High-content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries.
J Biotechnol
; 251: 189-200, 2017 Jun 10.
Article
en En
| MEDLINE
| ID: mdl-28445693
ABSTRACT
The CRISPR-Cas9 system has been utilized for large-scale, loss-of-function screens mainly using lentiviral pooled formats and cell-survival phenotypic assays. Screening in an arrayed format expands the types of phenotypic readouts that can be used to now include high-content, morphology-based assays, and with the recent availability of synthetic crRNA libraries, new studies are emerging. Here, we use a cell cycle reporter cell line to perform an arrayed, synthetic crRNAtracrRNA screen targeting 169 genes (>600 crRNAs) and used high content analysis (HCA) to identify genes that regulate the cell cycle. Seven parameters were used to classify cells into cell cycle categories and multiple parameters were combined using a new analysis technique to identify hits. Comprehensive hit follow-up experiments included target gene expression analysis, confirmation of DNA insertions/deletions, and validation with orthogonal reagents. Our results show that most hits had three or more independent crRNAs per gene that demonstrated a phenotype with consistent individual parameters, indicating that our screen produced high-confidence hits with low off-target effects and allowed us to identify hits with more subtle phenotypes. The results of our screen demonstrate the power of using arrayed, synthetic crRNAs for functional phenotypic screening using multiparameter HCA assays.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Ciclo Celular
Tipo de estudio:
Diagnostic_studies
/
Screening_studies
Límite:
Humans
Idioma:
En
Revista:
J Biotechnol
Asunto de la revista:
BIOTECNOLOGIA
Año:
2017
Tipo del documento:
Article
País de afiliación:
Estados Unidos