sgRNA production

DNA templates for each sgRNA were produced by PCR with specific primers followed by in vitro transcription. The steps are listed below:

PCR amplification of overlapping primers was performed with Platinum PCR SuperMix following manufacturer’s instructions (Invitrogen).

Purification of DNA was carried out with SpinPrep PCR Clean-up Kit (Millipore) and used as template for transcription reaction (500 ng of starting material per reaction) with the MEGAscript T7 Transcription Kit (Ambion).

Transcription reactions were run for 20 h followed by DNaseI treatment (20 min at 37 °C).

RNA concentration was determined by Nanodrop ND-1000 spectrophotometer.

sgRNAs were resuspended with DEPC-H₂0 and adjusted to 2500 ng/μl and stored at -80 °C in 10 μl single use aliquots.