Plug-and-Play Protein Modification Using Homology-Independent Universal Genome Engineering

Neuron. 2019 Aug 21;103(4):583-597.e8. doi: 10.1016/j.neuron.2019.05.047. Epub 2019 Jul 1.


Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.

Keywords: CRISPR; HiUGE; genomics; immunolabeling; knockin; proteomics.

Publication types

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

MeSH terms

  • Animals
  • Brain / cytology
  • Brain / metabolism
  • CRISPR-Cas Systems
  • Cells, Cultured
  • Dependovirus / genetics
  • Gene Editing / methods
  • Gene Knock-In Techniques / methods*
  • Genetic Vectors / genetics
  • Genomics / methods*
  • Humans
  • Immunochemistry / methods
  • Inteins
  • Mice
  • Mutagenesis, Insertional
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / genetics
  • Protein Engineering / methods*
  • Protein Processing, Post-Translational*
  • Proteomics
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Recombinant Fusion Proteins / genetics
  • Sequence Homology, Nucleic Acid


  • Nerve Tissue Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • Recombinant Fusion Proteins