BCMB 301B Lab 5 CRISPR Cas-9

What are the generalised steps in this lab?

• isolate plasmid from E. coli DH5a with alkaline lysis and silica spin column

• rolling circle amplification with phi29 polymerase and random hexamer primers

• 30 C for 48hr on LB+Kan+Mannose (the editing incubation)

• 50 C plasmid cure

• 42 C plasmic cure

• Differential Media (LB + X-gluc, LB + starch +iodine, LB + Kan)

• interpretation of results

Why is mannose used and at what step in the protocol is this critical?

Mannose is used to induce the Cas-9 expression that is under control of the Pmanp promoter that is mannose inducible. This is critical for the 30 C editing step and to only express Cas when needed as over expression for a long period of time is usually lethal.

What is the purpose of the Pmanp promoter?

It is a mannose inducible promoter that is placed upstream of cas9 to regulate when it is expressed in this experiment.

Why is KanR included in the plasmid?

It is used as a selection marker for cell that alone are KanS but if transformed with plasmid, will survive.

Where is the type II cas -9 system from in this lab? What does it contain?

S. pyogenes

It encodes the cas-9, guide RNA and modified depending on the experiment by adding the protospacer and homology template (based on the region you want to insert)

What bacteria do we isolate the plasmid from?

E.coli DH5a

What makes E.coli DH5a useful for plasmid storage?

• endA mutation: it is a mutation in an endonuclease to help minimise degradation of the plasmid

• recA mutation: a mutation in a recombinase used for homologous recombination (help prevent recombination events so plasmid stay separate)

endA mutation

• mutation in an endonuclease to help minimise degradation of the plasmid

recA mutation

• a mutation in a recombinase used for homologous recombination (help prevent recombination events so plasmid stay separate)

What makes RCA optimal for use in this experiment?

• small amount of starting material and significant yield

• random hexamer primers ( can anneal at random locations all over the plasmid during denature @ 94 C)

• phi29 polymerase can proofread and has high processivity (requires temp to be 30 C for function)

• branched structure (concatemer) is more easily transformed into B. subtilius

why way xylose added to the culture medium?

the extra comK gene is under the control of the xylose inducible promoter PxylA and this allows for the control of the over expression and competency

Two controls were plated on LB media containing kanamycin and 0.2% mannose as part of the transformation protocol: B. subtilis FN3 alone and a streak plate of B. subtilis FN3 transformed with pJOE8999. What were the expected results and what did the results allow you to conclude?

• FN3 alone no growth and therefore sensitive to Kan

• FN3 with empty plasmid should grow as there is KanR gene on plasmid for marker

• this allows us to confirm that resistance to Kan is due to the presence of the plasmid and can be used to select for transformed B. subtilis FN3 as it’s phenotype alone is KanS

The transformed B. subtilis FN3 was transformed with pJOE899-aA2 were plated on LB+Kan+Mannose. What is the purpose of each additive to the LB media?

• Kan is used to select for cells containing the plasmid

• Mannose is used to induce the Cas9 system as it is under control of the PmanP promotor which is mannose inducible.

What promotor is the sgRNA under?

the constitutive promotor PvanP

Why is it important to have the Cas9 system under a mannose inducible promoter PmanP?

• cas 9 forms dsDNA breaks and if not fixed the cells will die (can only fix with homologous recombination and non homologous end joining which bacteria don’t have a robust repair system)

What was happening in the 30 C for 75 min? What was the general mech?

The Editing of B. subtilis:

• homology template contains the gusA gene flanked by homology arms on the plasmid

• Cas9 expression is induced with mannose PmanP

• Cas 9 combines with the sgRNA and can reconize the protospacer sequence and cleave

• the double stranded break it repaired homologous repair (what we want ) or non-homologous end joining

• If it undergoes homologous repair, the gusA gene should be inserted,

What is inactivated during the 50 C incubation step?

The plasmid replication protein encoded by the rep pE194ts on the pJOE8999 plasmid.

What is the sgRNA composed of?

tracrRNA - trans activating crRNA

Linker

crRNA - for the target of interest protospacer sequence