Olga V. Volodina

Here we present a lightweight workflow designed to improve the robustness and scalability of high-throughput amplicon sequencing analysis while leveraging CRISPResso2. To handle large batch processing without sensitive runtime interruptions, our pipeline processes samples sequentially, allowing the analysis to resume from the last successfully completed sample if an error occurs. The workflow implements a fallback mechanism for failed runs, automated extraction and separate processing of control samples, and a background noise model derived from control libraries within defined editing windows. Validation using spike-in datasets across Cas9, base editing, and prime editing demonstrated that our noise-adjusted estimates deviated from the ground truth by only 0.1–0.6%, whereas native CRISPResso2 caused a 1.5–3.7% overestimation. Unlike existing tools such as CRISPECTOR, which focus primarily on CRISPR-Cas9, our approach supports multiple editing modalities and enables project-level organization of large datasets. Input complexity is reduced through configurable flags and modular metadata tables, minimizing manual parameter entry. Together, these features provide a flexible, fault-tolerant, and highly accurate framework for analyzing large genome editing datasets.

Gene editing therapy represents a promising strategy to permanently cure cystic fibrosis (CF). This paper presents the results of correcting the most common CF mutation, F508del, in the CFTR gene in airway basal cells (BCs) obtained from patients, using both classical CRISPR-Cas9-mediated homology-directed repair (HDR) and prime editing techniques. Our findings demonstrate that both methods achieve mutation correction with efficiencies reaching up to 18.9% of alleles in successfully transfected cells. However, the CRISPR-Cas9 approach results in insertions and deletions (indels) in approximately 15% of alleles at the editing site, whereas prime editing induced only minimal indel events. Functional analysis evaluating the effectiveness of gene editing revealed that prime editing of the F508del mutation in the CFTR gene—using the pegRNA4 combined with the PE2-NG variant—restores CFTR channel conductance, comparable to triple therapy with CFTR modulators (ivacaftor/tezacaftor/elexacaftor). While both prime editing and CRISPR-HDR resulted in successful correction of the CFTR-F508del mutation in patient BCs, prime editing exhibited a low frequency of unintended modifications both within and outside the editing locus, making this approach a promising candidate for the development of gene therapy strategies for CF.

Prime editing is a promising approach for correcting pathogenic variants, but its efficiency remains variable across genomic contexts. Here, we systematically evaluated 12 modifications of the PEmax system for correcting the CFTR F508del pathogenic variant that caused cystic fibrosis in patient-derived airway basal cells. We chose EXO1 and FEN1 nucleases to improve the original system. While all tested variants showed comparatively low efficiency in this AT-rich genomic region, 4-FEN modification demonstrated significantly improved editing rates (up to 2.13 fold) compared to standard PEmax. Our results highlight two key findings: first, the persistent challenge of AT-rich target sequence correction even with optimized editors, and second, the performance of 4-FEN suggests its potential value for other genomic targets.

Prime editing is a highly promising strategy for treating hereditary disorders due to its superior efficiency and safety profile compared to the conventional CRISPR-Cas9 systems. This study is dedicated to development of a causal therapy for cystic fibrosis by targeting the F508del variant of 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. Efficiency of the F508del variant correction reached 2.81% (without normalization for transfection efficiency) in the airway basal cells from the patients with homozygous F508del mutation. However, the average transfection efficiency was only 11.9%, emphasizing critical need for the advancements in delivery methodologies. These findings highlight potential of prime editing as an approach for treating cystic fibrosis, while also underscoring necessity for further optimization of both editing constructs and delivery vectors to achieve clinically relevant correction levels.

