RESUMEN
Lipidic nanocarriers are a broad class of lipid-based vectors with proven potential for packaging and delivering emerging nucleic acid therapeutics. An important early step in the clinical development cycle is large-scale screening of diverse formulation libraries to assess particle quality and payload delivery efficiency. Due to the size of the screening space, this process can be both costly and time-consuming. To address this, computational models capable of predicting clinically relevant physio-chemical properties of dendrimer-lipid nanocarriers, along with their mRNA payload delivery efficiency in human cells are developed. The models are then deployed on a large theoretical nanocarrier pool consisting of over 4.5 million formulations. Top predictions are synthesised for validation using cell-based assays, leading to the discovery of a high quality, high performing, candidate. The methods reported here enable rapid, high-throughput, in silico pre-screening for high-quality candidates, and have great potential to reduce the cost and time required to bring mRNA therapies to the clinic.
Asunto(s)
Simulación por Computador , ARN Mensajero , ARN Mensajero/genética , Humanos , Nanopartículas/química , Lípidos/química , Dendrímeros/química , Portadores de Fármacos/químicaRESUMEN
The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/genética , Eliminación de Gen , Edición Génica/métodos , Mutagénesis Insercional , Proteína 9 Asociada a CRISPR/metabolismo , Proliferación Celular , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , ADN/metabolismo , Células HEK293 , Células Hep G2 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Motivos de Nucleótidos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions.
Asunto(s)
Sistemas CRISPR-Cas , Epigénesis Genética , Biblioteca de Genes , ARN Guía de Kinetoplastida/genética , Animales , Clonación Molecular/métodos , Vectores Genéticos/genética , Células Hep G2 , Humanos , Lentivirus/genética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Tumors comprise functionally diverse subpopulations of cells with distinct proliferative potential. Here, we show that dynamic epigenetic states defined by the linker histone H1.0 determine which cells within a tumor can sustain the long-term cancer growth. Numerous cancer types exhibit high inter- and intratumor heterogeneity of H1.0, with H1.0 levels correlating with tumor differentiation status, patient survival, and, at the single-cell level, cancer stem cell markers. Silencing of H1.0 promotes maintenance of self-renewing cells by inducing derepression of megabase-sized gene domains harboring downstream effectors of oncogenic pathways. Self-renewing epigenetic states are not stable, and reexpression of H1.0 in subsets of tumor cells establishes transcriptional programs that restrict cancer cells' long-term proliferative potential and drive their differentiation. Our results uncover epigenetic determinants of tumor-maintaining cells.