Hrp conjugated streptavidin

Single-target Beads-ELISA detection reaction (20 µL) contained 1× Binding buffer, 15 nM sgRNAa, 15 nM sgRNAb, 30 nM dCas9 protein, 1-4×10 4 Beads@oligo, 0.5 µM oligo re-biotin (Table S3), 40 U RNA Enzyme inhibitor, and DNA sample (see figures for amount). Multi-target Beads-ELISA detection reaction (20 µL) contained 1× Binding buffer, 15 nM each of sgRNAa, 15 nM each of sgRNAb, 15× N nM dCas9 protein, 1-4×10 4 Beads@oligo, 0.5 µM oligo re-biotin, 40 U RNase inhibitor, and various amount of DNA sample (see figures); where N is the number of targets detected. The reaction was incubated at RT for 15 min in rotation. The beads were then washed three times with a washing buffer (1× dCas9 buffer contained 10 U of RNase inhibitor and 0.5% BSA), in which 1× dCas9 buffer can be purchased from New England Biolabs as NEBuffer 3.1 or prepared at home. The beads were incubated with 20 µl washing buffer containing of 8 ng of HRP-conjugated Streptavidin (Sangon Biotech, Shanghai) for 3 min. The beads were washed three times with the washing buffer and finally re-suspended in 30 µl of washing buffer. The beads were transferred into microplate and added 50 µl of TMP chromogenic solution for ELISA (P0209-100ml; Beyotime). The beads were incubated at RT for 10 min. The microplate was read at 630 nm and imaged with a BioRad gel imager in staining-free mode.