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

By Vijay Kumar MalesuReviewed by Danielle Ellis, B.Sc.Jul 15 2024

In a recent study published in the Nature Biomedical Engineering, a group of researchers optimized prime editing (PE) for the efficient correction of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) F508del (a three-nucleotide deletion that is the predominant cause of CF) mutation in human airway epithelial cells, achieving high correction efficiencies and functional restoration with minimal off-target effects.

Background 

PE allows for precise genome editing without the need for double-stranded deoxyribonucleic acid (DNA) breaks, reducing the risk of bystander and off-target editing. Unlike nuclease-mediated methods, PE minimizes uncontrolled indels, large deletions, p53 activation, retrotransposon insertion, and chromosomal defects. It does not require donor DNA templates and is effective in both mitotic and non-mitotic cells, with successful applications in vivo in mice and non-human primates.

By combining a programmable nickase, reverse transcriptase, and PE guide ribonucleic acid (RNA), PE efficiently corrects pathogenic mutations. Further research is needed to enhance its efficiency, applicability, and safety in clinical settings.

About the study 

Guide RNA expression plasmids, including Prime Editing Guide RNAs (pegRNAs), Engineered (e)pegRNAs, Nicking Guide (ng) RNAs, Dead Single Guide (dsg) RNAs, and Single Guide (sg) RNAs, were cloned and purified using standard kits. DNA polymerase chain reaction (PCR) amplification used Phusion U Green Multiplex PCR master mix. Synthetic pegRNAs and epegRNAs had specific modifications and were sourced from Agilent Research Labs, Synthego, and Integrated DNA Technologies.

Prime editor and associated messenger (m)RNAs were generated via in vitro transcription and purified. Human Embryonic Kidney 293T Cells (HEK293T) cells from the American Type Culture Collection were cultured in modified Eagle medium, while 16HBEge-F508del (Immortalized Human Bronchial Epithelial Cells Homozygous for CFTR F508del Mutation) cells from the Cystic Fibrosis Foundation were cultured in essential medium. Primary airway epithelial cells were isolated from donors, expanded, and differentiated in PneumaCult Air-Liquid Interface Medium (PneumaCult-ALI) medium.

HEK293T cells were transfected using Lipofectamine 2000 with specific plasmid amounts and incubated for 72 hours. 16HBEge-F508del and primary airway epithelial cells were electroporated with PE RNA reagents and cultured. Genomic DNA was extracted using custom lysis protocols, and fluorescence-activated cell sorting prepared cells for sorting.

High-Throughput Sequencing (HTS) of genomic loci involved two rounds of PCR with Illumina-barcoded primers, and CRISPResso2 was used to analyze editing efficiency and indels. A homozygous CFTR F508del HEK293T cell line was created via PE2 strategy transfection and limiting dilution.

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

Study results 

The team sought to correct the CFTR F508del mutation soon after PE was originally reported in 2019. They first generated a clonal HEK293T cell line homozygous for this deletion in the endogenous CFTR gene. Using this cell line, they screened pegRNAs with different Primer Binding Site (PBS) and Reverse Transcription Template (RTT) lengths at two F508del-proximal protospacers (NGG1 and NGG2). They observed no correction with NGG1 pegRNAs, likely due to a TTTT sequence acting as a transcriptional terminator. Correction was detected with NGG2 pegRNAs, but editing efficiencies did not exceed 0.2%.

To enhance correction, a PE3 screen of two NGG2 pegRNAs was performed against various ngRNA protospacers. While the best PE3 NGG2 pegRNA showed improvement over PE2, the maximum mean editing did not exceed 0.5%. It was hypothesized that inefficient correction might be due to chromatin state affecting the accessibility of the edit 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 in PE were developed, including epegRNAs with RNA pseudoknot motifs to protect against exonuclease degradation, the inhibition of DNA mismatch repair (MMR) using MLH1dn, and the PEmax prime editor protein. The PE6 suite, featuring laboratory-evolved RTs and prime editor Cas9 domains, was also introduced.

Combining these enhancements, the team screened 178 epegRNAs with various PBS and RTT lengths across seven protospacers, including NGG and Protospacer Adjacent Motifs Recognized by a Modified Cas9 Variant (NGA PAMs). The Specific Designation of a Prime Editing Guide RNA with a Primer Binding Site Length of 13 and a Reverse Transcription Template Length of 41 (NGG2 PBS13 RTT41) strategy showed a significant improvement in editing rates.

Further development included testing ngRNAs from the earlier PE3 experiments with the epegRNA NGG2 PBS13 RTT41 in a PE5max experiment. This led to identifying a +104 nick that improved editing efficiency to 0.86%. Adding silent edits that disrupted NGG2's PAM and installed additional edits around it further enhanced efficiency. The best strategy, SE2, resulted in a mean editing rate of 6.8%, a fivefold improvement over previous attempts.

The team also evaluated recently evolved PE6 variants, finding that PE6b and PE6c further enhanced F508del correction. Using the optimized epegRNA with MLH1dn and the +104 ngRNA, PE6c achieved the highest editing rates. In 16HBEge-F508del cells, combining epegRNAs, silent edits, PE6 variants, and dsgRNAs improved editing efficiency significantly. The most efficient strategy achieved a mean F508del correction rate of 51%.

Testing this strategy in primary airway epithelial cells from CF patients resulted in a mean correction rate of 25%, with substantial rescue of CFTR channel activity. On-target and off-target analyses indicated minimal unintended editing outcomes, demonstrating the strategy's precision and potential for therapeutic application.

Conclusions 

To summarize, initial PE3 experiments yielded 0.42% mean correction, while applying recent advancements, including engineered pegRNAs (epegRNAs), PEmax, MLH1dn, silent edits, PE6, and dsgRNAs, led to up to 11%, 58%, and 25% mean correction in HEK293T, 16HBEge-F508del, and primary airway epithelial cells from CF patients, respectively.

These enhancements resulted in minimal off-target effects (≤0.1%). The optimized PE system offers a high correction-to-indel ratio without double-stranded DNA breaks or DNA templates, simplifying potential clinical applications for cystic fibrosis treatment.