Journal: STAR Protocols

Article Title: Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons

doi: 10.1016/j.xpro.2025.103945

Figure Lengend Snippet: Schematic of CRISPR KI vector design and key elements hU6: Human U6 promoter, amplified from the pMJ117 plasmid (Addgene plasmid #85997; a gift from Jonathan Weissman), drives robust expression of the single guide RNA (sgRNA) in mammalian cells. gRNA Insertion Site: Site cleaved by the BbsI restriction enzyme to allow insertion of the sgRNA sequence of interest. Scaffold cr1: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ114 plasmid (Addgene plasmid #85995; a gift from Jonathan Weissman), forms the structural component of the sgRNA and facilitates Cas9 binding. mU6: Mouse U6 promoter, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman), drives expression of a second sgRNA cassette. gRNA (DRSR2): sgRNA sequence targeting the donor recognition sequence R2 (DRSR2): 5′-GCGATCGTAATCACCCGAGT-3′,used for homology-independent targeted integration.Scaffold cr2: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman, used in conjunction with the DRSR2-targeting gRNA to support Cas9 function. DRSR2: Target site recognized by the gRNA: DRSR2, 5′-GCGATCGTAATCACCCGAGTGGG-3′ to enable targeted cleavage and integration of the KI cassette. Linker: Flexible amino acid linker (N-GGGGSGGGGSGGGGS-C) inserted between the targeted protein and oScarlet to minimize steric hindrance and preserve protein function. Sequence Between the Linker and oScarlet: Distinct sequences were inserted between the linker and oScarlet to preserve the correct reading frame in each construct. The sequences are as follows: ORF-0, 5′-CCTCGA-3’; ORF-1, 5′-CTCGA-3’; ORF-2, 5′-CGCTCGA-3’.oScarlet: Codon-optimized red fluorescent protein (RFP) used as a KI tag, It enables visualization of the tagged endogenous protein in live or fixed cells, amplified from the pAAV-Ef1a-oScarlet plasmid (Addgene plasmid #137135; gift from Karl Deisseroth). X: Sequence containing multiple stop codons in all three reading frames to ensure translational termination where appropriate.

Article Snippet: Schematic of CRISPR KI vector design and key elements hU6: Human U6 promoter, amplified from the pMJ117 plasmid (Addgene plasmid #85997; a gift from Jonathan Weissman), drives robust expression of the single guide RNA (sgRNA) in mammalian cells. gRNA Insertion Site: Site cleaved by the BbsI restriction enzyme to allow insertion of the sgRNA sequence of interest.

Techniques: CRISPR, Plasmid Preparation, Amplification, Expressing, Sequencing, Binding Assay, Construct