Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing.
Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing.
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DOI：
10.1038/s41467-017-01875-9
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发表时间：
2017-11-23
影响因子：
16.6
通讯作者：
Saha K
中科院分区：
文献类型：
--
作者：
Carlson-Stevermer J;Abdeen AA;Kohlenberg L;Goedland M;Molugu K;Lou M;Saha K
将特定的DNA序列写入人类基因组是具有挑战性的非病毒基因编辑试剂，因为大多数编辑的序列包含各种不精确的插入或缺失。我们开发了一种模块化的RNA适体-链霉亲和素策略，称为S1 mplex，用于将CRISPR-Cas9核糖核蛋白与核酸供体模板以及其他生物素化分子（如量子点）复合。在人类细胞中，定制的S1 mplexes将精确编辑与不精确编辑的等位基因的比例提高到标准基因编辑方法的18倍，并富集含有多重精确编辑的细胞群高达42倍。这些多功能预组装试剂的进步可以大大减少精准医学和药物发现中体外或离体基因编辑应用的时间和成本，并有助于开发体内体细胞基因编辑的增加和连续给药方案。使用CRISPR编写特定的基因序列有时可能很困难，因为哺乳动物细胞倾向于使用NHEJ修复断裂。在这里，作者形成纳米颗粒将模板序列定位到核酸酶上，使修复转向有利于HDR。
Writing specific DNA sequences into the human genome is challenging with non-viral gene-editing reagents, since most of the edited sequences contain various imprecise insertions or deletions. We developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to complex CRISPR-Cas9 ribonucleoproteins with a nucleic acid donor template, as well as other biotinylated molecules such as quantum dots. In human cells, tailored S1mplexes increase the ratio of precisely edited to imprecisely edited alleles up to 18-fold higher than standard gene-editing methods, and enrich cell populations containing multiplexed precise edits up to 42-fold. These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro or ex vivo gene-editing applications in precision medicine and drug discovery and aid in the development of increased and serial dosing regimens for somatic gene editing in vivo. Using CRISPR to write specific genetic sequences can sometimes be difficult due to the preference of mammalian cells to repair breaks using NHEJ. Here the authors form nanoparticles to localize the template sequence to the nuclease, shifting repair in favor of HDR.
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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：
64.8
作者：
Konermann S;Brigham MD;Trevino AE;Joung J;Abudayyeh OO;Barcena C;Hsu PD;Habib N;Gootenberg JS;Nishimasu H;Nureki O;Zhang F
通讯作者：
Zhang F
影响因子：
8.8
作者：
Gundry MC;Brunetti L;Lin A;Mayle AE;Kitano A;Wagner D;Hsu JI;Hoegenauer KA;Rooney CM;Goodell MA;Nakada D
通讯作者：
Nakada D
影响因子：
3.7
作者：
Li K;Wang G;Andersen T;Zhou P;Pu WT
通讯作者：
Pu WT
影响因子：
12.3
作者：
Carpenter AE;Jones TR;Lamprecht MR;Clarke C;Kang IH;Friman O;Guertin DA;Chang JH;Lindquist RA;Moffat J;Golland P;Sabatini DM
通讯作者：
Sabatini DM