Detection of gene cis-regulatory element perturbations in single-cell transcriptomes

PLoS Comput Biol. 2021 Mar 12;17(3):e1008789. doi: 10.1371/journal.pcbi.1008789. eCollection 2021 Mar.

Abstract

We introduce poly-adenine CRISPR gRNA-based single-cell RNA-sequencing (pAC-Seq), a method that enables the direct observation of guide RNAs (gRNAs) in scRNA-seq. We use pAC-Seq to assess the phenotypic consequences of CRISPR/Cas9 based alterations of gene cis-regulatory regions. We show that pAC-Seq is able to detect cis-regulatory-induced alteration of target gene expression even when biallelic loss of target gene expression occurs in only ~5% of cells. This low rate of biallelic loss significantly increases the number of cells required to detect the consequences of changes to the regulatory genome, but can be ameliorated by transcript-targeted sequencing. Based on our experimental results we model the power to detect regulatory genome induced transcriptomic effects based on the rate of mono/biallelic loss, baseline gene expression, and the number of cells per target gRNA.

Publication types

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

MeSH terms

  • Algorithms
  • Animals
  • CRISPR-Cas Systems / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Computational Biology
  • Databases, Factual
  • Humans
  • Mice
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Regulatory Elements, Transcriptional / genetics*
  • Sequence Analysis, RNA / methods*
  • Single-Cell Analysis / methods*
  • Transcriptome / genetics*

Substances

  • RNA, Guide, CRISPR-Cas Systems