Frequent sgRNA-barcode recombination in single-cell perturbation assays.
Shiqi XieAnne CooleyDaniel A ArmendarizPei ZhouGary C HonPublished in: PloS one (2018)
Simultaneously detecting CRISPR-based perturbations and induced transcriptional changes in the same cell is a powerful approach to unraveling genome function. Several lentiviral approaches have been developed, some of which rely on the detection of distally located genetic barcodes as an indirect proxy of sgRNA identity. Since barcodes are often several kilobases from their corresponding sgRNAs, viral recombination-mediated swapping of barcodes and sgRNAs is feasible. Using a self-circularization-based sgRNA-barcode library preparation protocol, we estimate the recombination rate to be ~50% and we trace this phenomenon to the pooled viral packaging step. Recombination is random, and decreases the signal-to-noise ratio of the assay. Our results suggest that alternative approaches can increase the throughput and sensitivity of single-cell perturbation assays.
Keyphrases
- single cell
- high throughput
- dna repair
- dna damage
- rna seq
- genome wide
- sars cov
- randomized controlled trial
- crispr cas
- air pollution
- transcription factor
- high glucose
- clinical trial
- genome editing
- stem cells
- dna methylation
- diabetic rats
- bone marrow
- label free
- drug induced
- risk assessment
- gene therapy
- high resolution
- loop mediated isothermal amplification
- liquid chromatography
- phase iii