Part:BBa_K2361001

dCas9 VRER

This part is an adapted variant of dCas9sp. The preferred difference between dCas9 sp and dCas9 VRER is insertion of mutations resulting in a dCas9 protein recognizing a different PAM site. This was done to ensure that the appropriate binding of the protein-sgRNA complex to the complementary spacer.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1100
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3379
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental validation

This part was validated experimentally in the same experiment as spdCas9 by iGEM Groningen 2017. For this experiment we have designed four gRNA's that target GFP. Two of this gRNA's bind to sequences flanked by NGG (1&2) and two bind to a sequence flanked by NGCG (3&4). These gRNA's were inserted into the plasmid pSB3C5 containing a pLacGFP construct as well. For spdCas9 and dCas9VRER expression those parts were put into a pBad vector in which they are expressed behind an Arabinose inducible promoter.

In total 18 E. coli strains were produced containing pBad:dCas9, pBad:dCas9VRER or no pBad combined with pS3C5 with one of the gRNA's, pSB3C5 without a gRNA or no pSB3C5 (Table below).

min	dCas9	dCas9VRER
dH5α	dCas9:nopSB3C5	dCas9VRER
noPbad:nogRNA	dCas9:nogRNA	VRER:nogRNA
noPbad:gRNA1	dCas9:gRNA1	VRER:gRNA1
noPbad:gRNA2	dCas9:gRNA2	VRER:gRNA2
noPbad:gRNA3	dCas9:gRNA3	VRER:gRNA3
noPbad:gRNA4	dCas9:gRNA4	VRER:gRNA4

All strains were grown overnight and the next day they were dilute to an OD of 0.05 in the afternoon. At the end of the afternoon at ODof 0.4-0.6 all cultures were induced with a final concentration of 0.01% arabinose. All induced cultures were put back into the incubator (37C 220 rpm) and grown overnight. The next morning the OD of all cultures was measured and to an OD of 0.2 in LB (in final volume of 1mL). The OD and GFP (470/510) of all samples were measured in a plate reader in quadruplicate. The data obtained from the fluorescence measurement were converted to relative fluorescence and are shown in figure 5. To convert the fluorescence to relative fluorescence we first divided all measured fluorescence values were divided by the measured OD's. Next the fluorescence/OD of the negative controls (samples without pSB1C3) was subtracted from the other samples. Next all normalized fluorescence/OD values were divided by that of the positive control (no pBad:pSB3C5 without gRNA).

From figure 5 it can be seen that all fluorescence values are quite similar for the samples without gRNA. Further we can see a clear decrease in the relative fluorescence of dCas9 in combination with gRNA 2 and 3. For gRNA's 1 and 4 the relative fluorescence of the dCas9 is similar to that of the samples without the pBad vector. For dCas9VRER we showed CRISPR interference in combination with gRNA's 1,2, and 4.

From the data we cannot conclude that the VRER mutations had any effect on the PAM preference, since dCas9VRER shows the strongest repression for gRNA 1 and 4 of which one has a NGG flanked target. Also gRNA 3 whose target is flanked by NGCG seems to be repressed by dCas9 and not by dCas9 VRER. If these results are correct this would suggest that the PAM preference has not changed for dCas9VRER, however it is quite odd that the differences between dCas9 and dCas9VRER are so big for especially gRNA 1 and gRNA4. Another explanation could be that we mixed up gRNA1 and gRNA3 in one of the steps from gBlock to double transformants. If we would switch around these data (so gRNA1-> gRNA3 and gRNA3-> gRNA1) the data would show that only dCas9VRER can be directed by towards NGCG flanked targets.