A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.
A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.
复制标题
一种泛RNA酶抑制剂，使CRISPR-mRNA平台能够用于原代人单核细胞的工程化。
DOI：
10.3390/ijms23179749
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发表时间：
2022-08-28
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
--
作者：
单核细胞及其下游效应子是先天免疫系统的关键组成部分。单核细胞配备有趋化因子受体，允许它们迁移到各种组织，在那里它们可以分化成巨噬细胞和树突状细胞亚群，并参与组织稳态、感染、自身免疫性疾病和癌症。在单核细胞及其效应细胞中实现基因组工程将促进基础和转化研究的无数应用。在这里，我们证明了CRISPR-Cas9 RNP可以用于原代人单核细胞中的有效基因敲除。此外，我们证明了细胞内RNA酶可能是导致差的和异源的mRNA表达的原因，因为pan-RNA酶抑制剂的掺入允许在Cas9和碱基编辑酶的基于mRNA的递送之后进行有效的基因组工程。此外，我们证明了CRISPR-Cas9与rAAV载体DNA供体模板组合介导转基因在原代人单核细胞中的位点特异性插入和表达。最后，我们证明SIRPa敲除单核细胞衍生的巨噬细胞具有增强的抗癌细胞活性，突出了在细胞免疫疗法中应用的潜力。
Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.
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影响因子：
46.9
作者：
Hendel A;Bak RO;Clark JT;Kennedy AB;Ryan DE;Roy S;Steinfeld I;Lunstad BD;Kaiser RJ;Wilkens AB;Bacchetta R;Tsalenko A;Dellinger D;Bruhn L;Porteus MH
通讯作者：
Porteus MH
DOI：
10.1073/pnas.0809422106
发表时间：
2009-04-14
影响因子：
11.1
作者：
Huang, Xin;Venet, Fabienne;Ayala, Alfred
通讯作者：
Ayala, Alfred
影响因子：
5.4
作者：
CHALLITA, PM;SKELTON, D;KOHN, DB
通讯作者：
KOHN, DB
影响因子：
46.9
作者：
Klichinsky M;Ruella M;Shestova O;Lu XM;Best A;Zeeman M;Schmierer M;Gabrusiewicz K;Anderson NR;Petty NE;Cummins KD;Shen F;Shan X;Veliz K;Blouch K;Yashiro-Ohtani Y;Kenderian SS;Kim MY;O'Connor RS;Wallace SR;Kozlowski MS;Marchione DM;Shestov M;Garcia BA;June CH;Gill S
通讯作者：
Gill S
影响因子：
14.9
作者：
Brinkman EK;Chen T;Amendola M;van Steensel B
通讯作者：
van Steensel B