The selection of optimal pegRNAs to enhance the efficiency of prime editing in AT-rich genome regions

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Abstract

Prime editing is a highly promising strategy for treating hereditary disorders due to its superior efficiency and safety profile compared to conventional CRISPR-Cas9 systems. This study is dedicated to development of a causal therapy for cystic fibrosis by targeting the F508del variant in the CFTR gene using prime editing, as this specific deletion accounts for a substantial proportion of cystic fibrosis cases. While prime editing has shown remarkable precision in introducing targeted genetic modifications, its application in AT-rich genomic regions, such as the one containing the F508del variant, remains challenging. To overcome this limitation, we systematically evaluated 24 pegRNAs designed for two distinct prime editing systems, PEmax and PE2-NG. The correction efficiency of the F508del variant reached 2.81% in basal lung cells from homozygous F508del patients, without normalization for transfection efficiency. However, the average transfection efficiency was only 11.9%, emphasizing the critical need for advancements in delivery methodologies. These findings highlight the potential of prime editing as a therapeutic approach for cystic fibrosis, while also underscoring the necessity for further optimization of both editing constructs and delivery vectors to achieve clinically relevant correction levels.

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About the authors

O. V. Volodina

Research Centre for Medical Genetics

Author for correspondence.
Email: volodold@gmail.com
Russian Federation, 115522 Moscow

A. G. Demchenko

Research Centre for Medical Genetics

Email: volodold@gmail.com
Russian Federation, 115522 Moscow

A. A. Anuchina

Research Centre for Medical Genetics

Email: volodold@gmail.com
Russian Federation, 115522 Moscow

O. P. Ryzhkova

Research Centre for Medical Genetics

Email: volodold@gmail.com
Russian Federation, 115522 Moscow

V. A. Kovalskaya

Research Centre for Medical Genetics

Email: volodold@gmail.com
Russian Federation, 115522 Moscow

E. V. Kondrateva

Research Centre for Medical Genetics

Email: volodold@gmail.com
Russian Federation, 115522 Moscow

V. Y. Tabakov

Research Centre for Medical Genetics

Email: volodold@gmail.com
Russian Federation, 115522 Moscow

A. V. Lavrov

Research Centre for Medical Genetics

Email: volodold@gmail.com
Russian Federation, 115522 Moscow

S. A. Smirnikhina

Research Centre for Medical Genetics

Email: volodold@gmail.com
Russian Federation, 115522 Moscow

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Supplementary files

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2. Fig. 1. Ribonucleic acid complex for primed editing, detailed description in text

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3. Fig. 2. Scheme of assembly of plasmid construct with pegRNA

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4. Fig. 3. Experimental design for screening 24 pegRNA for lung CF in two patients with homozygous F508del variant in the CFTR gene.

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5. Fig. 4. Pathogenic variant correction efficiency after primed editing using 24 pegRNA in the lung BC of two patients with homozygous F508del variant in the CFTR gene. Genetic constructs were delivered by electroporation. The graph shows the average pathogenic variant correction efficiency in two experiments. Error bars on the Y axis represent the standard error of the mean; K is the untransfected control.

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6. Fig. 5. Efficiency of pathogenic variant correction after primed editing using the 7 most effective pegRNAs in the lung BC of two patients with the homozygous F508del variant in the CFTR gene. The graph shows the average editing efficiency of each pegRNA. Pooled data are shown for two biological and three technical replicates on the lung BC of two patients. Statistical analysis was performed using Dunn's test. Error bars on the Y axis represent the standard error of the mean; K – untransfected cells; * p < 0.05; *** p < 0.001

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7. Fig. 6. Evaluation of off-target DNA changes after primed editing in samples without a targeted CTT insert. The figure shows the analyzed DNA sequence (with the F508del variant), the corresponding amino acid sequence (the amino acid and its position in the polypeptide chain are indicated), and the location of the single-strand break introduced using pegRNA20, -21, and -22. A graph is also shown showing the distribution of single-nucleotide substitutions c.1528G>N, c.1529T>N, c.1531T>N, and c.1531delT introduced during editing using pegRNA20, -21, and -22. Error bars on the Y axis represent the standard error of the mean.

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8. Fig. 7. Evaluation of off-target DNA changes after primed editing in samples with the targeted CTT insertion. The figure shows the analyzed DNA sequence (without the F508del variant), the corresponding amino acid sequence (the amino acid and its position in the polypeptide chain are indicated), and the sites of single-strand break introduction using different pegRNAs. a – Introduction of changes to the targeted insertion during editing using pegRNA1, -17, -19, and -20; error bars on the Y axis reflect the standard error of the mean. b – Distribution of single nucleotide substitutions c.1528G>A, c.1529T>S, and c.1531T>C introduced during editing using pegRNA20, -21, and -22; error bars on the Y axis reflect the standard error of the mean.

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