Genome engineering

5.2.17.1 Transfer of knockouts using phage P1

Phage P1 is a temperate bacteriophage that can choose between the lysogenic cycle in which it exists inside the bacterial cell without producing progeny and the lytic cycle during which the virus produces new particles resulting in the host’s cell death. During lysogeny, the phage DNA exists inside the bacterial cell in an independent circular form. The desired phage P1 particles can be produced by infecting the donor strain with the phage in the presence of CaCl2. As phage P1 needs calcium for infectivity, the phage will enter the lytic cycle and produce viral particles in which occasionally random bacterial DNA is inserted instead of the viral genome. This is exploited in the lab to transfer genetic markers, like antibiotic resistance genes, from one strain to another strain. These particles are then

used to transduce the recipient strain. Cells infected with particles containing the viral genome will produce new particles and die. Those particles containing bacterial DNA are incapable of virus production. Once injected into the host, the bacterial DNA will insert into the host genome by homologous recombination, replacing the gene containing homologous regions. By cultivating the infected recipient strain e.g. on the appropriate antibiotic and in the presence of sodium citrate, cells transduced with the genetic marker can be selected. Citrate decreases the concentration of free calcium by chelation, thus inhibiting functional phage P1 particles to infect the surrounding cells. Only those cells transduced with the genetic marker will survive^538,539.

In this study, strains from the Keio collection³⁸⁶ were used as donor strains (supplied by the Coli Genetic Stock Center, CGSC). The Keio collection is a collection of strains containing single-gene deletions of non-essential genes, which were knocked out via the Datsenko and Wanner method⁵⁴⁰. In place of the target gene, these strains harbor a kanamycin resistance cassette for selection, which is flanked by FLP recognition target (FRT) sites. By expressing the FLP recombinase the resistance gene can later be eliminated through recombination of these FRT sites.

Preparation of Phages

5 mL of LB medium with the appropriate antibiotics were inoculated with 50 µL of an overnight culture of the donor strain. The cells were incubated at 37 °C and 200 rpm until an OD600 of about 0.6 was reached. After the addition of 5 mM CaCl2, the cells were incubated at 37 °C and 200 rpm for another 30 min. The donor cell suspension was then mixed 1:1 with a solution of phage P1:

100 µL phage P1 (10^-3) 100 µL donor cell suspension

Three microcentrifuge tubes were prepared. The mixtures were incubated at 37 °C (without shaking) for 20 min and then added to 4 mL of 0.6 % soft agar with 5 mM CaCl2. The soft agar mixture was subsequently poured on LB agar plates (4 mL soft agar per plate) and incubated at 37 °C overnight.

The soft agar containing plaques was removed with a drigalski spatula and 800 µL of chloroform was added per plate. The soft agar/chloroform mixture was then vortexed until only small agar pieces remained. It was centrifuged at 15,422 g for 10 min and the supernatant was transferred to a glass bottle with a lid. A few drops of chloroform were added and the phage solution was stored at 4 °C.

Transduction

5 mL of LB medium with the appropriate antibiotics were inoculated with 50 µL of an overnight culture of the recipient strain and incubated at 37 °C and 200 rpm until an OD600 of 0.6-0.7 was reached. After the addition of CaCl2 (end concentration of 5 mM), the culture was incubated at 37 °C and 200 rpm for another 30 min. The following mixtures were prepared:

1) 3 µL donor phage solution + 1 mL recipient strain suspension 2) 30 µL donor phage solution + 1 mL recipient strain suspension 3) 1 mL recipient strain suspension (control)

4) 10 µL donor phage solution + 1 mL LB medium + 5 µL 1 M CaCl2 (control)

The mixtures were incubated at 37 °C for 15 min (without shaking). They were vortexed, centrifuged at 7,000 rpm for 1 min, and resuspended in 1 mL LB medium with 0.1 M sodium citrate. The samples were incubated at 37 °C for 45 min and plated on LB agar plates with the appropriate antibiotics. The plates were incubated at 37 °C overnight and the knockout was verified via colony PCR.

The Kan resistance cassette was flp-out by the transformation of pCP20, which harbors the FLP recombinase. After successful KanR removal, the plasmid was cured by incubation at 42°C.

