RESUMEN
Genome-wide pooled CRISPR-Cas-mediated knockout, activation, and repression screens are powerful tools for functional genomic investigations. Despite their increasing importance, there is currently little guidance on how to design and analyze CRISPR-pooled screens. Here, we provide a review of the commonly used algorithms in the computational analysis of pooled CRISPR screens. We develop a comprehensive simulation framework to benchmark and compare the performance of these algorithms using both synthetic and real datasets. Our findings inform parameter choices of CRISPR screens and provide guidance to researchers on the design and analysis of pooled CRISPR screens.
Asunto(s)
Algoritmos , Sistemas CRISPR-Cas , Simulación por Computador , ARN Interferente Pequeño , Análisis de SecuenciaRESUMEN
We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Hibridación Fluorescente in Situ/métodos , Línea Celular Tumoral , ADN/análisis , Roturas del ADN de Doble Cadena , Vectores Genéticos , Células HEK293 , Humanos , Microscopía Fluorescente , Imagen Molecular , ARN/análisis , ARN Guía de Kinetoplastida/genética , Linfocitos TRESUMEN
Systems for CRISPR-based combinatorial perturbation of two or more genes are emerging as powerful tools for uncovering genetic interactions. However, systematic identification of these relationships is complicated by sample, reagent, and biological variability. We develop a variational Bayes approach (GEMINI) that jointly analyzes all samples and reagents to identify genetic interactions in pairwise knockout screens. The improved accuracy and scalability of GEMINI enables the systematic analysis of combinatorial CRISPR knockout screens, regardless of design and dimension. GEMINI is available as an open source R package on GitHub at https://github.com/sellerslab/gemini .
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas Genéticas , Programas Informáticos , Teorema de Bayes , Epistasis GenéticaRESUMEN
Pooled CRISPR screens allow researchers to interrogate genetic causes of complex phenotypes at the genome-wide scale and promise higher specificity and sensitivity compared to competing technologies. Unfortunately, two problems exist, particularly for CRISPRi/a screens: variability in guide efficiency and large rare off-target effects. We present a method, CRISPhieRmix, that resolves these issues by using a hierarchical mixture model with a broad-tailed null distribution. We show that CRISPhieRmix allows for more accurate and powerful inferences in large-scale pooled CRISPRi/a screens. We discuss key issues in the analysis and design of screens, particularly the number of guides needed for faithful full discovery.
