Part:BBa_K1129006

Cas 9 from Streptococcus thermophilus

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3202
  

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Background

The CRISPR Mechanism

Cas9, isolated from Streptococcus thermophilus LMD-9 is the hallmark of the type II system—a massive 163 kDa multifunctional endonuclease protein involved in the processing of pre-crRNA and the cleavage of exogenous DNA. The Cas9 gene was subcloned out of [http://www.addgene.org/39314/ pMJ824] and assembled under both the arabinose inducible pBAD BBa_I13453 and constitutive BBa_J23118 promoters. Cas9 was submitted to the registry as part BBa_K1129006. Expression of Cas9 under both arabinose induction and constitutive expression was confirmed with SDS-PAGE.

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci found in some bacterial and archaeal genomes that, together with associated Cas (CRISPR-associated) genes function as an adaptive immune system in prokaryotes. While the details of immunity-conferring “spacer” sequence acquisition are still being worked out, it is known that exogenous DNA is processed by Cas proteins into short (~30 base) sequences that are adjacent to the Protospacer Adjacent Motif (PAM) site. These short pieces of DNA are then incorporated into the host genome between repeat sequences to form the spacer elements. The repeat-spacer-repeat array is constitutively expressed (pre-CRISPR RNAs, pre-crRNAs) and processed by Cas proteins to form small RNAs (crRNAs). The small RNAs are then loaded into Cas proteins and guide them to initiate the sequence-specific cleavage of the target sequence (or the protospacer)

Experimental Data

SDS-PAGE Protein Expression of Cas9

The Cas9 construct was transformed into E. cloni® 10G, plated on LB agar plates made with chloramphenicol (33ug/mL), and grown overnight at 37°C.

A single colony was picked and grown overnight in LB (with chloramphenicol) at 30C and shaking (1000 rpm). Growth was very slow for the Cas9 culture and OD was only ~0.6 in the morning. Several aliquots of 175 uL were taken and placed in separate wells of a 96-well plate. They were induced with varying concentrations of arabinose (0, 0.5%, 1%, and 2%) for three hours at 37°C and 1000 rpm.

OD was measured and it was determined that there was a tradeoff between induction concentration and growth. The highest induction concentration that also had relatively good growth was 1% arabinose (OD=1.17 vs 0.98 for 2% induction and 1.67 for non-induced). This culture and the non-induced control was collected, centrifuged, resuspended in SDS-PAGE loading buffer, and boiled for 10 minutes. They were loaded in a 12% gel and run at 150V for 1 hour and 10 minutes.

The gels were stained with InstantBlue overnight and destained with water for several hours.

There is a large, dark band in the Cas9 lane at the approximate size of Cas9 (163 kDa) that is not present in the uninduced control (Figure 1). This would be consistent with the successful expression of the Cas9 protein.

FIGURE 1: 12% SDS-PAGE gel showing strong expression of a protein of approximately 163 kDa in the sample that was induced with 1% arabinose for three hours at 37°C. Consistent with Cas9 expression. No corresponding band found in the uninduced control.

T4 Phage infection

FIGURE 1: The effect of initial T4 bacteriophage concentration on E.coli GB10 (without any CRISPR/Cas9 cassettes) cell growth for various T4 phage inoculums. Overnight cultures of wild-type E. coli 10G cells were grown at 37 °C until an OD of 0.3 was obtained diluted to each inoculum, the cells counts of which were estimate by plate counts (CFU). The cultures were grown in a 96-well format and OD was measured every 5 min for 2 hrs (n = 2). At every CFU, the increasing titer of bacteriophage inoculated resulted in decreased growth of wild-type E. coli 10G cells as measured by their O.D600. The highest titer of phage resulted in the lowest amount growth of E.coli cells, while the no phage added resulted in the highest amount of growth.

Figure 2. Cas9, tracrRNA (sense/antisense) and the T4 spacer-repeat-spacer with inducible promoters were assembled in a combinatorial libraries of approximately 800 clones. Combinatorial libary clones were each inoculated with 10³ phage and incubated for 24 hours at 30 °C. The horizontal red line represents the threshold for selection of clones for sequencing and colored points correspond to characterization data in the subsequent figure 3.

