Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases

piggyPrime: High-Efficacy Prime Editing in Human Cells Using piggyBac-Based DNA Transposition

“…These unique properties of prime editing are based on the delivery of a prime editor (PE), consisting of a Cas9-reverse transcriptase fusion protein (hereafter referred to as PE2), along with the prime editing guide RNA (pegRNA) that specifies both the genomic target as well as the desired edit to be written directly into the genome. Prime editing has tremendous potential for treatment of disease-causing mutations, as well as generation of disease models, both in vitro and in vivo ( Schene et al, 2020 ; Jang et al, 2021 ; Kim et al, 2021 ; Liu et al, 2021 ; Park et al, 2021 ; Petri et al, 2021 ; Qian et al, 2021 ). However, the use of prime editing is currently challenged by low efficacy, leading to time-consuming optimization and/or screening approaches in order to achieve satisfactory editing activities ( Liu et al, 2020 ; Schene et al, 2020 ; Chemello et al, 2021 ; Kim et al, 2021 ; Petri et al, 2021 ).…”

Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain

“…To further improve prime-editing efficiency, a single-guide RNA (sgRNA) can be added to PE2, resulting in PE3 and PE3b. PE3 and PE3b often, but not always, exhibit higher prime-editing efficiency at the expense of a higher risk of generating unintended indels 1,[9][10][11][12] .…”

Broadening the reach and investigating the potential of prime editors through fully viral gene-deleted adenoviral vector delivery

“…In particular, a dual AAV protein trans-splicing PE3 system corrected 0.6%, 2.3% and 3.1% of defective alleles in livers of alpha-1 antitrypsin deficiency mice at 2, 6 and 10 weeks post-injection, respectively ( 57 ). Another study reports that, in the absence of positive selection for gene-corrected cells, dual AAV RNA trans-splicing PE2 and PE3 systems did not lead to detectable correction of defective alleles in livers of hereditary tyrosinemia type 1 mice ( 58 ). Notably, however, using the same AAV platform, gene correction levels ranging from 4.1% to 7.4% were measured in RPE65 -associated Leber congenital amaurosis mice after sub-retinal injections ( 58 ).…”

“…Another study reports that, in the absence of positive selection for gene-corrected cells, dual AAV RNA trans-splicing PE2 and PE3 systems did not lead to detectable correction of defective alleles in livers of hereditary tyrosinemia type 1 mice ( 58 ). Notably, however, using the same AAV platform, gene correction levels ranging from 4.1% to 7.4% were measured in RPE65 -associated Leber congenital amaurosis mice after sub-retinal injections ( 58 ). In another recent study, a dual AAV protein trans-splicing system installed a G > T transversion within Dnmt1 at a frequency of 1.71 ± 1.35% 6 weeks after sub-retinal injections in mice ( 59 ).…”