: The development of gene therapy using genome editing tools recently became relevant. With the invention of programmable nucleases, it became possible to treat hereditary diseases due to introducing targeted double strand break in the genome followed by homology directed repair (HDR) or non-homologous end-joining (NHEJ) reparation. CRISPR-Cas9 is more efficient and easier to use in comparison with other programmable nucleases. To improve the efficiency and safety of this gene editing tool, various modifications CRISPR-Cas9 basis were created in recent years, such as prime editing – in this system, Cas9 nickase is fused with reverse transcriptase and guide RNA, which contains a desired correction. Prime editing demonstrates equal or higher correction efficiency as HDR-mediated editing and much less off-target effect due to inducing nick. There are several studies in which prime editing is used to correct mutations in which researchers reported little or no evidence of off-target effects. The system can also be used to functionally characterize disease variants. However, prime editing still has several limitations that could be further improved. The effectiveness of the method is not yet high enough to apply it in clinical trials. Delivery of prime editors is also a big challenge due to their size. In the present article, we observe the development of the platform, and discuss the candidate proteins for efficiency enhancing, main delivery methods and current applications of prime editing.

BACKGROUND: Cystic fibrosis (CF) is the most common fatal and incurable genetic disease. The most frequent reason is the F508del mutation in the CFTR gene, which can theoretically be corrected by genome editing. Currently, pressing issue is the search for the most effective CRISPR/Cas9-based editing system for this mutation, as well as the selection of target cells that could support self-renewal and provide differentiated progeny of lung cells. Such promising targets can be presented by human induced pluripotent stem cells (hiPSCs) and human induced airway basal stem cells (hiBCs). AIM: To correct the F508del mutation in the CFTR gene in hiPSCs and hiBCs derived from CF patients using CRISPR/Cas9 technology. MATERIALS AND METHODS: We obtained three hiPSCs lines by reprogramming fibroblasts from patients with CF and three hiBCs lines by targeted differentiation of these hiPSCs. Based on three different variants of the Cas9 nuclease, three single guide RNAs, and two single-stranded oligodeoxyribonucleotides we created eight systems for editing the F508del mutation and transfected them into hiPSCs and hiBCs by electroporation. Then we analyzed the levels of non-homologous end joining, indels, and directed homologous repair in the transfected cells by deep target sequencing. RESULTS: Eight editing systems were tested on hiPSCs. The highest efficiency of non-homologous end joining, indels, and directed homologous repair was observed when using SpCas9(1.1). The mutation was significantly corrected using a combination of this nuclease with the guide RNA sgCFTR#sp1 (with an efficiency up to 6.6% of alleles in transfected cells). Only two editing systems), which seemed to be most effective on chiPSCs, were tested on hiBCs. The mutation was significantly corrected using both systems (with efficiency up to 20% of alleles in transfected cells). CONCLUSION: We demonstrated the possibility of highly effective correction of the F508del mutation in the CFTR gene in cells obtained from patients with CF using a designed system for editing this mutation based on the CRISPR/Cas9 system. Since hiBCs can be transfected and edited quite successfully, and can also be obtained in satisfactory quantities from hiPSCs, they are a promising platform for the development of gene therapy for cystic fibrosis.

The development of gene therapy based on genome editing has opened up new possibilities for the treatment of human genetic disorders. This field has developed rapidly over the past few decades, some genome editing-based therapies are already in phase 3 clinical trials. However, there are several challenges to be addressed before widespread adoption of gene editing therapy becomes possible. The main obstacles in the development of such therapy are safety and efficiency, so one of the biggest issues is the delivery of genetic constructs to patient cells. Approaches in genetic cargo delivery divide into ex vivo and in vivo, which are suitable for different cases. The ex vivo approach is mainly used to edit blood cells, improve cancer therapy, and treat infectious diseases. To edit cells in organs researches choose in vivo approach. For each approach, there is a fairly large set of methods, but, unfortunately, these methods are not universal in their effectiveness and safety. The focus of this article is to discuss the current status of in vivo and ex vivo delivery methods used in genome editing-based therapy. We will discuss the main methods employed in these approaches and their applications in current gene editing treatments under development. Graphical Abstract

Введение. В условиях современного информационного общества актуализируются вопросы понимания механизмов получения, освоения, переработки и осмысления знаний как продукта интеллектуальной деятельности. Когнитивная культура формируется в процессе активной вовлеченности в разнообразные виды информационной деятельности. Обновленная стратегия иноязычного образования связана с раскрытием интеллектообразующего потенциала обучения, воспитания и развития обучающихся средствами изучаемого языка. Целью данного исследования является выявление педагогические…