RNA generation for CRISPR/Cas9 injection to Drosophila embryos

Andrew Bassett Updated 13th June 2013

PCR of template
1. Order CRISPR_F oligonucleotide replacing ggn18 with target sequence
gaaattaatacgactcactataggnnnnnnnnnnnnnnnnnngttttagagctagaaatagc
2. Amplify guide RNA template with Pfusion polymerase (NEB) in 100 l volumes
Component
ddH2O
5x HF buffer
10 mM dNTPs
Pfusion DNA polymerase
CRISPR_F Primer (10 M)
CRISPRsgR (10 M)

98C
98C
60C
72C
72C
4C

x1
67 l
20 l
2 l
1 l
5 l
5 l
100 l
30 s
10 s
30 s
x 35 cycles
15 s
10 min
Hold

3. PCR purify with a Qiagen PCR purification kit, eluting in 30 l EB.

4. Measure concentration by absorbance at 260 nm on a Nanodrop. Should give
approximately 150 ng/l concentration
In vitro transcription of sgRNA
5. Perform in vitro transcription with a T7 MEGAscript kit (Ambion). Assemble the
following reaction in the order shown below at RT to avoid precipitation of the DNA
with the reaction buffer.
6 l
2 l
2 l
2 l
2 l
2 l
2 l
2 l
20 l

Nuclease free water

ATP
CTP
GTP
UTP
10x reaction buffer
PCR product (150 ng/l)
Enzyme mix
Total

6.
7.
8.
9.

Mix, and incubate at 37C for 4 h

Add 1 l turbo DNAse, mix and incubate for a further 15 min at 37C
Add 115 l nuclease free water and 15 l ammonium acetate stop solution
Add 150 l phenol:chloroform:isoamyl alcohol (24:24:1), pH 7 and vortex thoroughly
for 30 s
10. Spin at 10000 g for 3 min at RT
11. Remove upper layer to a fresh tube, and precipitate with 150 l isopropanol
12. Incubate at -20C for 15+ min, then centrifuge at 17000 g for 15+ min at 4C
13. Wash twice in 0.5 ml RT 70% ethanol, spinning at 17000 g for 3 min at 4C between
each wash
14. Resuspend in 30 l ddH2O and measure concentration on Nanodrop. Dilute to 1000
ng/l and run on a gel alongside Riboruler RNA ladder (Thermo scientific)
15. Coprecipitate with Cas9 mRNA at an appropriate ratio (20:1 (w/w) Cas9:sgRNA)
Cas9 mRNA production
1. Digest MLM3613 (Addgene 42251) Cas9 vector with Pme I to linearize
MLM3613 Cas9 plasmid
10x BSA
10x NEBuffer 4
Pme I (10 U/l)
ddH2O

10 g
5 l
5 l
5 l
to 50 l

2. Incubate at 37C for a minimum of 2 h (to overnight)

3. Stop linearization by adding
i. 1/20th volume 0.5 M EDTA (2.5 l)
ii. 1/10th volume 3 M sodium acetate pH 5.2 (5 l)
iii. 2 volumes ethanol (115 l)
4. Mix, and incubate at -20C for at least 10 min
5. Collect DNA at 17000 g for 15+ min at 4C
6. Respin, and remove residual fluid with a small pipette tip
7. Resuspend in 12 l ddH2O, and measure concentration by absorbance at 260 nm on
a Nanodrop. Aim for 500-1000 ng/l
8. Use the mMessagemMachine T7 kit (Ambion) and polyA tailing kit (Ambion) to
produce capped, polyadenylated mRNA
9. Remember to keep reaction buffer at room temperature to avoid precipitation, and
add to reaction later to prevent DNA precipitation
10. Add the following at room temperature in this order

i.
ii.
iii.
iv.
v.

1 g
To 20 l
10 l
2 l
2 l

linearized plasmid DNA (2 l)

ddH2O (4 l)
2x NTP/CAP
10x reaction buffer
enzyme mix

11. Mix and incubate at 37C for 2 h

12. Add the following components to polyadenylate the transcript. If desired, remove
2.5 l before and after incubation to analyse on an agarose gel
i.
ii.
iii.
iv.
v.

36 l
20 l
10 l
10 l
4 l

ddH2O
5x EPAP buffer
25 mM MnCl2
10 mM ATP
E-PAP

13. Mix and incubate at 37C for 30 min

14. Purify RNA with a RNeasy mini kit (Qiagen) using the following adapted protocol
15. Add 350 l buffer RLT and mix (do not add -mercaptoethanol; check there is no
precipitate)
16. Add 250 l 100% ethanol and mix by pipetting
17. Transfer sample to RNeasy mini spin column
18. Centrifuge for 15 s at 10000 g at RT, and discard flow through
19. Add 500 l RPE (make sure ethanol has been added) and centrifuge for 15 s at 10000
g at RT, and discard flow through
20. Add 500 l RPE and centrifuge for 30 s at 10000 g at RT
21. Transfer column to fresh 2 ml tube, and centrifuge for 2 min at 10000 g to dry
column
22. Transfer column to 1.5 ml collection tube, add 50 l RNAse free water (prewarmed
to 37C), incubate for 1 min, then centrifuge for 1 min at 10000 g at RT
23. Measure concentration by absorbance at 260 nm on a Nanodrop, and freeze 10 g
aliquots at -80C
24. Analyse 0.5 l Cas9 mRNA on a 1% agarose gel alongside the sgRNA
Co-precipitation of sgRNA and Cas9 mRNA
1. Mix 0.5 g sgRNA with 10 g Cas9 mRNA
2. Make up to 30 l with water, and add 3 l 3M NaOAc and mix thoroughly
3. Add 90 l ethanol, and incubate at -20C for a minimum of 15 min
4. Spin at 17000 g at 4C for 30+ min
5. Wash 2x 100 l RT 70% ethanol, spinning at 17000 g for 3 min at 4C between each
wash
6. Quickly spin down and remove last liquid with a small pipette tip
7. Dry at room temperature for a few minutes

8. Resuspend in 12 l ddH2O, and analyse 1 l on a gel, and the concentration of a

further 1 l by absorbance at 260 nm on a Nanodrop
Reagents
MLM3613 Cas9 vector
Pfusion High Fidelity DNA Polymerase
mMESSAGEmMACHINE T7 kit
Poly(A) tailing kit
MEGAscript T7 kit
RiboRuler High Range RNA ladder
RNeasy Mini kit
QIAquick PCR purification kit

Addgene
New England Biolabs
Life Technologies
Life Technologies
Life Technologies
Thermo Scientific
Qiagen
Qiagen

42251
M0530L
AM1344
AM1350
AM1333
SM1821
74104
28104