Highly Multiplexed Analysis of CRISPR Genome Editing Outcomes in Mammalian Cells

Methods Mol Biol. 2021:2312:193-223. doi: 10.1007/978-1-0716-1441-9_12.

Abstract

CRISPR-Cas-based genome editing has enabled efficient genetic engineering of a range of organisms and sparked revolutions in many fields of biology. After Streptococcus pyogenes Cas9 was first demonstrated for mammalian genome editing, many CRISPR-associated (Cas) protein variants have been isolated from different species and adopted for genome editing. Furthermore, various effector domains have been fused to these Cas proteins to expand their genome-editing abilities. Although the number of genome-editing tools has been rapidly increasing, the throughput of cell-based characterization of new genome-editing tools remains limited. Here we describe a highly multiplexed genome editing and sequencing library preparation protocol that allows high-resolution analysis of mutation outcomes and frequencies induced by hundreds to thousands of different genome-editing reagents in mammalian cells. We have successful experiences of developing several key genome-editing tools using this protocol. The protocol also is designed to be compatible with robotic liquid handling systems for further scalability.

Keywords: Amplicon sequencing; Base editing; CRISPR–Cas9; Genome editing; High-throughput sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Cells, Cultured
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Gene Editing*
  • Gene Expression Regulation*
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Multiplex Polymerase Chain Reaction*
  • Transfection

Substances

  • CRISPR-Associated Protein 9