Construction of CRISPR Libraries for Functional Screening.
Methods Mol Biol
; 1772: 139-150, 2018.
Article
in En
| MEDLINE
| ID: mdl-29754226
ABSTRACT
Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/CAS9 system. Using pooled libraries of guide RNA expression vectors that direct CAS9 to a specific genomic site allows identification of genes that are either enriched or depleted in response to a selection scheme, thus linking the affected gene to the chosen phenotype. The quality of the data generated by the screening is dependent on the quality of the guide RNA delivery library with regards to error rates and especially evenness of distribution of the guides. Here, we describe a method for constructing complex plasmid libraries based on pooled designed oligomers with high representation and tight distributions. The procedure allows construction of plasmid libraries of >60,000 members with a 95th/5th percentile ratio of less than 3.5.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Cloning, Molecular
/
CRISPR-Cas Systems
Type of study:
Diagnostic_studies
/
Screening_studies
Language:
En
Journal:
Methods Mol Biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2018
Document type:
Article
Affiliation country:
United States