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Frequent sgRNA-barcode recombination in single-cell perturbation assays.

ABSTRACT: 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.

SUBMITTER: Xie S

PROVIDER: S-EPMC5991360 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Frequent sgRNA-barcode recombination in single-cell perturbation assays.

Xie Shiqi S Cooley Anne A Armendariz Daniel D Zhou Pei P Hon Gary C GC

PloS one 20180606 6

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-circularizatio ...[more]

PMID: 29874289

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