INDEL detection, the 'Achilles heel' of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels.
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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DOI：
10.1093/nar/gkaa975
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发表时间：
2020-12-02
影响因子：
14.9
通讯作者：
Frödin M
中科院分区：
文献类型：
--
作者：
Bennett EP;Petersen BL;Johansen IE;Niu Y;Yang Z;Chamberlain CA;Met Ö;Wandall HH;Frödin M
基因组编辑技术的进步使得能够在单碱基水平上操纵基因组。这些技术基于可编程核酸酶（PN），包括大范围核酸酶、锌指核酸酶（ZFN）、转录激活因子样效应物核酸酶（TALEN）和规则间隔短回文重复序列（CRISPR）/CRISPR相关9（Cas9）核酸酶，并使研究人员能够在细胞、组织和整个生物体中删除、插入或替换基因组DNA。重新设计PN的基因组靶特异性的巨大灵活性极大地扩展了基因编辑在生命科学中的应用范围，并显示出开发下一代基因疗法的巨大前景。PN技术的共同原理是在基因组中用户指定的位点诱导DNA双链断裂（DSB），然后对诱导的DSB进行细胞修复。PN引起的DSB主要通过非同源末端连接（NHEJ）和微同源介导的末端连接（MMEJ）途径修复，其可以引起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插入/缺失并移动阅读框，则可以使用任何可用的PN轻松实现靶向基因敲除（KO）。尽管原则上可以容易地实现基因失活，但在实践中，成功的KO不仅取决于NHEJ和MMEJ修复的效率;它还取决于所用PN的设计和性质、所选择的递送形式、靶位点处的优选indel修复结果、靶位点的染色质状态和编辑细胞中修复途径的相对活性。这些变量排除了PN诱导的indel的性质和频率的准确预测。因此，任何基因KO实验的关键步骤变成在细胞、组织或整个生物体中的靶向基因组位点处诱导的indel的检测、表征和定量。在这篇综述中，我们简要回顾了自然发生的indel及其检测。接下来，我们回顾了已经开发的用于检测PN诱导的indels的方法。我们简要概述了实验步骤，并描述了各种方法的优点和缺点，以帮助用户决定一个合适的方法，他们的编辑应用程序。我们强调了最近的进展，使准确和灵敏的定量indel事件在细胞中，无论其基因组的复杂性，把一个复杂的池不同的indel事件到信息indel配置文件。最后，我们回顾了通过使用新方法对PN引发的indel形成的了解，以及这种见解如何有助于进一步推进基因组编辑领域。
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.
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影响因子：
19
作者：
Ceccaldi R;Rondinelli B;D'Andrea AD
通讯作者：
D'Andrea AD
影响因子：
14.9
作者：
Brinkman, Eva K.;Kousholt, Arne N.;van Steensel, Bas
通讯作者：
van Steensel, Bas
影响因子：
14.9
作者：
Gao H;Huang J;Barany F;Cao W
通讯作者：
Cao W
DOI：
10.1007/978-1-62703-968-0_4
发表时间：
2014
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Bonocora, Richard P;Belfort, Marlene
通讯作者：
Belfort, Marlene
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin