TITLE: Fungal gene knockout with Agrobacterium tumefaciens using Gibson assembly
AUTHORS: Johannes Wolfram Debler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This method uses Agrobacterium tumefaciens to transfer a piece of TDNA containing a resistance gene (hph for hygromycin in this particular case) flanked by homologous regions of the gene you want to knock out into the fungal spore. </div><div class = "text-block">The process is broken down into following steps:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Assembly of the knockout construct via Gibson assembly</li><li style = "counter-reset:ol0;list-style-type:disc;">Transformation into E.coli</li><li style = "counter-reset:ol0;list-style-type:disc;">Plasmid harvest and transformation into Agrobacterium tumefaciens</li><li style = "counter-reset:ol0;list-style-type:disc;">TDNA transfer into fungal spores</li><li style = "counter-reset:ol0;list-style-type:disc;">Verifying knockout</li></ul></div></div>

[STEPS]
?. [PCR of flanking regions]
Depending on your fungal organism you may have to try different sizes. A good starting length is 1000 bp up- and downstream of your gene of interest.Use a proof reading polymerase!The primers we use to amplify the 5'- and 3' UTRs will carry tails which overlap with our plasmid backbone and the selection marker cassette.Design your primers as 15-20 bp matching the region of interest which we will use as the homologous fragments (the 1000 bp up- and downstream of the sequence we want to replace) and add the following sequences as 5'-extensions to your primers:5'UTR Forward primer extension: CATTGCGGACGTTTTTAATGTACTGGGTACC (overlaps with the Left Border of the plasmid backbone)5'UTR Reverse primer extension: AGTGCTCCTTCAATATCATCTTCTGGGTAC (overlaps with the PtrpC promoter of the resistance cassette)3'UTR Forward primer extension: AATGCACAGGTACACTTGTTTAGAGGGTAC (overlaps with the TtrpC terminator of the resistance cassette)3'UTR Reverse primer extension: ACCCAAATCAAGTTTTTTGGGGTCGGGTACC (overlaps with the Right Border of the plasmid backbone)Use your polymerase manufacturers manual for PCR conditions.10-20 ul assays should give you enough fragment for cloning.Example primers:5' UTR Forward: CATTGCGGACGTTTTTAATGTACTGGGTACAGGACGAACCATGTTTGCAT5' UTR Reverse: AGTGCTCCTTCAATATCATCTTCTGGGTACGATGAGTTTGGTGTGTGTGABold: 5' primer extensionItalics: gene (UTR) specific sequence where primer binds
?. [Digestion of Backbone with KpnI]
We need to open the plasmid up to separate the backbone from the resistance cassette (PtrpC-hph-TtrpC). The plasmid was designed to contain two KpnI restriction sites flanking the resistance cassette in order to accomplish this.Follow the restriction enzyme manufacturers recommendations.Digest about 1ug of plasmid.Plasmid in genbank and geneious format:
?. [Cleanup of flanking regions, plasmid backbone and resistance cassette]
After PCR of flanking regions and plasmid digestion run all reactions on a gel.We do this for two reasons:To check if the PCR of the flanking regions actually workedTo separate the digested plasmid fragments from any undigested plasmid (which would otherwise lead to false positive colonies later on which do not contain the assembled flanking regions).Use your preferred gel extraction kit for this and elute fragments in as small a volume as you can get away with.
?. [Gibson assembly of fragments into backbone]
We use Gibson assembly to create our knockout construct. As shown in the picture below, we want a construct that looks as follows:plasmid backbone - LB - 5'UTR - resistance cassette - 3'UTR - RB - plasmid backbone.Follow your Gibson enzyme mix manufacturer's instructions. In the case of NEB's NEBuilder®  HiFi DNA Assembly Master Mix/NEBuilder HiFi DNA Assembly Cloning Kit use the NEBioCalculator and following table as a guide. The manual suggests to use 50 fmol per fragment for the assembly of 4 or more fragments.  ABCD12x NEBuilder mix52hph cassette ( 31 ng/ul)0.722 ng50 fmol3backbone (125 ng/ul)0.575 ng50 fmol45'UTR 500 bp (25 ng/ul)0.410 ng50 fmol53'UTR 500 bp (24 ng/ul)0.410 ng50 fmol6H2O3We should end up with a plasmid like this:
ABCD12x NEBuilder mix52hph cassette ( 31 ng/ul)0.722 ng50 fmol3backbone (125 ng/ul)0.575 ng50 fmol45'UTR 500 bp (25 ng/ul)0.410 ng50 fmol53'UTR 500 bp (24 ng/ul)0.410 ng50 fmol6H2O3
?. [Transformation of assembly mix into E.coli]
Transform 1 - 5 ul of the Gibson assembly mix into 50 ul thermocompetent E.coliCombine 1 - 5 ul NEBuilder mix with 50 ul thermocompetent E.coliIncubate on ice for 10-30 minutesheat shock for 30-40 seconds at 42°C in a waterbathIncubate on ice for 5-10 mintuesadd 750 ul SOC mediumIncubate for 1 hour on a shaker incubator at 37°C and 250 rpm
?. [Plate transformants]
Concentrate cells by centrifugation at 2000 x g for 2-5 minutesdiscard most of the supernatantresuspend cell pellet in 100-200 ul leftover supernatantplate cell mixture on LB plates containing 30 ug/ml kanamycin (try different amounts to avoid ending up with too many colonies, for example: one plate with 5 ul, one with 20 ul and one with the rest)incubate overnight at 37°CDepending on the competency of your E.coli cells, you may be able to use them directly without spinning and concentrating them first.
