Download 1. Select the sgRNA target site according to online tools provided by

1. Select the sgRNA target site according to online tools provided by Feng Zhang’s laboratory
(http://crispr.mit.edu/). The typical target sequence is "NNNNNNNNNNNNNNNNNNNNXGG"
2: Design the primers as
5’ sgRNA primer: GGACGAAACACCG(N20)GTTTTAGAGCTA
3’ sgRNA primer: TTTCTAGCTCTAAAAC(n20)CGGTGTTTC
Note: N20 and n20 should be complementary matched.
GGACGAAACACCG(N20)GTTTTAGAGCTA
CTTTGTGGC(n20)CAAAATCTCGATCTTT
3: Anneal 5’ sgRNA primer and 3'gRNA primer.
a: Dissolve/Resuspend the primers using RNase/DNase-Free water to final concentration 200uM
b: Add the following reagent into 0.5ml EP tube at room temperature.
ddH2O
8 ul
10X oligo Annealing
2 ul
Buffer
5’ sgRNA primer
5 ul
(200M)
3’ sgRNA primer
5 ul
(200M)
Total Volume
20 ul
10X Oligo Annealing Buffer
(1M NaCl, 100 mM Tris-HCl, pH=7.4)
c: Heat the EP tube at 950C for 4 min.
d: Take the EP tube at room temperature to cool down about 30 min. The final concentration of the double
strand Oligos (dsOligos) is 50 M
e: Short spin the dsOligo for 5 sec, gently mix.
f: Dilute the 50M dsOligos to 5nm (1:5000)
Add 1l 50uM dsOligos to 99ul water. Mix (vortex). Get the final concentration of 500nM
50μM dsOligo
1μL
ddH2O
99μL
Total Volume
100μL
Add 1l 500nM dsOligos to 49ul 1X Oligo Anneal Buffer. Mix (vortex). Get the final concentration of
10nM.
500nM dsOligo
1μL
10×Oligo Annealing Buffer 5μL
ddH2O
44μL
Total Volume
50μL
g: Aliquot the 10nM dsOligo, store at -20℃.
4: Cut the Cas9 sgRNA vector using BbsI. Incubate at 37℃ for 2 hours.
BbsI (NEB R0539)
Cas9 sgRNA vector
10X NEBuffer 2
ddH2O
5. Gel purify the vector and obtain the concentration.
3 l
1 g
5 l
To 20 l
6: Insert the annealed products into BbsI-digested Cas9 sgRNA vector.
10×T4 Ligase Buffer
dsOligo (10nM)
Cas9 sgRNA vector
T4 DNA Ligase
ddH2O
2μL
4μL
50ng
1μL
To 20μL
Incubate ligation at 16oC overnight (16 hours).
7. Transform into E. coli and select using Kanamycin-containing agarose plates.
8. Select clones and sequence using T7 or SP6 primer.
Typically only 2-3 clones are required for obtaining the correct plasmid
NOTE:
1. The sequence information could be found in the related document on the website of Addgene and Table S4
in the manuscript.
2. If the target sequence begins with "G"(GNNNNNNNNNNNNNNNNNNNXGG), the primer could be
designed as
5’ sgRNA primer: GGACGAAACACCG(N19)GTTTTAGAGCTA
3’ sgRNA primer: TTTCTAGCTCTAAAAC(n19)CGGTGTTTC