Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
The Beijer laboratory and Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Nat Commun. 2022 Feb 2;13(1):627. doi: 10.1038/s41467-022-28244-5.
CRISPR-Cas9 genome editing has potential to cure diseases without current treatments, but therapies must be safe. Here we show that CRISPR-Cas9 editing can introduce unintended mutations in vivo, which are passed on to the next generation. By editing fertilized zebrafish eggs using four guide RNAs selected for off-target activity in vitro, followed by long-read sequencing of DNA from >1100 larvae, juvenile and adult fish across two generations, we find that structural variants (SVs), i.e., insertions and deletions ≥50 bp, represent 6% of editing outcomes in founder larvae. These SVs occur both at on-target and off-target sites. Our results also illustrate that adult founder zebrafish are mosaic in their germ cells, and that 26% of their offspring carries an off-target mutation and 9% an SV. Hence, pre-testing for off-target activity and SVs using patient material is advisable in clinical applications, to reduce the risk of unanticipated effects with potentially large implications.
CRISPR-Cas9 基因组编辑技术具有治疗目前尚无治疗方法的疾病的潜力，但治疗方法必须是安全的。在这里，我们表明 CRISPR-Cas9 编辑可以在体内引入意外的突变，这些突变会传递给下一代。通过使用体外选择的针对脱靶活性的四个向导 RNA 编辑受精的斑马鱼卵，然后对来自两代超过 1100 个幼虫、幼鱼和成鱼的 DNA 进行长读测序，我们发现结构变体（SVs），即 50bp 以上的插入和缺失，代表创始幼虫中编辑结果的 6%。这些 SV 发生在靶标和非靶标位点。我们的结果还表明，成年创始斑马鱼的生殖细胞呈嵌合状态，其 26%的后代携带一个脱靶突变，9%携带一个 SV。因此，在临床应用中，使用患者材料进行脱靶活性和 SVs 的预先测试是可取的，以降低具有潜在重大影响的意外影响的风险。
