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首页> 美国卫生研究院文献>Nucleic Acids Research >INDEL detection the ‘Achilles heel’ of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels
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INDEL detection the ‘Achilles heel’ of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels

机译：indel检测精确基因组编辑的achilles脚后跟：对基因编辑诱导诱导的准确分析的方法调查

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摘要

Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.
机译：基因组编辑技术的进步使得能够在单个基础上操纵基因组。这些技术基于可编程核酸酶（PNS），其包括Meganuclase，锌 - 手指核酸酶（ZFN），转录活化剂样效应器核酸酶（TALENS）和聚类，通常间隔的短语重复（CRISPR）/ CRISPR相关的9（CAS9）核酸酶并给研究人员删除，插入或替换细胞，组织和整个生物中的基因组DNA的能力。重新设计PNS基因组靶特异性的巨大灵活性大大扩大了生命科学的基因编辑应用的范围，对下一代基因疗法的发展表现出很大的承诺。 PN Technologies在基因组中诱导DNA双链断裂（DSB）的原理，其次是诱导的DSB的细胞修复。 PN引发的DSB主要由非同源终端连接（NHEJ）和微噬菌学介导的终端连接（MMEJ）途径修复，其可以引出各种小的插入或缺失（Indel）突变。如果在蛋白质编码序列中引发吲哚并移位读取框，则可以使用可用的PNS中的任一种可以容易地实现靶向基因敲除（KO）。尽管在实践中可以实现基因失活的基因失活，但成功的KO不仅取决于NHEJ和MMEJ修复的效率;它还取决于所使用的PN的设计和性质，所选择的递送格式，靶位位点的优选的吲哚修复结果，靶位点的染色质状态以及编辑细胞中的修复途径的相对活性。这些变量能够准确地预测PN诱导的indels的性质和频率。因此，任何基因KO实验的关键步骤都成为在细胞，组织或全生物中靶向基因组位点诱导的吲哚的检测，表征和定量。在本调查中，我们简要介绍了天然存在的诱惑及其检测。接下来，我们审查了用于检测PN诱导的indels的方法。我们简要概述了实验步骤，并描述了帮助用户决定其编辑应用程序的合适方法的各种方法的优缺点。我们突出了最近的进展，使得能够准确和敏感细胞中的诱导事件，无论其基因组复杂程度如何，将复杂的不同indel事件池转化为信息诱导型材。最后，我们通过使用新方法以及如何帮助进一步推进基因组编辑领域的新方法以及如何帮助进一步提高基因组编辑领域的PN引发的indel形成。

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