Frequent sgRNA-barcode recombination in single-cell perturbation assays

PLoS One. 2018 Jun 6;13(6):e0198635. doi: 10.1371/journal.pone.0198635. eCollection 2018.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics
  • Genetic Vectors / genetics
  • HEK293 Cells
  • Humans
  • K562 Cells
  • Lentivirus / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • Sequence Analysis, RNA
  • Single-Cell Analysis / methods*
  • Transfection / methods*

Substances

  • RNA, Guide, CRISPR-Cas Systems