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CRISPR-Cas9介导在细胞系和脐带血造血干细胞及祖细胞中诱导KMT2A重排的方案。

Protocol for CRISPR-Cas9-mediated induction of KMT2A rearrangements in cell line and umbilical cord blood hematopoietic stem and progenitor cells.

作者信息

Benz Tamara, Larghero Patrizia, Meyer Claus, Müller Marcel, Brüggmann Dörthe, Hentrich Anna-Elisabeth, Louwen Frank, Erkner Estelle, Fitzel Rahel, Schneidawind Corina, Marschalek Rolf

机构信息

Institute Pharmaceutical Biology/DCAL, Goethe-University, 60438 Frankfurt am Main, Germany.

Institute Pharmaceutical Biology/DCAL, Goethe-University, 60438 Frankfurt am Main, Germany.

出版信息

STAR Protoc. 2025 Mar 21;6(1):103481. doi: 10.1016/j.xpro.2024.103481. Epub 2024 Dec 18.

DOI:10.1016/j.xpro.2024.103481
PMID:39700011
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11721537/
Abstract

KMT2A rearrangements are associated with a poor clinical outcome in infant, pediatric, and adult acute lymphoblastic and myeloid leukemia. Here, we present a protocol to reconstruct chromosomal translocations with different partner genes of KMT2A in vitro. We describe steps for patient-specific single guide RNA (sgRNA) design, optimized sgRNA in vitro transcription, detailed purification of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB), and CRISPR-Cas9 editing of the test cell line K562 as well as UCB HSPCs. The provided methodology is donor independent.

摘要

KMT2A重排与婴儿、儿童和成人急性淋巴细胞白血病及髓系白血病的不良临床预后相关。在此，我们展示了一种在体外重建KMT2A与不同伙伴基因的染色体易位的方案。我们描述了患者特异性单向导RNA（sgRNA）设计步骤、体外转录优化的sgRNA、从脐带血（UCB）中详细纯化造血干细胞和祖细胞（HSPC），以及对测试细胞系K562和UCB HSPC进行CRISPR-Cas9编辑的步骤。所提供的方法不依赖供体。

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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8f6d/11721537/49bc360e20a6/gr6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8f6d/11721537/a05f862ab13b/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8f6d/11721537/4125e36b6926/gr8.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8f6d/11721537/0cd3b45b2c26/gr10.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8f6d/11721537/84479bfe9eab/gr11.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8f6d/11721537/07e392b96703/gr12.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8f6d/11721537/d40d1ebbd62e/gr13.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8f6d/11721537/264e87e21398/gr14.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8f6d/11721537/7035d91fa926/gr15.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8f6d/11721537/04d7f3017566/gr18.jpg

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本文引用的文献

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Flow Cytometric Enumeration of Peripheral Blood CD34 Cells Predicts Bone Marrow Pathology in Patients with Less Than 1% Blasts by Manual Count.流式细胞术对外周血CD34细胞进行计数可预测手工计数原始细胞比例低于1%的患者的骨髓病理情况。
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