Abstract
Genome engineering has been tremendously affected by the appearance of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based approach. Initially discovered as an adaptive immune system for prokaryotes, the method has rapidly evolved over the last decade, overtaking multiple technical challenges and scientific tasks and becoming one of the most effective, reliable, and easy-to-use technologies for precise genomic manipulations. Despite its undoubtable advantages, CRISPR/Cas9 technology cannot ensure absolute accuracy and predictability of genomic editing results. One of the major concerns, especially for clinical applications, is mutations resulting from error-prone repairs of CRISPR/Cas9-induced double-strand DNA breaks. In some cases, such error-prone repairs can cause unpredicted and unplanned large genomic modifications within the CRISPR/Cas9 on-target site. Here we describe the largest, to the best of our knowledge, undesigned on-target deletion with a size of ~293 kb that occurred after the cytoplasmic injection of CRISPR/Cas9 system components into mouse zygotes and speculate about its origin. We suppose that deletion occurred as a result of the truncation of one of the ends of a double-strand break during the repair.
Highlights
Nowadays, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system, known as the most powerful tool for gene editing, is intensively used in a wide range of biological studies, for conducting various screenings on cell cultures [1–4] and creating unique genome-modified animal models [5–8]
We describe the largest, to the best of our knowledge, undesigned on-target deletion that occurred after the injection of CRISPR/Cas9 system components into mouse zygotes
Genome editing directed by the CRISPR/Cas9 system is in widespread use in molecular biology and in therapeutic and clinical trials, making the editing process relatively easy and allowing for the introduction of various genomic modifications into the genome, with high accuracy and precision
Summary
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system, known as the most powerful tool for gene editing, is intensively used in a wide range of biological studies, for conducting various screenings on cell cultures [1–4] and creating unique genome-modified animal models [5–8]. Numerous molecular biology strategies were described to significantly reduce off-target genome activities: alternative editing enzymes, such as dimeric fusions with FokI [26,27], CpfI [28,29], small Cas orthologs [30], CasX [31], and modified spCas enzymes [32,33]; improved delivery strategies, such as adeno-associated viral vectors packaging the CRISPR-Cas system [34,35], inorganic and lipid nanoparticles [36–39], and preassembled CRISPR/Cas RNPs [40]; protocol modifications with an intracellular concentration of the nuclease and duration of a nuclease activity [41,42]; truncated gRNAs [43]; and double-nicking strategy [44]. We describe the largest, to the best of our knowledge, undesigned on-target deletion that occurred after the injection of CRISPR/Cas system components into mouse zygotes
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