?. [Colony PCR to screen for correct transformants]
Growing colonies after overnight incubation point to transformants carrying the resistance gene. But in order to make sure those are not just contamination from carried over plasmid backbone we will run two PCR reactions on each colony (multiplex works as well, but is better for shorter flanking regions) to check for presence of assembled flanking regions.  ABC1IDsequencebinds at2JD281GGCAGTGACGTCATCGplasmid backbone (RB)3JD333CCGTAACACCCAATACGCTtrpC4JD306AGACTGAGGAATCCGCTCTtrpC (optional primer for multiplex)5JD288GGCTGATCTGACCAGTTGPtrpC6JD445GCCTTACAACGGCTCTCplasmid backbone (LB) For a 500 bp flanking region we expect following bands:JD281 + JD333 = 828 bp(JD281 + JD306 = 1124 bp - use this pair if you want to multiplex all 4 primers to get fragments of more different sizes - JD281, JD306, JD288, JD445)JD288 + JD445 = 848bp
ABC1IDsequencebinds at2JD281GGCAGTGACGTCATCGplasmid backbone (RB)3JD333CCGTAACACCCAATACGCTtrpC4JD306AGACTGAGGAATCCGCTCTtrpC (optional primer for multiplex)5JD288GGCTGATCTGACCAGTTGPtrpC6JD445GCCTTACAACGGCTCTCplasmid backbone (LB)
?. [Harvest plasmid and sequence]
If colony PCR gives you bands of the expected size grow the respective colonies in 5 ml LB medium containing kanamycin at a final concentration of 30 ug/ml  overnight at 37°C at 250 rpm.The next morning combine 700 ul overnight culture with 300 ul 50% glycerol and store as backup in the -80°C freezer.Perform a mini prep on the other 4 ml with your favourite kit or method. Adjust plasmid concentration after prep to about 100 ng/ul.Sequence your assembled plasmid across the flanking regions using following primers:  ABC1JD445GCCTTACAACGGCTCTCfrom backbone into 5' flanking region2JD288GGCTGATCTGACCAGTTGfrom PtrpC into 5' flanking region3JD333CCGTAACACCCAATACGCfrom TtrpC into 3' flanking region4JD281GGCAGTGACGTCATCGfrom backbone into 3' flanking region
ABC1JD445GCCTTACAACGGCTCTCfrom backbone into 5' flanking region2JD288GGCTGATCTGACCAGTTGfrom PtrpC into 5' flanking region3JD333CCGTAACACCCAATACGCfrom TtrpC into 3' flanking region4JD281GGCAGTGACGTCATCGfrom backbone into 3' flanking region
?. [Electroporation into Agrobacterium tumefaciens]
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?. [Electroporate plasmid into Agrobacterium]
Electroporate your sequence verified plasmid into your Agrobacterium tumefaciens strain of choice.Electroporation of Agrobacterium tumefaciensYou may want to run another colony PCR on Agrobacterium colonies to verify that the colony contains your knockout construct.
?. [Electroporation into Agrobacterium tumefaciens]
?. [Fungal transformation]
Grow transformed Agrobacterium (strain AGL1) on a plate (LB + rifampicin 50 ug/ml + kanamycin 30 ug/ml) at 25°C until colonies appear.Then inoculate a colony from that plate into 50 ml LB + rifampicin 50 ug/ml + kanamycin 30 ug/ml and grow at 27°C at 250 rpm overnight.
