Highly Multiplexed Analysis of CRISPR Genome Editing Outcomes in Mammalian Cells.
Methods Mol Biol
; 2312: 193-223, 2021.
Article
em En
| MEDLINE
| ID: mdl-34228292
ABSTRACT
CRISPR-Cas-based genome editing has enabled efficient genetic engineering of a range of organisms and sparked revolutions in many fields of biology. After Streptococcus pyogenes Cas9 was first demonstrated for mammalian genome editing, many CRISPR-associated (Cas) protein variants have been isolated from different species and adopted for genome editing. Furthermore, various effector domains have been fused to these Cas proteins to expand their genome-editing abilities. Although the number of genome-editing tools has been rapidly increasing, the throughput of cell-based characterization of new genome-editing tools remains limited. Here we describe a highly multiplexed genome editing and sequencing library preparation protocol that allows high-resolution analysis of mutation outcomes and frequencies induced by hundreds to thousands of different genome-editing reagents in mammalian cells. We have successful experiences of developing several key genome-editing tools using this protocol. The protocol also is designed to be compatible with robotic liquid handling systems for further scalability.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Regulação da Expressão Gênica
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Reação em Cadeia da Polimerase Multiplex
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
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Sistemas CRISPR-Cas
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Edição de Genes
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Proteína 9 Associada à CRISPR
Limite:
Humans
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article