Composition and method for efficient gene screening through tagged guide RNA constructor

Abstract

The invention provides a composition, reagent kit and method for gene screening by one or more sets of guide DNA constructors with internal tags ("iBAR"). Each set has three or more guide RNA constructors targetting same genomes and gene loci, and different iBAR sequences are inlaid.

Description

technical field [0001] The present invention relates to compositions, kits and methods for genetic screening using guide RNA constructs with internal tags ("iBARs"). Background technique [0002] The CRISPR / Cas9 system enables editing at targeted genomic loci with high efficiency and specificity 1-2 . One of its many uses is to identify the function of coding genes, non-coding RNAs, and regulatory elements by combining high-throughput pooled sequencing with next-generation sequencing ("NGS") analysis. By introducing pooled libraries of single guide RNAs (“sgRNA”) or paired guide RNAs (“pgRNAs”) into cells expressing Cas9 or catalytically inactive Cas9 fused to an effector domain (dCas9), researchers can Perform multiplex genetic screens by generating multiple mutations, large genomic deletions, transcriptional activation, or transcriptional repression. [0003] In order to generate high-quality gRNA cell banks in any given pooled CRISPR screen, low multiplicity of infecti...

AI Technical Summary

Problems solved by technology

To further reduce FDR and improve data reproducibility, deep coverage of gRNAs and multiple biological replicates are often required to obtain hits with high statistical significance, which results in increased workload

Additional difficulties may arise when performing extensive genome-wide screens, when cellular material for library construction is limited, or when it is difficult to obtain experimental replicates or control the MOI when performing more challenging screens (e.g., in vivo screens)

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