TITLE: Genotyping for specific genomic insertions with Cas12a ssDNA cleavage
AUTHORS: David Booth

[DESCRIPTION]
Based on the DETECTR assay, this protocol screens for precise genomic insertions by programming a CRISPR RNA (crRNA) to direct LbCas12a to recognize an amplified genomic locus. This sensitive assay enables the detection of low frequency targets (limit of detection ~0.1-1%) in a plate-based format for high throughput screening. The protocol begins by seeding each well of a 96-well plate with clones or small populations of cells that have been genomically edited. After growing cells to saturation, genomic DNA is rapidly extracted with a buffer that is compatible for direct use in PCR reactions. LbCas12a is directly added to the PCR reaction to detect the presence of specific sequences from the amplified locus. Upon binding to the dsDNA target, LbCas12a can indiscriminately cleave single-stranded DNA (ssDNA). The conjugation of a fluorophore and quencher dye to an ssDNA substrate produces a fluorogenic probe to detect the LbCas12a nuclease activity that depends on dsDNA target recognition. A fluorogenic ssDNA probe is commercially available as a DNase Alert Substrate from Integrated DNA Technologies. This protocol was adapted by David Booth and his Lab at UCSF with special input from Fredrick Leon and Jesus Espinoza-Esparza, building on foundational work from Vicki Deng to optimally extract genomic DNA and to perform plate-based screens. 

[BEFORE_START]
1. Validate all primers targeting the edited genomic locus prior to conducting any genome editing. Establish your melt temperature and cycles to make amplicons, confirming that your primer only amplify the desired locus and not any off-targets.
2. Perform genome editing with your preferred protocol. We use this one for S. rosetta:
dx.doi.org/10.17504/protocols.io.j8nlk86o5l5r/v1

[STEPS]
SECTION: Set-up Cas12a ssDNA cleavage assay
1. 1440 minafter transfection count the cell density using a haemocytometer

SECTION: Set-up Cas12a ssDNA cleavage assay
2. Dilute cells to a density of 45 cells/ml in a 50 mLconical tube.

SECTION: Extract DNA from cell populations
5. Once cells have grown to saturation, remove 12 µL  from each well and then transfer that volume into each well of the PCR plate containing DNAzol direct. 
 


SECTION: Extract DNA from cell populations
6. Seal plate with foil lid, vortex, and briefly centrifuge liquid to bottom of wells.
SECTION: Extract DNA from cell populations
7. Heat DNAzol plates at80 °C 

SECTION: Set-up PCR reaction
8. In a fresh PCR plate, aliquot2 µL into each will. This is the PCR template.

SECTION: Set-up PCR reaction
9. Aliquot 23 µL into each well of the PCR plate containing templates. 
 

SECTION: Set-up PCR reaction
10. Seal plate with foil lid, tap to mix (DO NOT VORTEX!), briefly centrifuge, and cycle as predetermined for your primer set.
SECTION: Set-up Cas12a ssDNA cleavage assay
11. As your PCR is finishing , prepare 20 µL:  
13 µL
 2 µL
5 µL 
2 µL 


 

SECTION: Set-up Cas12a ssDNA cleavage assay
12. And then prepare, 600 µL per plate:  
486 µL 
60 µL 
18 µL 
36 µL 

 

SECTION: Set-up Cas12a ssDNA cleavage assay
13. After the PCR has completed amplifying the target sequence, dispense 5 µL into each well of the 96-well plate.

SECTION: Set-up Cas12a ssDNA cleavage assay
14. Firmly cover PCR plate, gently mix, and lightly centrifuge to collect the liquid at the bottom of the well.  
16. Replace PCR cover with an optically clear film cover and place plate in the qPCR instrument (or fluorescent plate reader if the plate is compatible).
SECTION: Set-up Cas12a ssDNA cleavage assay
3. Plate 100 µL  into each well of several 96-well plates, increasing the number of plates if the expected editing efficiency is low.

 


SECTION: Extract DNA from cell populations
4. Aliquot 36 µL into each well of a 96-well PCR plate.

 

17. Read the fluorescence signal in the channel for VIC (Ex 526 nm / Em 543 nm). 

 

SECTION: Set-up Cas12a ssDNA cleavage assay
15. Incubate the plate for 60 min at 37 °C. Lightly centrifuge the plate afterwards to collect the liquid at the bottom.