Department of Medicine and Vascular Biology Center, Medical College of Georgia at Augusta University, Augusta, Georgia, USA.
Department of Neuroscience and Regenerative Medicine, Medical College of Georgia at Augusta University, Augusta, Georgia, USA.
CRISPR J. 2023 Apr;6(2):163-175. doi: 10.1089/crispr.2022.0099.
Microinjected transgenes, both large and small, are known to insert randomly into the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. As the vast majority of transgenic mouse lines remain unmapped, we developed CRISPR-Cas9 Long-Read Sequencing (CRISPR-LRS) to ascertain transgene integration loci. This novel approach mapped a wide size range of transgenes and uncovered more complex transgene-induced host genome re-arrangements than previously appreciated. CRISPR-LRS offers a facile, informative approach to establish robust breeding practices and will enable researchers to study a gene without confounding genetic issues. Finally, CRISPR-LRS will find utility in rapidly and accurately interrogating gene/genome editing fidelity in experimental and clinical settings.
微注射的转基因，无论是大的还是小的，都已知随机插入到小鼠基因组中。传统的转基因定位方法具有挑战性，因此使繁殖策略和表型的准确解释变得复杂，特别是当转基因破坏关键编码或非编码序列时。由于绝大多数转基因小鼠品系仍未被定位，我们开发了 CRISPR-Cas9 长读测序（CRISPR-LRS）来确定转基因整合位点。这种新方法能够定位广泛的转基因大小范围，并揭示了比以前认识到的更复杂的转基因诱导的宿主基因组重排。CRISPR-LRS 为建立稳健的繁殖实践提供了一种简单、信息丰富的方法，并将使研究人员能够在没有遗传问题干扰的情况下研究一个基因。最后，CRISPR-LRS 将在实验和临床环境中快速、准确地检测基因/基因组编辑保真度方面具有实用价值。