?. [PCR of flanking regions]
OPTIONAL (2 step PCR to save money on primers)If you are going to do this on a regular basis, ordering long primers like those above becomes expensive quickly. We therefore split the PCR process into two rounds. The first uses a gene specific primer with a shorter tail. That PCR product then becomes the template for a second PCR with universal primers which bind to the short tail introduced during PCR 1  and carry their own tail to complete the full overlap sequence.Example:5'UTR forward primer (gene specific) for PCR1:5'UTR universal primer for PCR2:Full overlap after PCR2:As above, you design your respective primers with 15-20 bp gene specific sequence and then add the following as 5' extensions:5'UTR forward primer extension: TAATGTACTGGGTAC5'UTR reverse primer extension: TCATCTTCTGGGTAC3'UTR forward primer extension: TTGTTTAGAGGGTAC3'UTR reverse primer extension: TTTGGGGTCGGGTACUniversal 5'UTR forward primer: CATTGCGGACGTTTTTAATGTACTGGGTACUniversal 5'UTR reverse primer: AGTGCTCCTTCAATATCATCTTCTGGGTACUniversal 3'UTR forward primer: AATGCACAGGTACACTTGTTTAGAGGGTACUniversal 3'UTR reverse primer: ACCCAAATCAAGTTTTTTGGGGTCGGGTACBold: 5' primer extensionItalics: binds to 5'extension of first round primerExample 5'UTR after PCR 2 with full overlaps:
?. [Cleanup of flanking regions, plasmid backbone and resistance cassette]
OPTIONAL (but improves efficiency)Cleanup of gel extracted fragmentI have had mixed results using the gel extracted fragments directly with Gibson assembly. The Nanodrop results are usually not that great. I therefore now use the Zyppy Clean and Concentrator kit to clean up the gel extracted fragment. This also allows you to elute in as little as 5 ul efficiently.
?. [Electroporate plasmid into Agrobacterium]
Verify your Agro colonies by PCR using the same primers as in step 7 above.
?. [Fungal transformation]
The next morning check if the inoculated media is turbid and if so, transfer to a 50 ml falcon tube and spin at 3000 x g at 4°C for 20-30 minutes.Resuspend pellet in 50 ml Induction Medium (IM) Measure OD600spin at 3000 x g at 4°C for 20-30 minutes.Resuspend pellet in IM so the final OD600 is ~0.5 (if it was 0.3 at first measurement, resuspend in 30 ml IM, if it was 0.4, resuspend in 40 ml etc.)Add 0.2 M Acetosyringone to a final concentration of 0.2 mM (for 50 ml add 50 ul, etc.)Incubate at 150 rpm at 27°C for about 5 hours
?. [Fungal transformation]
Harvest fungal spores the way you usually harvest them. In our case, we flush a plate containing spore producing fungus with sterile H2O and pass the water through a syringe stuffed with sterile cotton wool. The filtrate then gets spun at 3000 - 5000 x g for 20 minutes at 4°C. The spore pellet, depending on how big it is, get resuspended in sterile H2O and spores are counted using a haemocytometer.
?. [Fungal transformation]
mix 100 ul spores (you'll have to find out what concentration works best for you) with 100 ul Agrobacterium solution (OD600 ~0.5). Put a nitrocellulose membrane (Whatman cellulose nitrate membrane, 0.45 um, 47 mm diameter) onto an IM Agar plate containing 0.2 mM Acetosyringone.Spread 200 ul Spore / Agrobacterium solution onto membrane (Use 3 - 10 plates per transformation)Wrap plate with parafilm or clingwrap and store at 25°C in the dark for 2 - 5 daysTransfer membrane to 1/2 PDA hyg 50 ug/ml cef 50 ug/ml strep 30 ug/ml plates, wrap and put back at 25°C in the darkObserve plates and wait for colonies to emergeFungusspore concentrationmarkerA. rabiei me142 x 10^6 spores/mlHygromycinA. lentis Kewell2 x 10^7 spores/mlHygromycin
Fungusspore concentrationmarkerA. rabiei me142 x 10^6 spores/mlHygromycinA. lentis Kewell2 x 10^7 spores/mlHygromycin
?. [Verify transformants]
Once Colonies appear on your selective plates you need to transfer them to fresh selective plates. One plate per colony. Let them grow for a while and then do a Quick DNA extraction followed by PCR across your flanking regions (5UTR seq + JD288, 3UTR seq + JD333) as well your gene of interest (which should now be replaced).
?. [Verify transformants]
OPTIONAL (but suggested) - sequence KOAmplify a region starting outside the UTR sequences used to create your KO clone and sequence from there into the marker cassette (Primers 5UTR seq  + JD288 and 3UTR seq + JD333 as denoted in the diagram above.