Experiment Summary

Recently developed gene editing technologies based on engineered CRISPR/Cas9 systems enables researchers to disrupt genes in a cell type-specific manner in the adult mouse brain. Using these technologies, we recently showed that the dopamine beta-hydroxylase gene in Locus Coeruleus (LC) norepinephrine neurons plays a vital role in the maintenance of wakefulness. Our method consists of four steps, (1) crossing Cre-dependent spCas9 knockin mice with a Cre-driver mouse line to express spCas9 in the target neural populations, (2) cloning of sgRNA, (3) construction of an AAV (adeno associated virus) vector expressing dual sgRNA, and (4) virus packaging and stereotaxic injection of the virus into the target brain area. Here, we describe a detailed protocol of AAV vector construction for cell type-specific CRISPR gene editing in the adult mouse brain. The method adopts a dual-sgRNA strategy for efficient disruption of the target gene. At first, a few different sgRNAs targeting the same gene are cloned into a plasmid expressing spCas9. After evaluation of the sgRNAs by a T7 endonuclease assay, the two most efficient sgRNAs are cloned in tandem into an AAV vector using the Gibson Assembly method.

Materials and Reagents

1. Pipette tips
2. 24-well plate (coated)
3. 1 ml plastic tube
4. NEB10-beta Competent E. coli
5. pX458
6. pAAV EF1α DIO mCherry
7. BbsI-HF
8. MluI-HF
9. DpnI
10. Alkaline Phosphatase, Calf Intestinal (CIP)
11. Guide oligonucleotides
12. Quick ligation kit
13. Gibson Assembly Master Mix
14. PrimeSTAR HS DNA polymerase
15. dNTP Mix 10 mM each
16. Gel Extraction kit
17. Spin Miniprep kit
18. Tris-EDTA, (1x Solution)
19. LB Broth EZMix Powder
20. LB Agar
21. Ampicillin
22. Primer to verify the cloning of sgRNA into pX458: 5'-gactatcatatgcttaccgt-3'
23. Primer to verify the tandem cloning of sgRNAs into pAAV: 5'-actgacgggcaccggagcca-3'
24. Primers for Gibson Assembly:
    Primer S1: 5'-taggggttcctgcggccgcacgcgtgagggcctatttc-3'
    Primer AS1: 5'-ataggccctctctagaaaaaaagcaccgactc-3'
    Primer S2: 5'-tttttctagagagggcctatttcccatg-3'
    Primer AS2: 5'-atccatctttgcaaagcttacgcgtaaaaaagcaccgac-3'
25. NIH3T3 cell
26. AAV vector (pX458-sgRNA or pX458 empty as negative control )
27. Primers: You can design the primers to amplify the target exons by CHOPCHOP (see Procedure C)
28. PrimeSTAR HS DNA polymerase
29. FuGENE6 Transfection Reagent
30. DMEM (1x)
31. Fetal Bovine Serum
32. Penicillin/Streptomycin
33. PBS, pH 7.4
34. TrypLE Select (1x)
35. DNA Extraction Solution
36. T7 Endonuclease I

Equipment

1. Pipettes
2. Centrifuge
3. Incubator
4. Thermocycler
5. Electrophoresis chamber
6. (Optional) CO₂ Incubator
7. (Optional) Clean bench
8. (Optional) Centrifugation

Procedure

A. Design of sgRNA

Single guide RNAs (sgRNAs) with high efficiency and specificity can be designed by using gRNA design webtools. Cloning of sgRNAs into the spCas9 expressing plasmid (pX458).

1. Digestion of pX458 with BbsI:

   X μl	pX458 (10 μg)
   5 μl	10x NEB cut smart
   1 μl	BbsI-HF
   24 - X μl	MilliQ water
   30 μl	in total

2. Incubate at 37 °C overnight.
3. Gel purify the plasmid using Gel extraction kit. Elute the plasmid with 50 μl of 1x Tris-EDTA buffer.
4. Annealing of oligonucleotides encoding sgRNAs:

   1 μl	oligonucleotide, sense (100 μM)
   1 μl	oligonucleotide, antisense (100 μM)
   8 μl	1x Tris-EDTA buffer
   10 μl	in total

5. Incubate at 95 °C for 5 min. Then cool down to room temperature by switching off the thermocycler (30 min).
6. Prepare the ligation reaction as following:

   1 μl	diluted (1:100 with 1x Tris-EDTA buffer) annealed oligonucleotides
   1 μl	BbsI-digested pX458 (50 ng/μl)
   5 μl	2x ligation buffer
   1 μl	Quick ligase
   2 μl	MilliQ water
   10 μl	in total

7. Incubate at room temperature for 10 min.
8. Add 5 μl of the ligation reaction to 50 μl of NEB10-beta.
9. Next place on ice for 10 min.
10. Heat shock at 42 °C for 30 s.
11. Transfer onto ice for 3 min.
12. Plate the reaction on an LB-Agar plate containing ampicillin (50 μg/ml).
13. Incubate at 37 °C overnight.
14. Inoculate 2 ml LB broth containing ampicillin (50 μg/ml) from a single colony and incubate at 37 °C overnight with agitation.
15. Purify the plasmid from 1 ml of the overnight culture using Spin Miniprep kit.
    Elute the plasmid with 50 μl of buffer EB and determine the concentration
16. Verify the plasmid by sequencing with a primer (5'-gactatcatatgcttaccgt-3')

B. (Optional) Evaluation of sgRNA by T7 endonuclease I ass

1. Plate NIH3T3 cells at the density of 2.5 x 104 cells (500 μl/well, DMEM/10% FBS/1% Penicillin /1% Streptomycin) onto a well of 24-well plate.

