Gene Knockout Mediated byCRISPR(Cas9-2hitKO) 5.0

Gene Knockout Mediated by CRISPR-Cas9 System

(Cas9-2hitKO)

What is the CRISPR-Cas9 system?

Figure 1. Overview of CRISPR-Cas9 system

CRISPR-Cas9 system is a widely used genome editing tool, which derives from the bacteria adaptive immune mechanism. As an engineered version, nowadays CRISPR-Cas9 system contains one enzyme Cas9 and one guide RNA. This guide RNA can direct cas9 to specific genome locus for further digestion. As shown in Figure 1.

So far, CRISPR-Cas9 system has two main applications: Gene knock-out and Gene knock-in. For gene knockout, once DNA double strand breaks are induced, the cells will repair the break through NHEJ DNA repair pathway which will cause tiny deletion or insertion. For gene knock in. with the homolog recombination template, the cells will be inserted one specific foreign sequence in genome.

Fragment deletion (Gene knockout) mediated by two digesting events

Compared with previous strategy, this protocol uses a different approach to achieve deletion of DNA fragment in specified size. In this protocol, two guide RNAs targeting adjacent sites of certain region are needed, and two digesting events mediated by Cas9 will cause the degradation of internal fragment. Then the ends will be ligated through NHEJ pathway. In the end, this strategy can cause fragment deletion in specified size. With this protocol, one or some exons can be deleted, or whole genes can be deleted. The diagram is shown in Figure 2.

To achieve higher knockout efficiency, vector carrying one cas9 protein and two guide RNA expressing cassettes is generated.

Figure 2. Diagram of Cas9-2hitKO system

Plasmids for this protocol

To achieve higher knockout efficiency, vector carrying one cas9 protein and two guide RNA expressing cassettes is generated. In this protocol, three plasmids are involved. PX458M and PX459M contain cas9 and one Guide RNA expressing cassette. Additionally the MCS is for the inserting of the second Guide RNA expressing cassette. EZ-GuideXH is for the preparation of the second guide RNA expressing cassette.

PX458M contains EGFP for fluorescent marking; PX459M contains puromycin resistant gene for drug selection.

Figure 3. Map for PX458M

Figure 4. Map for PX459M

Figure 5. Map for EZ-GuideXH

PX458M and PX459M are reconstructed from PX458 and PX459 plasmids from Zhang Feng’s lab in MIT through addgene. (PX458 and PX459 plasmids are gift from Prof. Xi Qiaoran’s lab) The main alteration is addition of one multiple cloning site for the second guide RNA expressing cassette.

EZ-GuideXH is generated based on EZ-T cloning vector. The main purpose of this plasmid is for preparation of the second guide RNA.

Assembly of Guide RNA

According to Zhang Feng lab’s protocol, guide RNA can be easily assembled through an ingenious strategy. This strategy is shown in Figure 6. When Guide RNA scaffold is digested by BbsI, an annealed duplex with specific sticking ends (as 20 Nucleotides’ target) will be ligated in this plasmid through T4 DNA ligase.

Figure 6. Strategy for guide RNA assembly

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Procedure

I. Pick Cas9 target with minimal off-targets potentials

Website for target prediction

http://crispr.mit.edu/. The brief manual is as below (Figure 7.)

1. Input no more than 250 nts’ genomic DNA sequence to the input box, and pick the corresponding species. Then click button ‘submit’.

2. Click button ‘Guides & offtargets’.

3. Choose targets whose scores > 90. The targets can be in both orientations.

Figure 7. Submission of prediction of guide RNA target

II. Pick a pair of Cas9 targets to delete the objective DNA fragment

To delete a genomic DNA fragment, two targets recognized by guide RNA are indispensable. According to previous test, as long as 1M bps sequence can be deleted efficiently. However, fragment deletion of <10 kbs should be fine and efficient enough. The criteria of choice on target pair is as followed, ( Figure 8 and Figure 9 are two examples)

a. If the interested gene is short (about 10 kbs, or a little larger), just delete the whole gene including promoter region.

b. If the interested gene is large, deleting the most important exon or exons should be a good choice.

c. If the interested gene is large, an alternative is deleting a part of the gene including whole promoter region

d. If the interested gene was knockout by traditional strategy previously, just delete the same region.

Figure 8. Cdkn2a Cas9-targeting

Figure 9. PTEN Cas9-targeting

III. Construct CRISPR-Cas9 vector carrying two Guide RNA expressing cassettes

Figure 10. Overview of construction

1. Synthesis target oligos.

Oligo-Forward: CACC-(N)20

Oligo-Reverse: AAAC-(N)20R

If the first nucleotide of the target sequence is not ‘G’, just add one ‘G’ to the 5’ end. The oligos become

Forward: CACC-G(N)20

Reverse: AAAC-(N)20RC

2. Annealing

On thermal cycler with heat lid.

37 ℃, 30 min

95 ℃, 5 min

Ramp down to 25℃ at 0.1℃/sec. (when step 25℃ is highlighted, click option, set Ramp as 0.1℃/sec)

25 ℃, 5 min

4 ℃, 5 min

Put on ice, or keep in -20℃ freezer for storage.

