Establishing combinatorial screening platform coupled to single-cell analysis

建立与单细胞分析结合的组合筛选平台

• 批准号: 10702875
• 负责人: Michael Aregger
• 金额: $ 11.13万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702875
• 关键词: Ablation; Binding Sites; Biological Process; Biomedical Research; CRISPR screen; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Coupled; DNA cassette; Data; Disease; Engineering; Genes; Genetic; Genetic Screening; Genomics; Guide RNA; Hybrids; Link; Modification; Phenotype; Poly A; Proteins; Protocols documentation; Reagent; Role; System; Technology; Therapeutic Intervention; Transcript; Work; combinatorial; cost; cost effective; design; experimental study; expression vector; gene function; genetic element; genome-wide; genomic platform; improved; indexing; insight; loss of function; novel; prevent; response; screening; single cell analysis; single-cell RNA sequencing; therapeutic candidate; transcriptome; transcriptomics

Hybrid guide RNA cassettes have been engineered to harbor capture sequences within Cas9 and Cas12a guide RNAs, which make them amenable to capture using 10x Genomics protocols and reagents during single-cell analysis. Pilot experiments have been performed to assess capture efficiency of hgRNAs along with polyadenylated transcripts. Current work includes further modifications of the hgRNA expression cassettes to improve the simultaneous capture of Cas9 and Cas12a guides while retaining efficient editing efficiency of our combinatorial system. Once the guide design is fully optimized we will perform genetic screening with single-cell transcriptomic readouts thereby allowing us to gain mechanistic insights into the cellular rewiring in response to single or combined genetic perturbations.

混合引导 RNA 盒经过精心设计，可在 Cas9 和 Cas12a 引导 RNA 中包含捕获序列，这使得它们能够在单细胞分析过程中使用 10x Genomics 方案和试剂进行捕获。已经进行了初步实验来评估 hgRNA 和聚腺苷酸化转录本的捕获效率。目前的工作包括进一步修改 hgRNA 表达盒，以提高 Cas9 和 Cas12a 向导的同时捕获，同时保留我们的组合系统的高效编辑效率。 一旦指南设计完全优化，我们将使用单细胞转录组读数进行遗传筛查，从而使我们能够深入了解细胞重新布线以响应单个或组合的遗传扰动。