Cas9实验步骤教程

1.       Copy the target sequence (<500bp), paste in “query sequence:” of the webpage:

https://crispr.cos.uni-heidelberg.de/

2.       Set two parameters:

①   target site length: 20

②   species: Arabidopsis thaliana TAIR10

3.       Click “Submit”, pick the best 20-bp “target_seq”, which has the least mismatch with other genes.

4.       Primers: the part highlighted in red in the following sequence is an example target sequence, which is replaced by the picked target sequence.

Target-Cas9-F: GaaaattctggcgtcgtgaggGTTTTAGAGCTAGAAATAGCAAGTTAA

Target-Cas9-R: CcctcacgacgccagaattttCAATCACTACTTCGACTCTAGCTG

Target-check-R: cctcacgacgccagaatttt

U6-26P-common-F: GAGAAGCAGGCCCATTTATATGGGA

U6-26T-common-R: CCAGAAATTGAACGCCGAAGAACAG

pEG100-Seq-R: caacgtgcacaacagaattga

5.       Construction of pCas9-T1-target as the following protocol.

①  Digestion of pCas9-T1: SmaI

②  PCR: Target

ü  Template: plasmid pCas9-T1 (100x dilution)

ü  Primers:

l  Target-UP: U6-26P-common-F, Target-Cas9-R→~170 bp

l  Target-DN: Target-Cas9-F, U6-26T-common-R→~160 bp

ü  Taq: PrimeStar GXL

③  Seamless cloning: pCas9-T1+Target-UP+Target-DN=pCas9-T1-target

④  Colony PCR: U6-26P-common-F, Target-check-R→~170 bp

⑤  Sequencing primers: pEG100-Seq-R

The same strategy is used for construction of multiple targets. If there are more than 2 targets, two of the target sequences are integrated into pCas9-T2 first. The rest of the targets are integrated into pEG100-T2, which is amplified by PCR as a whole cassette and integrated into pCas9-T2.

CRISPR-Cas9原理图