5.2.17.2 CRISPR/Cas9

The CRISPR/Cas system is an ancient immune system in bacteria as a protection against foreign DNA (e.g. from viruses). After the survival of an infection, bits of the foreign DNA (called spacer) are stored in a CRISPR array, which serves as a library. Interspersed between each spacer in the CRISPR array are repeat regions. After the entire CRISPR array is transcribed into one long CRISPR RNA, another RNA molecule, the trans-activating CRISPR RNA binds to the repeat regions of the CRISPR array and recruits Cas9. The CRISPR array is then processed by RNase III, yielding functional CRISPR/Cas9 units containing a single spacer (and tracrRNA). Upon a recurring infection with the same pathogen, its DNA will be targeted by the CRISPR/Cas9 unit bearing the homologous RNA fragment (spacer) and the foreign DNA will be degraded. The region of the foreign DNA that is homologous to the spacer is called protospacer and requires a protospacer adjacent motif (PAM) to be targeted by CRISPR/Cas9. This is a defense system against self-cleavage⁴⁸³.

The CRISPR/Cas system can be exploited in the laboratory for genome engineering. For this purpose, scientists have developed a single guide RNA (gRNA or sgRNA), which combines the spacer and the attached tracrRNA into one molecule. Various genome engineering techniques make use of the CRISPR/Cas system, in this study the CAGO technique⁴⁸⁶ was chosen, which employs a universal gRNA.

This universal gRNA is encoded on the pCAGO plasmid and targets the N20PAM fragment, which is inserted into the editing cassette. Also encoded on the pCAGO plasmid is the Cas9 protein, as well as the λ Red system. It is comprised of the genes γ, β, and exo from the bacteriophage λ. The γ gene product, Gam, inhibits the host RecBCD exonuclease V which would otherwise degrade the linear editing cassette. The gene products from β and exo, Bet and Exo, can then bind to the ends of the PCR product to promote recombination and replace the gene of interest with the editing cassette harboring an antibiotic resistance gene for selection. After successful recombination, the selection marker is removed. Cas9 bound to the universal gRNA targets the N20PAM sequence embedded in the editing cassette and induces a double-strand break, which is repaired by λ Red-mediated recombination of the R short region of the editing cassette with its homologous region in the right homology arm of the cassette. Thus, the selection marker between these regions is cut out.

Figure 56 I Schematic overview of processes involved in the CRISPR/Cas9 response. Figure supplied by © Johan Jarnestad / The Royal Swedish Academy of Sciences.

The editing cassette was assembled from four PCR fragments in a one-pot golden gate assembly according to the following program:

N20PAM_marker 100 ng

L_homo 75 ng

Insert 140 ng

R homo 68 ng

T4 buffer 1.5 µL

FD Eco31I 1 µL

T4 DNA ligase 1 µL

ddH2O ad 15 µL

Step Temperature Time

25 Cycles: Digestion Ligation

37 °C 16 °C

3 min 4 min

Inactivation of enzymes 80 °C 5 min

Store 8 °C forever

The editing cassette was amplified via PCR and purified via gel extraction as described above. 5 mL of LB^Amp were inoculated 1:100 with an overnight culture of ∆metEH::FRT transformed with pCAGO. The culture was incubated at 30 °C until an OD600 of approximately 0.2 was reached and the λ Red system was induced with 1 mM IPTG. The cells were further cultivated at 30 °C until an OD600 of approximately 0.6 was reached. The cells were made electrocompetent as described above, resuspended in 100 µL of cold ddH2O and 50 µL aliquots were prepared. Approximately 400 ng of the editing cassette were transformed via electroporation and the cells were recovered for 1 h 45 min at 30 °C. After recovery, the cells were spread on LB agar plates containing the appropriate antibiotics and 1% glucose to suppress leaky expression of Cas9 from the arabinose promoter. Single colonies were picked and the recombination of the editing cassette was verified via sequencing. Positive clones were incubated overnight at 30 °C in 5 mL LB^Amp containing 1 mM IPTG for expression of the λ Red system and 10 mM arabinose for Cas9 expression. The cultures were spread on LB agar plates containing Amp and incubated for 30 °C overnight. Once again, single colonies were picked and the removal of the selection marker was verified via sequencing. The plasmid pCAGO was cured by incubation at 42 °C until no more growth on Amp could be observed.