3. Digest Px458M(or PX459M) and EZ-Guide-XH with BbsI

Purify the enzyme digested product with PCR product purification kit, and make sure that the concentration is more than 50 ng/ul.

4. Ligation

Dilute the annealed target oligos 200 times for further ligation

Ligate for more than 2 hours at 16 ℃. (Overnight is better.)

5. Transformation and identification of positive clones

Pick single clones and culture for several hours in 37℃ incubator, and perform PCR to identify the positive clones.

For Px458M and Px459M, use Oligo-Forward and CAG-R as primers; for EZ-GuideXH, use Oligo-Forward and M13F as primers.

6. Digest Guide RNA Target2 expressing cassette in the EZ-GuideXH and PX458M-Target1(or PX459M-Target1)

7. Gel extraction and purification

For EZ-GuideXH-Target2, cut and purify the smaller DNA band about 360 bps; for the PX458M-Target1, cut the unique main band about 3300 bps.

8. Ligation

Ligate overnight at 16 ℃.

9. Transformation and identification of positive clones

Use Oligo-Forward (target1) and CAG-R as primers, and the PCR product is about 750 bps.

10. Plasmid extraction and sequencing validation.

Culture the positive clone in 10 ml Amp+ LB medium, and extract plasmid.

Use CAG-R as sequencing primer.

IV. Assess knockout efficiency in 293FT cells (or other cells)

1. Transfect PX458M plasmid carrying Guide RNA expressing cassettes in 293FT cells for 36-48 hours. For mouse genes, MEF, 3T3 and some other easy-transfect cells can be used.

2. Detect the GFP Fluorescence through fluorescent microscopy or examine the transfection efficiency through Flow cytometer.

3. Extract genomic DNA from transfected cells. And perform genotyping with these gDNA samples. If the mutant PCR band is obvious and strong enough, the pair of Guide RNAs can be regarded as efficient. Figure 11 show the reglular strategy for knockout genotyping; and Figure 12 show the example of PPM1A Knockout.

Figure 11. Diagram of genotyping

Figure 12. Genotyping of PPM1A knockout

V. Knockout in interested cells

Theoretically, any cells transfected with plasmid should be KO cells. However, to get pure KO clones, single clones culture are necessary. Culture of single clones is a little difficult for some cell types. To improve the expansion of single cells, gelatin pre-coating and conditional medium can increase the survival rate of single cells.

1. Plate the cells in 6-well plate one day before, and make sure the cell confluency reach about 70-80%.

2. Transfect the interested cells with validated px459M based plasmid and culture for 24 hours.

3. Treat the transfected cells with 2ug/ml puromycin for 3 or 4 days. Set one mock un-transfected well as control.

Cautions!: before treatment, a pilot experiment is needed to explore the optimized puromycin concentration.

4. Withdraw the puromycin, and culture the cells with 20% FBS medium.

5. Several days later, if independent clones expand, pick the clones and transfer them into 24-well plate for culture. The remained cells keep growing until about 50% cell confluency.

6. When the cells in 24-well plate reach more than 30% confluency, trypsin-digest these wells, take out 80% cells for genotyping, and remain 20% cells for further culture.

7. Quick-spin the cell suspension at 14000rpm for 30s. Remove the supernatant, and lyse the cell pellet with 20-60ul solution A. Heat the lysis at 95 ℃ for 10 min, then add the same volume solution B, vortex for a while, and centrifuge at 13000rpm for 10min. remove the supernatant for PCR template.

8. Do the genotyping, and keep the clones containing knocked-out cells

9. When the clones containing knocked-out cells reach full confluency, digest the cells with trypsin, and count the cell number. Serially dilute the cells in 40 cells/10ml medium by 10-fold.

10. The medium for the step 9 should be the conditional medium. That is, coll ecting medium supernatant of the same cell type (culture time is less than or about 24 hours, avoiding the over-proliferating’s medium supernatant). Add 20% FBS in this supernatant and filter the medium with 0.22 ul filter.

11. Take out 96-well plates, add 100 ul autoclaved 0.1% gelatin solution per well, then incubate in 37 ℃ for at least 30 min.

12. Aspirate the gelatin, and aliquot 100 ul diluted 0.4 cell containing conditional medium per well. Each 8 or 10 wells are added, shake the cell suspension and continue.

13. Add another 100ul conditional medium into the 96-well plate on the fourth or fifth day, and no later than the fifth day.

14. When the cell confluency reach more than 30%, or the clones expand large enough, digest the cells and transfer them into 24-well plate, repeat the step6-8. Keep pure knocked-out clones for further experiment.

15. For the remained cells in step 5. When cell reach 50% confluency, they can also be used for limiting dilution for single clones. Or this mixture can be frozen for later isolation.

VI. Assess off targets effect

Once some clones are identified KO ones. The potential off-target effect should be detected. Website (http://crispr.mit.edu/.) will list all potential off-targets site. Amplify those high-risky off-target site, and examine whether off-target occur through sanger sequency. If any obvious off-targets are not detected, then these clones can be used for further functional experiment.

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