CRISPR-UMI: single-cell lineage tracing of pooled CRISPR-Cas9 screens.
Nat Methods
; 14(12): 1191-1197, 2017 Dec.
Article
en En
| MEDLINE
| ID: mdl-29039415
ABSTRACT
Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ARN Guía de Kinetoplastida
/
Linaje de la Célula
/
Análisis de la Célula Individual
/
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas
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Sistemas CRISPR-Cas
/
Edición Génica
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Nat Methods
Asunto de la revista:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Año:
2017
Tipo del documento:
Article
País de afiliación:
Austria