In this section, we describe a method to clone a 20-bp taret sequence into our gRNA expression vector pBFv-U6.2.
Finding a target sequence and ordering oligonucleotides
First, you need to select a 20-bp target sequence in your gene.
The sequence needs to start with a "G", followed by a "NGG" sequence. The first "G" is required for transcription initiation from the U6 promoter while the "NGG" tail is the PAM sequence recognized by Cas9 protein.
We developed a web tool for finding such sequences.
Choose one of the target sequences suggested by the web tool and order oligonucleotides.
Digest 1µg of pBFv-U6.2 vector with 10U of BbsI (NEB) and gel-purify the cut vector using QIAquick Gel Extraction Kit (Qiagen) and elute in 30µl.
To anneal oligos, mix 4 µl each of the two oligos (100 µM) and 2 µl of 5x Phusion HF Buffer (NEB).
Heat the mixture to 95℃ on a heat block, turn it off and let it cool down to room temperature.
Ligation, transformation and plasmid purificatioin
Ligate 1µl of the digested vecter and 1µl of the annealed oligonucleotides using 200U of T4 ligase (NEB) in a 5µl reactin volume.
Transform the whole reaction into bacteria. We use the DH5alpha strain as a host.
Pick a single colony and purify plasmid DNA using QIAprep Spin Miniprep Kit (Qiagen).