DNA templates with blocked long 3' end single-stranded overhangs (BL3SSO) promote bona fide Cas9-stimulated homology-directed repair of long transgenes into endogenous gene loci.
DNA templates with blocked long 3' end single-stranded overhangs (BL3SSO) promote bona fide Cas9-stimulated homology-directed repair of long transgenes into endogenous gene loci.
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DOI：
10.1093/g3journal/jkab169
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发表时间：
2021-08-07
期刊：
影响因子：
--
通讯作者：
Lau NC
中科院分区：
文献类型：
--
作者：
Bandyopadhyay S;Douglass J;Kapell S;Khan N;Feitosa-Suntheimer F;Klein JA;Temple J;Brown-Culbertson J;Tavares AH;Saeed M;Lau NC
通过Cas9介导的同源定向修复（HDR）敲入大型转基因是一个极其低效的过程。尽管使用单链寡核苷酸（ssODN）作为HDR供体已经改善了较小转基因的整合，但它们不支持大DNA序列的有效插入。为了深入了解HDR介导的较大转基因整合的机制并改进技术，我们进行了靶向人EMX 1基因座的敲入实验，并在人HEK 293细胞系中应用了严格的基因组PCR分析。该实验揭示了由在单个同源臂处起始的转基因HDR和随后的质粒骨架序列的基因组整合引起的意想不到的分子复杂性。为了围绕这个问题，我们设计了一种新的PCR构建的模板，其含有阻断的长3'单链突出端（BL 3SSO），其大大提高了在EMXl基因座处真正Cas9刺激的HDR的效率。我们进一步完善了BL 3SSO技术，并成功地将GFP转基因插入到两个重要的干扰素刺激基因（ISGs）位点，Viperin/RSAD 2和ISG 15。本研究证明了BL 3SSO平台用于将长DNA序列插入组成型和诱导型内源基因座以产生用于重要生物过程研究的新型人类细胞系的实用性。
Knock-in of large transgenes by Cas9-mediated homology-directed repair (HDR) is an extremely inefficient process. Although the use of single-stranded oligonucleotides (ssODN) as an HDR donor has improved the integration of smaller transgenes, they do not support efficient insertion of large DNA sequences. In an effort to gain insights into the mechanism(s) governing the HDR-mediated integration of larger transgenes and to improve the technology, we conducted knock-in experiments targeting the human EMX1 locus and applied rigorous genomic PCR analyses in the human HEK293 cell line. This exercise revealed an unexpected molecular complication arising from the transgene HDR being initiated at the single homology arm and the subsequent genomic integration of plasmid backbone sequences. To pivot around this problem, we devised a novel PCR-constructed template containing blocked long 3' single-stranded overhangs (BL3SSO) that greatly improved the efficiency of bona fide Cas9-stimulated HDR at the EMX1 locus. We further refined BL3SSO technology and successfully used it to insert GFP transgenes into two important interferon-stimulated genes (ISGs) loci, Viperin/RSAD2, and ISG15. This study demonstrates the utility of the BL3SSO platform for inserting long DNA sequences into both constitutive and inducible endogenous loci to generate novel human cell lines for the study of important biological processes.
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影响因子：
14.9
作者：
Gaj T;Staahl BT;Rodrigues GMC;Limsirichai P;Ekman FK;Doudna JA;Schaffer DV
通讯作者：
Schaffer DV
影响因子：
3.7
作者：
Hwang WY;Fu Y;Reyon D;Maeder ML;Kaini P;Sander JD;Joung JK;Peterson RT;Yeh JR
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Yeh JR
影响因子：
14.9
作者：
He X;Tan C;Wang F;Wang Y;Zhou R;Cui D;You W;Zhao H;Ren J;Feng B
通讯作者：
Feng B
影响因子：
46.9
作者：
通讯作者：
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影响因子：
7.7
作者：
Gutierrez-Triana JA;Tavhelidse T;Thumberger T;Thomas I;Wittbrodt B;Kellner T;Anlas K;Tsingos E;Wittbrodt J
通讯作者：
Wittbrodt J