CRISPR/Cas9-mediated genome editing

From ActinoBase

CRISPR/Cas9 Genome editing

CRISPR/Cas9 genome editing can be used for precise CRISPR/Cas9-mediated genome engineering of Streptomyces strains. Mutations made have ranged from 1-100kbp deletions, precise codon changes to alter amino acids and insertions to add Flag-tags to proteins encoded at their native loci. Several different plasmids have been constructed and published and are listed below. Click the links to find out more.



For a comprahensive guide for CRISPR/Cas knockouts in Streptomyces using pCRISPomyces-2 see the link below to the 'Hutchings Lab protocol for generating CRISPR/Cas Knockouts using pCRISPomyces-2', written by Dr. Rebecca Devine.

Hutchings Lab protocol for generating CRISPR/Cas Knockouts using pCRISPomyces-2


This protocol has been confrimed to work for the following-

Workflow Overview

1. Design flanking primers and gRNA

2. Optimise primer conditions using gradient PCR on flanking primers using WT genomic DNA (gDNA)

3. Anneal guide RNA oligos (gRNA) oligos using oligo spacer programme

4. Golden gate assembly of annealed gRNA into plasmid

5. Transform vector into E. coli using heat shock

6. Select for successful transformants using blue/white screening to select successful insertion of the gRNA into the vector

7. Recover plasmid by miniprep

8. Amplify up flanks using gDNA and optimised primer conditions

9. Gel extract flanks

10. Linearize pCRISPomyces-2 vector + gRNA generated above using XbaI digestion

11. Gel purify vector

12. Gibson Assembly of flanks into digested vector

13. Transform into chemically competent E. coli NEB DH5alpha cells

14. Grow cells on LB + Apr plates to select for transformants

15. Confirm transformation using colony PCR (or XbaI digestion of plasmid prep)

16. Extract plasmid DNA from successful colony(s) by plasmid prep

17. Transform finished construct into ET cells using electroporation

18. Conjugate ET into Streptomyces and antibiotic treat to select for ex-conjugants

Designing flanking primers

1. Find the sequence for your target gene

2. Keep the start (ATG/GTG) and stop (TGA) codon (to keep the deletion in frame), and delete KO region and insert linker sequence (GCGAGCTCGCCTGGTCGCAGCAGC)

3. Go back ~20NT to generate internal primers with similar Tm to help annealing and ~70% GC

4. Design ~20NT external flanking primers with 60-70% GC and ~68°C Tm

5. Insert XbaI and gibson overlap sequence on end (not essential but increases efficiency of Gibson assembly and allows templates to be digested out/cloned back in)

Designing gRNA

1. Open a new (unedited) copy of the target gene sequence

2. Look at the REVERSE STRAND

3. Find NGG (PAM) within gene to be removed (search for ANGG or better TANGG to find unique sequences in high GC genomes)

4. Design ~20 NT gRNA with a Tm > 60°C

5. Blast ALL 4 POSSIBLE gRNAs (I.E where N is either A,C,T, or G) against the full genome to check gRNA specificity. It is more important to not have matching sequences on the 3’ end

6. Add BbsI cut site sticky ends for golden gate assembly

The website CRISPY-web can also be used to gRNA design, simply upload your target genome and define a target region. The software will then provide a list the most unique PAM sequences & gRNAs it can find within the target region.

Anneal gRNA using oligo spacer programme

1. Mix 5 μl 100μM Forward gRNA and 5 μl 100μM Reverse gRNA oligo with 90 μl HEPES buffer

2. Heat to 95°C in a thermocycler and then ramping down to 4°C at 0.1°C per second using spacer oligos programme

Golden gate assembly of annealed gRNA into pCRISPomyces-2

Set up 20 μl reactions with:

• 100 ng backbone

• 0.3 μl gRNA

• 2 μl T4 ligase buffer (NEB)

• 1 μl T4 ligase (NEB)

• 1 μl BbsI (NEB)

• dH2O

Incubate using golden gate programme (as below)

1. 10 minutes at 37°C

2. 10 minutes at 16°C

3. Go to step 1, 9 times

4. 1 cycle of 5 minutes at 50°C

5. 1 cycle of 20 minutes at 65°C

6. 4°C hold

Transform Golden Gate plasmid into E. coli using heat shock

1. Thaw chemically competent NEB5/Top10 cells on ice

2. Transfer 50 μl of competent cells to a 1.5 ml microcentrifuge tube

3. Add 2 μl assembled vector and mix gently (tip mix, DO NOT VORTEX!!)

4. Incubate on ice for 30 minutes (DO NOT MIX!!)

5. Heat shock cells at 42°C for 30 seconds

6. Transfer tubes on ice for 2 minutes

7. Add 950 μl SOC media and incubate at 37°C for 1 hour at 250 rpm to allow recovery

8. Plate onto pre-warmed LB + Apramycin + X-gal plates and incubate at 37°C overnight

Select for successful transformants using blue/white selection

Golden gate cloning into pCRISPomyces-2 uses blue white screening to select for successful transformants (See AddGene blog here for an explaination of blue-white screening). Pick white colonies and grow overnight in 10 ml LB + Apramycin then extract plasmid DNA from successful colony(s).

