Parallel genetics of regulatory sequences using scalable genome editing in vivo

Cell Rep. 2021 Apr 13;35(2):108988. doi: 10.1016/j.celrep.2021.108988.

Abstract

How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. Regulatory elements must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3' UTRs of 16 genes in C. elegans. Our software crispr-DART analyzes indel mutations in targeted DNA sequencing. We quantify the impact of mutations on expression and fitness by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3' UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression independently of each other. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of regulatory sequences directly in animals.

Keywords: CRISPR-Cas9; Caenorhabditis elegans; animal phenotype; gene regulation; gene-regulatory elements; indel mutations; in vivo; let-7 microRNA; lin-41 3′ UTR; parallel genetics.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems
  • Caenorhabditis elegans / genetics*
  • Caenorhabditis elegans / growth & development
  • Caenorhabditis elegans / metabolism
  • Caenorhabditis elegans Proteins / genetics*
  • Caenorhabditis elegans Proteins / metabolism
  • Gene Editing / methods
  • Gene Expression Regulation, Developmental
  • Genetic Association Studies*
  • Genome, Helminth*
  • Genotype
  • INDEL Mutation*
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Phenotype
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Regulatory Sequences, Nucleic Acid
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism

Substances

  • Caenorhabditis elegans Proteins
  • LIN-41 protein, C elegans
  • MicroRNAs
  • RNA, Guide, CRISPR-Cas Systems
  • Transcription Factors
  • let-7 microRNA, C elegans
  • CRISPR-Associated Protein 9