Single-stranded oligo DNA (ssODN) templates, ALT-R XT CRISPR RNA (crRNA), ALT-R trans-activating crRNA (tracrRNA) and ALT-R Cas9 Nuclease V3 were acquired from Integrated DNA Technologies (IDT).
Prior to use, the crRNA and the tracRNA were resuspended in pH 7.5 TE buffer (IDT # 11-01-02-02) to a final concentration of 100
µM. To prepare crRNA:tracRNA duplexes at 50
µM, equal volumes of each RNA were mixed and heated for 5 minutes at 95 °C, followed by controlled cool-off to 25 °C at ramp rate 0.1 °C/second. The formed duplexes were placed on ice until ready to use.
Ribonucleoproteins were prepared by mixing 5
µl each of crRNA:tracRNA duplex (50
µM) and recombinant Cas9 enzyme (61
µM), followed by incubation at room temperature for 20 minutes. Next, 200 pmol of each HDR template was added to the RNPs prior to delivery into iPSCs.
Prior to use, the crRNA and the tracRNA were resuspended in pH 7.5 TE buffer (IDT # 11-01-02-02) to a final concentration of 100
µM. To prepare crRNA:tracRNA duplexes at 50
µM, equal volumes of each RNA were mixed and heated for 5 minutes at 95 °C, followed by controlled cool-off to 25 °C at ramp rate 0.1 °C/second. The formed duplexes were placed on ice until ready to use.
Ribonucleoproteins were prepared by mixing 5
µl each of crRNA:tracRNA duplex (50
µM) and recombinant Cas9 enzyme (61
µM), followed by incubation at room temperature for 20 minutes. Next, 200 pmol of each HDR template was added to the RNPs prior to delivery into iPSCs.