Protocols · Benchling

Materials

- Nanodrop
- Thermocycler
- Nucleofector (e.g. Amaxa IIb)
- Nucleofector kit for cell line of interest
- Vacuum centrifuge
- Tris-EDTA buffer (pH = 8)
- TAE buffer
- Agarose
- 5X DNA loading dye
- DNA ladder
- Nucleic acid stain (e.g. SYBR Safe)
- Phusion High-Fidelity DNA polymerase (NEB #M0530)
- 10mM dNTPs
- gBlocks_F_primer: TGAGTATTACGGCATGTGAGGGC
- gBlocks_R_primer: TCAATGTATCTTATCATGTCTGCTCGA
- PCR clean-up kit (e.g. QIAGEN MinElute Reaction Cleanup Kit #28206)
- Test cells (e.g. HEK293)
- HDR template
- Cas9-encoding plasmid (e.g. PX459)
- gBlocks template (gene fragment encoding U₆ and FE-modified sgRNA for increased stability once expressed): AGTATTACGGCATGT GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATAC AAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACAC AAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCT TGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCA TATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATAT ATCTTGTGGAAAGGACGAAACACC G[N₁₈–20] GTTTAAGAGCT ATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGTTTTAG AGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTTTAGCG CGTGCGCCAATTCTGCAGACAAATGGCTCTAGAGGTACGGCC GCTTCGAGCAGACATGATAAGATACATTGA

NB: It is very important to work as carefully and clean as possible because the final products of this work will be added to your cell culture.

Design sgRNAs for targeting

Replace [N₁₈-20] in the gBlocks template above with your sgRNA sequence. Delete the extra 5'G if your sgRNA starts with a G already

Order gBlock from IDT

Resuspend lyophilised 500ng gBlock in 100µL TE (final concentration 5ng/µL)

Set-up High Fidelity PCR reaction to amplify gBlock (perform minimum 3X 50µL reactions per targeting)

Component	50 ul reaction	Final concentration	Thermocycling conditions
NS-free H2O	32.25		STEP	TEMP	TIME
5X Phusion HF Buffer	10	1X	Initial Denaturation	98C	30s
10 mM dNTPs	1	200 uM	30 cycles	98C	15s
5 uM gBlock-CRISPR_fw (PR032)	2.5	0.25 uM		60C	30s
5 uM gBlock-CRISPR_rv (PR033)	2.5	0.25 uM		72C	30s
5 ng/ul gBlock	1.25	1.25 ng/ul	Final extension	72C	5 minutes
Phusion DNA Polymerase	0.5	1.0 units/50 ul PCR	Hold	4C	forever
Total	50

Following cycling, it is recommended to run 2µL of each PCR reaction onto a 2 % TAE-agarose gel to confirm the successful amplification and generation of a 500bp amplicon.

Perform PCR clean-up once you have confirmed successful amplification. I routinely use the QIAGEN MinElute Reaction Cleanup Kitaccording to manufacturer's instructions except that I elute in 30µL elution buffer per 100-250µL of PCR reaction; vacuum centrifugation will concentrate the sample later. It is best not to reduce this volume because you would like to use 1-2µL for nanodropping without reducing your yield substantially (different from original protocol by Arbat et al.).

I have decided to Nanodrop the cleaned product and calculate backwards how many ul I need for 3µg of each gBlock (different from original protocol by Arbab et al.). Transfer the amount to a new tube and mix with other components to be nucleofected (5µg Cas9 plasmid and 200pmol HDR template).

Concentrate the gBlocks PCR product <15µL using a vacuum centrifuge.

NB: Wipe vacuum centrifuge clean with 70 % ethanol and concentrate samples until <15µL. It is not a problem if you end up drying the DNA as it will be resuspended subsequently. Usually, it takes about 5 minutes to evaporate 15µL of TE/H₂O at 60C. Check volume every 5 minutes to prevent overdrying (although your experiment should still work once you resuspend the DNA in your nucleofection mix; I tried when I accidentally overdried some samples).

Nucleofect 1,000,000 cells in a 100µL reaction with 3µg gBlock, 5µg Cas9 and 200pmol HDR template. I use the Amaxa Nucleofector IIb.

Harvest cells 48h later for downstream genotyping.