow thunder), RNP or mRNA electroporation or LV/AAV transduction. BEs can cause RNA and DNA off-target effects in a sgRNA-independent (red dots) or -dependent (blue dots) manner. Off-target activity can be reduced by modifying the deaminase and/or the Cas9. Current methods used to predict and detect DNA and RNA off-targets are indicated in the table. WGS, Whole Genome Sequencing; WES, Whole Exome Sequencing.

Figure 4

Novel base and prime editors. Novel BEs with a modified deaminase, such as dual functioned BE and CGBE, convert AC-to-GT or CA-to-TG, and C-to-G, respectively. In the prime editing system, a reverse transcriptase uses a pegRNA to install substitutions, insertions, and deletions.