In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9

Swiech et al. (2015). Nat Biotechnol DOI: 10.1038/nbt.3055 Citations: 804

Parts used

PE5 Prime EditorPE4 Prime EditoreSpCas9 NucleaseCas12f NucleaseU6 Promoter

Canonical designs

{U6-(gRNA)}*3-hSyn-mCherry-KASH-hGHpA U6-(gRNA)-CMV-ZsGreen-pA

Key findings

  • First demonstration of dual-AAV CRISPR delivery system for in vivo genome editing in mammalian brain. Separated SpCas9 nuclease and sgRNA into independent AAV vectors (pX551 and pX552, Addgene #60957/#60958), enabling flexible promoter selection and multi-guide delivery. Achieved efficient somatic genome editing in adult mouse neurons, validating dual-vector approach for applications requiring tissue-specific expression or multiple guide RNAs that exceed single-AAV packaging constraints.