2. Incubate in a 37 °C CO2 incubator overnight.

3. Transfection

a) Add 1.5 μl of FuGENE6 reagent to 50 μl of serum-free DMEM in a 1 ml plastic tube and mix by vortexing.

b) Incubate the mixture for 5 min at room temperature.

c) Add 0.5 μg of pX458-sgRNA or control vector to the mixture and mix by vortexing.

d) Incubate the mixture for 30 min at room temperature.

e) Add the mixture to each well of a 24-well plate containing cells.

f) Incubate in a 37 °C CO₂ incubator for 24 h.

g) Replace the cell culture media with fresh one (DMEM/10% FBS/1% Penicillin /1% Streptomycin).

h) Repeat Steps C3d-C3e.

i) Incubate in a 37 °C CO₂ incubator for 48 h.

4. Remove the supernatant of cells by pipetting and wash cells with 500 μl of 1x PBS.

5. Add 100 μl of QuickExtract to each well and collect the lysate containing genomic DNA to a 1 ml plastic tube.

6. Incubate at 65 °C for 15 min and then at 98 °C for 10 min.

7. Centrifuge at 20,000 x g for 10 min.

8. Collect 60 μl of the supernatant (can be stored at -20 °C).

9. PCR to amplify 200-300 bp fragments around the CRISPR binding site. The primers can be designed by CHOPCHOP.

10. Start PCR with the following settings:

a) Denaturation at 95 °C for 2 min.

b) 35 cycles of

95 °C for 10 s

55 °C for 5 s

72 °C for 45 s

c) Final elongation at 72 °C for 2 min.

d) Store at 4 °C.

11. Gel purify PCR product using Gel extraction kit. Elute PCR product with 50 μl of buffer EB.

a) Denaturarion and annealing of PCR product:

5 μl PCR product (200 ng)

2 μl 10x NEB buffer 2

12 μl MilliQ water

19 μl in total

b) 95 °C for 5 min.

c) Cool down to room temperature by switching off the thermocycler (30 min).

12. Add 1 μl of diluted (1:5) T7 endonuclease I (2.5 U/μl).

13. Incubate at 37 °C for 30 min.

14. Run on 2% agarose gel and verify bands in sgRNA transfected-wells compared with control wells (Figure 2).

Figure 2. Agarose gel electrophoresis of T7 endonuclease I-digested PCR products derived from NIH3T3 cells transfected with sgRNA and Cas9.

C. Tandem cloning of two sgRNAs into pAAV EF1α DIO mCherry

In Procedure A, you clone several sgRNAs targeting the same gene. For the efficient gene disruption, you tandemly clone two different sgRNAs driven by independent human U6 promoters into an AAV vector (Figures 3B and 3C) using the Gibson Assembly method.

Figure 3. Construction strategy of a dual sgRNA viral vector.

1. Digestion, dephosphorylation and purification of pAAV EF1α DIO mCherry with MluI.

a) Digestion:

b) Incubate at 37 °C overnight.

c) Add 1 μl of CIP, and then incubate at 37 °C for 60 min.

d) Gel purify the plasmid using Gel extraction kit.

e) Elute the plasmid with 50 μl of buffer EB.

2. Amplification of U6 promoter plus sgRNA#1 or sgRNA#2.

a) PCR reaction#1 (Expected size is 393 bp):

b)PCR reaction#2 (Expected size is 388 bp):

3. Start PCR with the following settings.

a) Denaturation at 95 °C for 2 min.

b) 35 cycles of

95 °C for 10 s

62 °C for 5 s

72 °C for 45 s

c) Final elongatation at 72 °C for 2 min.

d) Store at 4 °C.

4. Run on a 2% agarose gel.

5. Gel purify the PCR product using Gel extraction kit. Elute the PCR product with 50 μl of MilliQ water.

6. Digestion of the residual template plasmid:

7. Incubate at 37 °C for 30 min, 80 °C for 30 min.

8. Gibson assembly:

9. Incubate at 50 °C for 15 min.

10. Add 2 μl of ligation reaction to 50 μl of NEB10-beta.

11. Follow Steps B9-B15.

12. Verify the plasmid by sequencing with a primer (e.g., 5'-actgacgggcaccggagcca-3').

13. Proceed to virus packaging.

14. Keep the virus stock at -80 °C.