A Tandem Guide RNA-Based Strategy for Efficient CRISPR Gene Editing of Cell Populations with Low Heterogeneity of Edited Alleles
| Autor: | Marcel Paulmann, Gerard Joberty, Aaron T. Cheng, Maria Fälth-Savitski, Paola Grandi, Carola Doce, Markus Bösche, Gerard Drewes |
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| Rok vydání: | 2020 |
| Předmět: |
Cell
Computational biology Biology Gene Knockout Techniques Genome editing INDEL Mutation Genetics medicine CRISPR Animals Humans Clustered Regularly Interspaced Short Palindromic Repeats Guide RNA Gene Silencing Allele Gene Gene knockout Research Articles Gene Editing Cas9 DNA Hep G2 Cells medicine.anatomical_structure CRISPR-Cas Systems Biotechnology RNA Guide Kinetoplastida |
| Zdroj: | The CRISPR Journal |
| ISSN: | 2573-1602 |
| Popis: | CRISPR/Cas9–based gene knockouts (KOs) enable precise perturbation of target gene function in human cells, which is ideally assessed in an unbiased fashion by molecular omics readouts. Typically, this requires the lengthy process of isolating KO subclones. We show here that KO subclones are phenotypically heterogenous, regardless of the guide RNA used. We present an experimental strategy that avoids subcloning and achieves fast and efficient gene silencing on cell pools, based on the synergistic combination of two guide RNAs mapping at close (40–300 bp) genomic proximity. Our strategy results in better predictable indel generation with a low allelic heterogeneity, concomitant with low or undetectable residual target protein expression, as determined by MS3 mass spectrometry proteomics. Our method is compatible with nondividing primary cells and can also be used to study essential genes. It enables the generation of high confidence omics data which solely reflect the phenotype of the target ablation. |
| Databáze: | OpenAIRE |
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