One-time prime editing targets a common genetic cause of cystic fibrosis

In a recently published study, Nature Biomedical EngineeringA group of researchers optimized prime editing (PE) to efficiently correct the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) F508del (a tri-nucleotide deletion that is the primary cause of CF) mutation in human airway epithelial cells, achieving high correction efficiency and functional recovery while minimizing off-target effects.

background

PE enables precise genome editing without the need for double stranding. Deoxyribonucleic Acid PE prevents DNA breaks, reducing the risk of bystander and off-target editing. Unlike nuclease-mediated methods, PE minimizes uncontrolled indels, large deletions, p53 activation, retrotransposon insertions, and chromosomal defects. It does not require a donor DNA template, is effective in both mitotic and non-mitotic cells, and has been successfully applied in vivo in mice and non-human primates.

Combining programmable nickase, reverse transcriptase and PE guides Ribonucleic Acid (RNA), PE efficiently corrects pathogenic mutations. Further research is needed to improve its efficiency, applicability, and safety in clinical settings.

About the Research

Guide RNA expression plasmids, including primed edited guide RNA (pegRNA), engineered (e)pegRNA, nicked guide (ng) RNA, dead single guide (dsg) RNA, and single guide (sg) RNA, were cloned and purified using standard kits. Phusion U Green Multiplex PCR Master Mix was used for DNA polymerase chain reaction (PCR) amplification. Synthetic pegRNA and epegRNA, with specific modifications, were obtained from Agilent Research Labs, Synthego, and Integrated DNA Technologies.

Prime editors and associated messenger (m)RNAs were generated by in vitro transcription and purified. Human embryonic kidney 293T cells (HEK293T) from the American Type Culture Collection were cultured in modified Eagle's medium, and 16HBEge-F508del (immortalized human bronchial epithelial cells homozygous for the CFTR F508del mutation) cells from the Cystic Fibrosis Foundation were cultured in essential medium. Primary airway epithelial cells were isolated from donors and grown and differentiated in PneumaCult air-liquid interface medium (PneumaCult-ALI) medium.

HEK293T cells were transfected with the specified plasmid amounts using Lipofectamine 2000 and cultured for 72 h. 16HBEge-F508del and primary airway epithelial cells were electroporated with PE RNA. reagent Genomic DNA was extracted using a custom lysis protocol and cells were prepared for sorting by fluorescence-activated cell sorting.

High-throughput sequencing (HTS) of genomic loci included two rounds of PCR using Illumina barcoded primers, and editing efficiency and indels were analyzed using CRISPResso2. Homozygous CFTR F508del HEK293T cell lines were generated by PE2 strategy transfection and limiting dilution.

pegRNA sequences were designed, synthesized, and tested using DeepPrime and Prime Editing Rational Design and Implementation Computational Tool Version 2.0 (PRIDICT 2.0). CFTR channel activity was assessed by Ussing chamber assay. Off-target site assignment was performed using Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq), and top sites were analyzed for indels and substitutions using HTS.

research result

The research team attempted to correct the CFTR F508del mutation shortly after PE was first reported in 2019. First, they generated a clonal HEK293T cell line homozygous for this deletion in the endogenous CFTR gene. They used this cell line to screen PEGRNAs with different lengths of primer binding site (PBS) and reverse transcription template (RTT) in the two F508del proximal protospacers (NGG1 and NGG2). No correction was observed in NGG1 PEGRNA, likely because the TTTT sequence functions as a transcription terminator. Although a correction was detected in NGG2 PEGRNA, the editing efficiency did not exceed 0.2%.

To enhance correction, a PE3 screen of two NGG2 pegRNAs was performed against various ngRNA protospacers. The best PE3 NGG2 pegRNA showed improvement over PE2, but the maximum average editing did not exceed 0.5%. It was hypothesized that the inefficient correction may be due to chromatin conditions affecting the accessibility of the editing site to Cas effectors. Efficient A•T to G•C editing using an adenine base editor (ABE) confirmed that NGG1 and NGG2 were accessible by pegRNA-guided Cas effectors.

Several advances have been developed in PE, including epegRNA with an RNA pseudoknot motif to protect against exonuclease degradation, inhibition of DNA mismatch repair (MMR) using MLH1dn, and the PEmax prime editor protein, while the PE6 suite with laboratory-evolved RT and prime editor Cas9 domains has also been introduced.

Combining these enhancements, the team screened 178 epegRNAs with various PBS and RTT lengths across seven protospacers, including NGG and the protospacer adjacent motif (NGA PAM) recognized by engineered Cas9 variants. A specific designation of primed editing guide RNA with a primer binding site length of 13 and reverse transcription template length of 41 (NGG2 PBS13 RTT41) strategy significantly improved the editing rate.

Further development included testing ngRNA from the previous PE3 experiment with epegRNA NGG2 PBS13 RTT41 in a PE5max experiment. This identified a +104 nick that improved the editing efficiency to 0.86%. Efficiency was further improved by adding a silent edit that disrupted the PAM of NGG2 and installed additional edits around it. The best strategy, SE2, resulted in an average editing rate of 6.8%, a 5-fold improvement over previous attempts.

The research team also evaluated recently evolved PE6 variants and found that PE6b and PE6c further enhanced F508del correction. Using optimized epegRNA together with MLH1dn and +104 ngRNA, PE6c achieved the highest editing rate. In 16HBEge-F508del cells, the combination of epegRNA, silent editing, PE6 variants, and dsgRNA significantly improved editing efficiency. The most efficient strategy reached an average F508del correction rate of 51%.

This strategy was tested in primary airway epithelial cells from CF patients, resulting in an average correction rate of 25% and significant restoration of CFTR channel activity. On-target and off-target analysis demonstrated minimal unintended editing outcomes, demonstrating the precision and potential therapeutic application of this strategy.

Conclusion

In summary, while initial PE3 experiments yielded an average correction of 0.42%, application of recent advances including engineered pegRNA (epegRNA), PEmax, MLH1dn, silent editing, PE6, and dsgRNA has yielded average corrections of up to 11%, 58%, and 25% in HEK293T, 16HBEge-F508del, and primary airway epithelial cells from CF patients, respectively.

With these enhancements, off-target effects were minimized (≤0.1%), and the optimized PE system provided a high correction-to-indel ratio without double-stranded DNA breaks or DNA templates, simplifying its potential clinical application for cystic fibrosis treatment.