Edit r crispr cas9 synthetic tracrrna

In this study, dCas9-VPR plasmids were utilized to generate cells expressing dCas9-VPR. SP-dCas9-VPR was originally generated by George Church (Addgene plasmid # 63798; http://n2t.net/addgene:63798; RRID:Addgene_63798) [21 (link)]. Transient transfection was performed with SP-dCas9-VPR plasmid using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s protocols. Expression of the dCas9 was confirmed by Western blot. These cells were then transfected by individual synthetic Edit-R CRISPRa Human CEBPA crRNA (CA-006422-01-0002, CA-006422-02-0002, CA-006422-03-0002, CA-006422-04-0002, Dharmacon, Lafayette, CO, USA) and Edit-R CRISPR-Cas9 Synthetic tracrRNA (U-002005-05, Dharmacon, Lafayette, CO, USA), which was carried out by lipid transfection at the recommended crRNA:tracrRNA working concentration (25 nM:25 nM). After 3–5 days, RT-qPCR was used to confirm target gene activation.

MCF10A cells were harvested with 0.25% trypsin/EDTA centrifugation at 500 rpm, 4 °C. The cells were then resuspended in 37 °C Opti-MEM Reduced Serum Medium (Gibco).
Both crRNA (Dharmacon) and tracrRNA (Dharmacon Edit-R CRISPR-Cas9 Synthetic tracrRNA-U-002005-20) stock solutions (200 μM each) were prepared by adding the appropriate volume of RNase-free water. Then, the 100 μM solution of crRNA:tracrRNA duplex was created by combining 200 μM stock solutions in a 1:1 ratio. The solution was gently mixed for 10 min and stored at −20 °C for future experiments. The project also utilised HS17 crRNA (5′-CAGACAGGCCCAGATTGAGG-3′) from Berg et al. [16 (link)]. The Cas9 ribonucleoprotein (RNP) complex was created by combining 1.5 μM Cas9 protein and 3 μM RNA final concentration and kept on ice until being mixed with the resuspended cells in Opti-MEM medium.
Electroporation of the Cas9:RNA complex was achieved using Gene Pulser/MicroPulser Electroporation Cuvettes with 0.2 cm gap cuvettes at in the Gene Pulser Xcell Electroporation System and an exponential pulse at 300 V and 300 μF. Complete cell culture media was then added to the Opti-MEM in a 1:1 ratio. Electroporated MCF10A cells were seeded on to coverslips pre-coated with 50 μg/mL poly-D-lysine (Sigma, St. Louis, MO, USA) and incubated at 37 °C in 5% CO₂.

Four CRISPR sgRNAs per gene designed by Dharmacon (GE Healthcare Dharmacon, Inc.) were used together to target the zebrafish wdr45 or atg7 genes (sequences in Supplementary Table 3). To maximize the knockdown efficiency, 100 ng of each sgRNA was mixed with 4 µl TRACR RNA (Dharmacon Edit-R CRISPR–Cas9 synthetic tracrRNA; Dharmacon, U002005) and 1.6 μl nuclease-free water^{42 (link)}. The mixtures were incubated at room temperature for 10 min before the addition of 2.64 μl of 2 M KCl and stored at −80 °C as 1.5 μl aliquots. On the day of injections, 5 μg Cas9 nuclease protein NLS (Horizon Discovery, CAS12206) was added to each thawed aliquot and incubated for 5 min at 37 °C, after which 0.2 μl phenol red was added to the samples to visualize the injected droplet.
Embryos were collected immediately after spawning and injected at the one-cell stage. the volumes injected were calibrated to a final amount of 0.32 ng CRIPSPR sgRNAs targeting atg7 and/or 0.64/0.96 ng for wdr45. For the double-knockdown experiments, CRISPR injections were performed sequentially—wdr45-targeting CRISPR–Cas9 solution was injected first, followed by atg7-CRISPR–Cas9 solution. Uninjected siblings were kept from each clutch in every experiment for comparison.