CRISPR PX330 sgRNA质粒克隆构建方法

1. sgRNA design 

Figure 1. From Staying on target with CRISPR-Cas . Dana Carroll , Nature Biotechnology 31,807–809 (2013). 

1. Use website https://www.dna20.com/eCommerce/cas9/input, put the interested gene name to get the higher score sgRNA, select the species, cas 9 taye( wt).   Choose the top three target sequences on the 5’ end, and the top three for the 3’

Such as axin 1, click √   to  select “Search only in the first common exon”.  https://www.dna20.com/eCommerce/cas9/results

2. Design the primers

To clone the guide sequence into the sgRNA scaffold, synthesize two oligos of the form:

                     5’ – CACCGNNNNNNNNNNNNNNNNNNN     – 3’

                     3’ –     CNNNNNNNNNNNNNNNNNNNCAAA – 5’

To clone in your target sequence, synthesize two partially complementary oligos with 4nt overhangs compatible for cloning into the vector. “N” and “n” represent complementary base pairs.  PAY CAREFUL ATTENTION TO THE 5′ > 3′ ORIENTATIONS

                          5’ –CACCGNNNNNNNNNNNNNNNNNNN –3’      5’ –AAACnnnnnnnnnnnnnnnnnnnC -3’

              When annealed oligos form double stranded DNA with overhangs for cloning into BbsI site in px330.

5’ –CACCGNNNNNNNNNNNNNNNNNNN –    3’

3’     –CNNNNNNNNNNNNNNNNNNNCAAA –5’

             Forward primer:    5’ –CACCGNNNNNNNNNNNNNNNNNNN –3’

                Reverse primer:     5’ –AAACnnnnnnnnnnnnnnnnnnnC -3’

  Note: 1) make sure The PAM site is not included in the sgRNA sequence. 

2) Add CACCG to forward oligo(if there is G in the 5’ site, just add CACC).

3) Design the oligo 2, Get reverse complementary sequence of forward oligo, then add aaac to 5’, c to 3’ site.

Design note for expressing sgRNA in cells from the U6 promoter in pX330: Please note that for the pX330 cloning backbone, the example guide sequence one base ‘G’ followed by 19 Ns. Because it needs U6 promoter to have a ‘G’ base at the transcription start site. Hence, we recommend finding a 20bp genome target starting with the base ‘G’. If you have to use other bases at the starting position of your genome target, you could add an additional ‘G’ to the front of your target. If you are going to use the construct simply to make RNA for microinjection into mouse embryos, then you can ignore this issue.

 sgRNA克隆构建 

Oligo annealing and cloning into backbone vectors:

1. PX330载体酶切体系配制:

X µl pX330   (1- 2 µg)

2 µl 10X NEBuffer 2.1

1 µl BbsI (NEB) Use more if cutting more DNA

H₂O to final Volume of 20 µl

Incubate the digestion reaction at 37^oC for at least 1hr.

Heats inactivate (65°C for 20 min) and columns purify linearized plasmid. 

2. 胶回收，线性化载体回收.
3. oligos退火:

       1 ul  oligo 1 (100μM)

       1 ul  oligo 2 (100μM)

            1 ul       10X T4 Ligation Buffer (NEB)

       6.5 ul      ddH₂O

       0.5 ul      T4 PNK (NEB)             

       10 ul total

PCR反应条件，梯度降温：

       37^oC 30 min

            95^oC        5 min and then ramp down to 25^oC at 5^oC/min

• after anneal the oligo, the dilution is around 200, and check the concentration with nano drop, the

concentration is around 50-100 ng/ul.

4. Oligo和载体连接反应and incubate at room temperature for 10 min:

       X ul BbsI digested plasmid

              from step 2 (50ng)

       1 ul  phosphorylated and annealed

              oligo duplex from step 3 (1:150–1:200 dilution)

       5 ul  2X Quickligation Buffer (NEB)

       X ul ddH₂O                         

       10 ul subtotal

       1 ul  Quick Ligase (NEB)      

       11 ul total

可选，T4连接酶反应配制

X µl pX330 BbsI digested vector (50ng)

2 µl annealed oligo duplex from step 1 (1:250 dilution)

2 µl 10x DNA ligase buffer (make sure fresh, else ATP or DTT may be shot)

1 µl T4 ligase

Y µl H₂O to 20 µl final volume

5.  2 ul连接产物转化感受态。
6. 挑选克隆，使用U6测序引物测序。

2.Useful sites for sgRNA design and off-target testing

• http://www.genome-engineering.org/crispr/?page_id=41
• http://www.addgene.org/CRISPR/guide/
• http://crispr.mit.edu/