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Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9.
Giuliano, Christopher J; Lin, Ann; Girish, Vishruth; Sheltzer, Jason M.
Afiliação
  • Giuliano CJ; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
  • Lin A; Stony Brook University, Stony Brook, New York.
  • Girish V; Massachusetts Institute of Technology, Cambridge, Massachusetts.
  • Sheltzer JM; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Curr Protoc Mol Biol ; 128(1): e100, 2019 09.
Article em En | MEDLINE | ID: mdl-31503414
CRISPR/Cas9 technology enables the rapid generation of loss-of-function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of biological reagents. However, the successful derivation of knockout clones can be technically challenging and may be complicated by multiple factors, including incomplete target ablation and interclonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation of true knockout clones and are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells. © 2019 The Authors.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Células Clonais / Técnicas de Inativação de Genes / Sistemas CRISPR-Cas Limite: Animals / Humans Idioma: En Revista: Curr Protoc Mol Biol Ano de publicação: 2019 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Células Clonais / Técnicas de Inativação de Genes / Sistemas CRISPR-Cas Limite: Animals / Humans Idioma: En Revista: Curr Protoc Mol Biol Ano de publicação: 2019 Tipo de documento: Article