TITLE: CRISPR knock-in (endogenous tagging)
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
CRISPR knock-in (endogenous tagging)

[STEPS]
SECTION: Plasmid construction
1. 



For the C-terminus knock-in experiment, the sgRNAs and the homology-independent donor templates were generated
following strategies similar to those described previously 
 
Briefly, the coding sequence of SNCA1 –the gene encoding α-syn at exon 6 was targeted by a sgRNA, and the oScarlet tag (a variant of mScarlet that is engineered to reduce
aggregation; a gift from Karl Deisseroth, Addgene plasmid #137135) was knocked
in using a homology-independent mechanism .





SECTION: Plasmid construction
2. The donor sequence was designed following the SATI knock-in vector 
with slight modifications for the intron knock-in experiment. Briefly, the
following sequences were directly conjugated as a donor tag; SNCA1 intron 4
(only the sequence after the gRNA targeting site), SNCA1 exon 5 (with wild type
or mutation sequences; S129A or S129D), SNCA1 exon 6 (coding site until stop
codon), 3X GGGGS linker, oScarlet (without a start codon and with a stop codon
at the end), and SNCA1 3’ UTR. The SNCA1 intron 4 was targeted by a
sgRNA (TTCTAAGTGTACCAAACCAC),
and the donor tag was homology-independently knocked in. The plasmid PX552
(RRID:Addgene_60958) (a
gift from Feng Zhang) was digested with a NotI restriction enzyme (New England
Biolabs, Cat#R3189L) and used as a plasmid backbone.



SECTION: Plasmid construction
3. Oligonucleotides were ordered from Sigma. The genomic sequence of SNCA1
was partially synthesized by Twist Bioscience, and for the rest, it was PCR
amplified from a purified genome of a C57BL/6J mouse. DNA fragments were
ligated together using the In-Fusion Snap Assembly Master Mix (Takara, Cat# 638947) or DNA Ligation Kit Mighty Mix (Takara, Cat#6023). The sgRNA sequence for knock-in is listed in the supplemental materials. The AAV-SpCas9 plasmid PX551 RRID:Addgene_60957 (a gift from Feng Zhang) was modified by removing the HA tag. 
SECTION: Adeno-associated virus (AAV) production, neuronal transduction, and quantification of synapses
4. 



Small-scale AAV cell lysates were produced
using the AAVpro Purification Kit (All Serotypes) (Takara, Cat# 6666) with slight
modifications. Briefly, HEK293T cells (RRID:CVCL_0063)
were triple transfected with the AAV targeting plasmid, helper plasmid (Agilent Technologies, Cat # 240071), and serotype PHP.S plasmid pUCmini-iCAP-PHP.eB (RRID:Addgene_103005)(a gift from Viviana Gradinaru) with PEI Max (Polysciences,
Cat#24765). 




SECTION: Adeno-associated virus (AAV) production, neuronal transduction, and quantification of synapses
5. The medium was changed the next day of transfection, and cells were
incubated for 3 days after transfection. HEK cells were then collected and
lysed with the AAV Extraction Solution A plus. 
SECTION: Adeno-associated virus (AAV) production, neuronal transduction, and quantification of synapses
6. The extracted solution was
centrifuged at 10,000 x g for 10 min to remove debris and mixed with Extraction
Solution B. This small-scale AAV solution was stored at -80 until use. For
viral transduction, hippocampal
neurons were plated at 60,000 cells/cm2 density and infected 4 hours
later with 10ul of the AAVs expressing SpCas9 and sgRNAs/donor. 
SECTION: Adeno-associated virus (AAV) production, neuronal transduction, and quantification of synapses
7. The media was replaced 2 days after infection. Before imaging or genomic analysis, the
transduced neurons were cultured to maturity (DIV-17-DIV-21). 


SECTION: Adeno-associated virus (AAV) production, neuronal transduction, and quantification of synapses
8. For quantification of the knock-in α-syn:o-Scarlet fluorescence at synapses, images
were first background-corrected, small ROIs were manually placed over ~20-30
synapses on each image, and average intensities were calculated – all using
dropdown menus in MetaMorph Microscopy Automation and Image Analysis Software (RRID:SCR_002368)(https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#gref). The resulting datasets were
statistically analyzed using GraphPad Prism [(RRID:SCR_002798)
http://www.graphpad.com/].