2. Grow up successful (white) colonies in LB at 37°C overnight

3. Recover plasmid using the QIAprep Spin Miniprep kit (Qiagen) (or a plasmid miniprep kit of choice)

4. Pellet ~3 ml cells by centrifugation

5. Resuspend cells in 250 μl Buffer P1 and 250 μl Buffer P2

6. After 2-3 minutes of lysis reaction add Buffer N3 and centrifuge samples at 13000 rpm for 10 minutes

7. Apply supernatant to a BLUE QIAprep spin column and centrifuge for 30 seconds

8. Wash the column with 750 µl Buffer PE

9. Discard the flow through and elute plasmid DNA in 50 μl dH2O

10. Quantify using Nanodrop and take note of concentration for use in the next steps

Linearize gRNA vector using XbaI digestion

1. Incubate or 1 μg vector with 1 μl XbaI, 5 μl buffer H and 40 μl dH2O at 37°C for approximately 2 hours

2. Dephosphorylate with shrimp alkaline phosphatase

3. Run product on a gel against undigested vector and visualise using UV

4. Excise band using a scalpel and extract DNA using the QIAquick gel extraction kit

Gibson Assembly of flanks into digested vector

1. PCR Amplify flanking DNA using Q5 (high fidelity – less errors, good for cloning) (Note: See High GC PCR for tips on how to perform PCR amplifications off of high GC genomes such as Streptomyces.)

2. Gel purify using Qiagen kit

3. Incubate digested vector with 2x flanks (aim for roughly 3:1 vector:insert) and 10 μl Gibson Assembly master mix (NEB) at 50°C for at least 15 minutes (longer gives better efficiency – I usually leave it for 1 hour)

4. Transform into chemically competent E. coli using heat shock (see above)

Confirm transformation using colony PCR

Conduct under STERILE conditions!!

1. Take ~8 1.5 ml microcentrifuge tubes (less if fewer colonies) and fill with ~500 µl LB

2. Make up Biotaq Red master mix with 1% v/v primer

3. Using a p10 tip, pick colony and touch tip to empty PCR tube before discarding into LB tube

4. Add 10 μl master mix to each PCR tube and amplify (55°C for pCRISP-2 test primers)

5. Run products on gel to see insert

6. Innoculate successful colonies in 10 ml LB (using the 500 µl LB plus tip)

7. Recover plasmid by same method as previously

Transform finished plasmid into ET cells using electroporation

1. Streptomyces strains contain a methyl-sensing restriction system therefore disrupted cosmids must initially be passaged through a non-methylating E. coli strain

2. Thaw ET cells on ice

3. Cool cuvette on ice

4. Electropotate using appropriate settings using 1 μl cosmid DNA in 50 μl of electrocompetent ET12567 cells containing the driver plasmid pUZ8002 and “pulse”

5. Take care not to hold the metal parts of the cuvette to warm it up

6. Reading should be around 5.4

7. Add 900 μl LB and pipette tip mix

8. Transfer to eppendorf & grow at 37ºC up for 1 hour to recover

11. Plate onto LB agar containing kanamycin, apramycin and chloramphenicol to select for the incoming plasmid

12. Incubate plates at 37ºC overnight

13. Pick colonies, grow overnight in liquid culture and use for conjugation and glycerol stock

Conjugate ET into Streptomyces treat with antibiotics to select for ex-conjugants

Also see Conjugation using ET12567/pUZ8002

1. Grow E. coli colonies selected from transformation plates in 10 ml LB broth plus Kanamycin, Chloramphenicol and Apramycin at 37°C overnight rotating at 250 rpm

2. Sub-culture E. coli under the same conditions for approximately 4 hours the following day

3. Once sub-cultures have reached OD600 0.6-0.8, wash the cells twice in LB to remove antibiotics that might inhibit the Streptomyces by spinning at 4000 rpm for 5 minutes.

4. Pre-germinate Streptomyces spores in 2X YT at 50°C for 10 minutes, leave to cool

5. Combine cells, centrifuge and resuspended in 200 μl LB

6. Plate dilutions of approx. 200 μl onto SFM agar + MgCl2 and incubated at 30°C for 16-20 hours (I increase the time for CRISPR conjugations compared to other vectors e.g. integrative plasmids i.e. use 20 hours rather than 16)

7. Overlay with 1 ml of dH2O + 0.5 mg Nalidixic acid + 1.25 mg Apramycin to selectively kill the E. coli

8. Return plates to the incubator at 30°C for four days or until colonies appeared (Often takes longer for CRISPR ex-conjugants to appear compared to other vectors e.g. integrative plasmids i.e. if normally takes 3-4 days expect to leave plates for 5-6 days AT LEAST!)

Protocol developed & written by Dr. Rebecca Devine, University of East Anglia, adapted for ActinoBase from Hutchings Lab protocol for generating CRISPR/Cas Knockouts using pCRISPomyces-2.