Genotyping for specific genomic insertions with Cas12a ssDNA cleavage v1

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.