Department of Internal Medicine, Carver College of Medicine, University of Iowa, 375 Newton Rd, 5206 MERF, Iowa City, IA, 52246, USA.
Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA, USA.
Sci Rep. 2023 Sep 8;13(1):14798. doi: 10.1038/s41598-023-41109-1.
CRISPR/Cas9 gene editing has evolved from a simple laboratory tool to a powerful method of in vivo genomic engineering. As the applications of CRISPR/Cas9 technology have grown, the need to characterize the breadth and depth of indels generated by editing has expanded. Traditionally, investigators use one of several publicly-available platforms to determine CRISPR/Cas9-induced indels in an edited sample. However, to our knowledge, there has not been a cross-platform comparison of available indel analysis software in samples generated from somatic in vivo mouse models. Our group has pioneered using CRISPR/Cas9 to generate somatic primary mouse models of malignant peripheral nerve sheath tumors (MPNSTs) through genetic editing of Nf1. Here, we used sequencing data from the in vivo editing of the Nf1 gene in our CRISPR/Cas9 tumorigenesis model to directly compare results across four different software platforms. By analyzing the same genetic target across a wide panel of cell lines with the same sequence file, we are able to draw systematic conclusions about the differences in these software programs for analysis of in vivo-generated indels. Surprisingly, we report high variability in the reported number, size, and frequency of indels across each software platform. These data highlight the importance of selecting indel analysis platforms specific to the context that the gene editing approach is being applied. Taken together, this analysis shows that different software platforms can report widely divergent indel data from the same sample, particularly if larger indels are present, which are common in somatic, in vivo CRISPR/Cas9 tumor models.
CRISPR/Cas9 基因编辑已从一种简单的实验室工具演变为体内基因组工程的强大方法。随着 CRISPR/Cas9 技术的应用不断发展，对编辑产生的插入缺失（indels）广度和深度进行特征描述的需求也在不断扩大。传统上，研究人员使用几种公开可用的平台之一来确定编辑样本中由 CRISPR/Cas9 编辑产生的 indels。然而，据我们所知，目前还没有对来自体内活体小鼠模型生成的样本中可用的 indel 分析软件进行跨平台比较。我们小组率先使用 CRISPR/Cas9 通过 Nf1 的基因编辑来生成恶性外周神经鞘瘤（MPNST）的体内原代小鼠模型。在这里，我们使用来自我们的 CRISPR/Cas9 肿瘤发生模型中 Nf1 基因体内编辑的测序数据，直接比较四个不同软件平台的结果。通过在具有相同序列文件的广泛细胞系面板上分析相同的遗传靶标，我们能够对这些软件程序在分析体内生成的 indels 方面的差异得出系统的结论。令人惊讶的是，我们报告了每个软件平台报告的 indels 的数量、大小和频率存在高度可变性。这些数据强调了根据基因编辑方法应用的上下文选择特定的 indel 分析平台的重要性。总之，这项分析表明，不同的软件平台可以从同一样本报告差异很大的 indel 数据，特别是如果存在较大的 indels，这在体内活体 CRISPR/Cas9 肿瘤模型中很常见。
