RESUMEN
The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.
Asunto(s)
Sistemas CRISPR-Cas , Biblioteca Genómica , Proteína 9 Asociada a CRISPR , Streptococcus pyogenesRESUMEN
Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.
Asunto(s)
Sistemas CRISPR-Cas/genética , Epistasis Genética/genética , Pruebas Genéticas , ARN Guía de Kinetoplastida/genética , Apoptosis/genética , Técnicas de Inactivación de Genes , Marcación de Gen , Humanos , Aprendizaje Automático , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Transducción de Señal/genética , Staphylococcus aureus/genética , Streptococcus pyogenes/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
CRISPR/Cas9 screening has proven to be a versatile tool for genomics research. Based on unexpected results from a genome-wide screen, we developed a CRISPR/Cas9-mediated approach to mutagenesis, exploiting the allelic diversity generated by error-prone non-homologous end-joining (NHEJ) to identify novel gain-of-function and drug resistant alleles of the MAPK signaling pathway genes MEK1 and BRAF. We define the parameters of a scalable technique to easily generate cell populations containing thousands of endogenous allelic variants to map gene functions. Further, these results highlight an unexpected but important phenomenon, that Cas9-induced gain-of-function alleles are an inherent by-product of normal Cas9 loss-of-function screens and should be investigated during analysis of data from large-scale positive selection screens.
Asunto(s)
Sistemas CRISPR-Cas , MAP Quinasa Quinasa 1/genética , Mutagénesis , Ingeniería de Proteínas/métodos , Proteínas Proto-Oncogénicas B-raf/genética , Alelos , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Reparación del ADN por Unión de Extremidades , Biblioteca de Genes , Células HEK293 , Humanos , Mutación INDEL , Indoles/farmacología , MAP Quinasa Quinasa 1/química , Fenotipo , Proteínas Proto-Oncogénicas B-raf/química , ARN Guía de Kinetoplastida/genética , Proteínas Recombinantes de Fusión/química , Alineación de Secuencia , Sulfonamidas/farmacología , Transducción Genética , VemurafenibRESUMEN
CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.