Cas9prot 250ug

DNA oligos of sgRNAs and x-gRNAs were designed according to the EnGen sgRNA Synthesis Kit (NEB #E3322) to add 5’- T7 RNA polymerase promoter sequence and 3’- Cas9 crRNA sequence and were purchased from Integrated DNA Technologies IDT then resuspended to a stock concentration of 100 μM. If the (x-)gRNA did not have an initial 5’- dG necessary for T7 RNA polymerase transcription, one was added in the DNA oligo sequence. For sgRNA synthesis, oligos were diluted 100x (1 μM) then used with the EnGen sgRNA Synthesis Kit per manufacturer’s instructions. Cas9 RNPs were formed following the IDT Alt-R CRISPR-Cas9 System – In vitro cleavage of target DNA with ribonucleoprotein complex protocol (Option 2). Cas9 enzyme (Sigma Aldrich, #CAS9PROT-250UG), eCas9 enzyme (Sigma Aldrich #ESPCAS9PRO-50UG), or dCas9 enzyme (IDT Alt-R^® S.p. dCas9 Protein V3 #1081066) and sgRNA were combined in equimolar amounts in Phosphate buffered saline, pH 7.4 - PBS (ThermoFisher, #10010023) and incubated at room temperature for 10 minutes. Following incubation, RNPs were stored at −80°C or immediately used for in vitro digestion reactions.

10 pmol of purified sgRNA (IDT, Alt-R CRISPR-Cas9 sgRNA) alone, or 10 pmol of sgRNA and 10 pmol of purified spCas9 nuclease (Sigma-Aldrich, CAS9PROT-250UG), were incubated at room temperature for 10 min in a 20 μl reaction in 1 × 3.1 Buffer (B7203S, NEB) to form RNP. Specified dose of TAMRA dye labelled PNA was diluted into 1 μl of water, added to wells, and incubated in a thermocycler for 30 min at 37°C. A BIORAD 5% TBE polyacrylamide gel was pre-run for 30 min at 10 mA. 5 μl of final binding reactions were loaded onto gels with BlueJuice loading buffer (Invitrogen) and run at 10 mA. Gels were first imaged using BIORAD GelDoc XRS+ with a Green Epi (BIORAD #170-8284) 605/50 filter to acquire TAMRA signal and then incubated in 1× SYBR Gold (Invitrogen) in 1× TBE buffer for 5 min before imaging by standard UV transillumination (Raw images in Supplementary Figure S1). For band density analysis, three independent experiments were loaded onto a single 26-well gel and run according to the specifications above. Integrated densities for TAMRA-Cas9 co-localized bands were determined across wells using ImageJ analyses, and background signal from each no PNA (0 pmol) control was subtracted for each experiment.