TITLE: Designing sgRNA Oligos and Inserting Guides into the GEARBOCS Vector 
AUTHORS: Luke Bradley

[DESCRIPTION]
This protocol describes designing sgRNAs for genes of interest using the GEARBOCS vector backbone. This method allows researchers to utilize the GEARBOCS system for astrocyte-specific genetic manipulation in CRISPR/Cas9 mice for in vivo assays

[STEPS]
SECTION: Designing sgRNA oligos
1. Use UCSC to get gene's exons and introns. Make sure the area you are targeting is in all isoforms.
SECTION: Designing sgRNA oligos
2. Before designing your own guides, check the literature for guides that you can use.
SECTION: Designing sgRNA oligos
3. Second, try CRISPick with the exon you want to target.
Reference Genome: Mouse GRCm38 (NCBI RefSeq v.108.20200622)
Mechanism: CRISPRko
Enzyme: SpyoCas9(NGG) and Hsu (2013) tracrRNA
Target(s): Bulk/Advanced targets – enter up to 2000 bp
CRISPick Quota: 10
SECTION: Designing sgRNA oligos
4. Make sure these aren't targeting helical structures or motifs by searching uniprot (https://www.uniprot.org/uniprotkb/P55088/entry).
SECTION: Designing sgRNA oligos
5. Ordering oligos for sgRNA annealing:
SECTION: Designing sgRNA oligos
6. Add the following bolded sequences to 20bp sgRNAs to add restriction enzyme recognition sites. We have written a script to add the overhangs for you here. It requires you to enter your gRNA names and sequences and click run on that website.
SECTION: Designing sgRNA oligos
7. Example:
SECTION: Designing sgRNA oligos
8. Aqp4 gRNA FW: CACCG ATTGTCTTCCGTATGACTAGAGG
SECTION: Designing sgRNA oligos
9. Aqp4 gRNA RV: AAAC CCTCTAGTCATACGGAAGACAAT C
SECTION: Designing sgRNA oligos
10. ^non-bolded sequence to add the SapI restriction enzyme recognition sites
SECTION: Insert guides into GEARBOCS vector
12. Set up the digestion reaction with the following components in a 0.6µL tube

GEARBOCS Vector -> 2ug
SapI Enzyme -> 1uL
NEB rCutSmart buffer -> 5uL
UltraPure H2O -> XuL (Volume will vary based on volume of vector)
Total Volume = 50uL
SECTION: Insert guides into GEARBOCS vector
16. sgRNA Oligo Annealing:
SECTION: Insert guides into GEARBOCS vector
13. Incubate @37˚C for 1 hour and run the sample in 1% agarose gel.
SECTION: Insert guides into GEARBOCS vector
14. Elute the linearized vector in 25µL water using Qiagen Gel purification kit.
SECTION: Insert guides into GEARBOCS vector
15. Use the eluted sample for ligation or preserve in -20˚C
SECTION: Insert guides into GEARBOCS vector
17. Set up the annealing reaction with the following components in a 0.2µL tube.

UltraPure Water -> 7µl
Forward Primer, 100uM -> 1µl 
Forward Primer, 100uM -> 1µl 
NEB Buffer 2.1 -> 1µl 
Total = 10µL
SECTION: Insert guides into GEARBOCS vector
18. Heat to 95'C for 5 min (use heat block). Cool to room temp over 1-2 hours
SECTION: Insert guides into GEARBOCS vector
19. Once the annealing is completed, add 90µL water to the tube and mix well
SECTION: Insert guides into GEARBOCS vector
20. Use the annealed sample for ligation or preserve the rest in -20˚C
SECTION: Insert guides into GEARBOCS vector
21. Ligation:
SECTION: Insert guides into GEARBOCS vector
11. GEARBOCS Vector digestion:
SECTION: Insert guides into GEARBOCS vector
22. Take 6µL of annealed sample for the ligation into pUGC Vector and keep the reaction as below

Insert- Annealed Oligo -> 6uL
GEARBOCS-SapI digested -> 2µl 
T4 Ligase Buffer -> 1µl 
T4 Ligase -> 1µl 
Total -> 10uL
SECTION: Insert guides into GEARBOCS vector
23. Incubate at 4˚C overnight and transform 4 µL into Stbl3 cells the next day. Plate 50 uL of S.O.C. medium + cells.
SECTION: Insert guides into GEARBOCS vector
24. Pick clones for minicultures.
SECTION: Insert guides into GEARBOCS vector
25. Save 0.5-1 mL for glycerol stock and mini-prep the rest for sequencing with the U6 primer.