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Figure 3. Measuring final OD of select combinatorial library clones under different arabinose concentrations after incubation for 24 hours at 30 °C. Well IDs correspond with the precending figure. 10³ phage were added to each well.

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Figure 4. The effect of phage predation on clones from combinatorial libraries. Clones were arrayed into a 96-well plate and grown for 6 hours at 30 °C. 10³ phage were then added to each well and OD was monitored overnight. The results seem to resolve two groups, one showing increased final OD over the other (blue and red lines respectively (n=2).

Figure 5. Monitoring growth of two select combinatorial library clones under high cell inoculum. 10³ phage were then added to each well and OD was monitored overnight (n=2).

Testing host background dependance

To ensure that the survival phenotype we were observing was in-fact dependant on the expected constructs, we re-isolated plasmid from "survivors" and re-assayed their growth under phage predation. We also sent these plasmids for sequencing as to assign a genotype that resulted from our combinatorial assembly. In a new host background, the growth advantage was not maintained, suggesting that the phenotype was dependant on changes occurring in the host background (Figure 9). Furthermore, the plasmid sequences showed that the repeat-spacer regions was not present, showing that the combinatorial screening strategy did now work as expected. There could, however, still be something interesting happening where the host strain is able to acquire resistance more readily while expressing Cas9. There was also only a slight correlation between the presence of tracrRNA and survival. We are looking at this more closely. More importantly, we need to be careful looking using high-throughput approaches as this increases the rate at which spontaneous processes can confound the results.

Figure 5. The effect of phage predation on clones from combinatorial libraries after re-transformation and genotyping. Change in OD in culture in a 96-well plate grown for 11 hours at 30 °C. 10³ phage were then added to each well and OD was monitored overnight (n=3). Clones containing either tracrRNA or spacers are shown in green.

Testing the standard assembled construct

We then finished the standard assembly of our designed CRISPR construct and tested growth under T4 phage predation. The construct contained cas9, the repeat-spacer region and both orientations of the tracrRNA. This showed an interesting phenotype where the entire construct seems to cause enhanced recovery after a cycle of phage predation (starting at approximately 25 hrs). The initial infection cycle (from 0 to 15 hrs) showed that cas9 caused a significant hindrance in growth compared to an empty vector and the repeat-spacer/tracrRNA control (Figure 6).

Figure 6. The effect of phage predation on clones from assembled CRISPR constructs all under inducible promoters: cas9/repeat-spacer/tracrRNA-sense (AAS), repeat-spacer/tracrRNA-sense (SS), and empty vector (control). Clones were arrayed into a 96-well plate and grown for 6 hours at 30 °C. 10³ phage and 1 ug/ml of arabinose was then added to each well and OD was monitored for 46 hrs (n=2).

We then monitored this trend by taking final OD after 46 hrs and ensured that the phenotype was consistent under various arabinose and phage concentrations. Figure 10 shows final OD with a higher phage inoculum.

Figure 7. The effect of phage predation on clones from assembled CRISPR constructs all under inducible promoters: cas9/repeat-spacer/tracrRNA-sense (ASS), repeat-spacer/tracrRNA-sense (SS), and empty vector (control). Clones were arrayed into a 96-well plate and grown for 6 hours at 30 °C. 10⁴ phage and 1 ug/ml of arabinose was then OD was measured after 46 hrs.

Transformation assay

While the phage phenotypes show promise for providing a growth advantage under phage, we also wanted to show some evidence for the sequence specificity of our CRISPR construct. The experimental approach is also described above in the toxic plasmids section. We designed a transformation assay which mixed a RFP containing control plasmid and a decoy plasmid which contained the targeted T4 and PAM sequence (both on PSB1A3, to be maintained along with PSB1C3 under both ampicillin and chloramphenicol selection). We would then expect that if the decoy plasmid was being cleaved, the ratio of red/white colonies counted on a plated would increase as the decoy plasmid was being cleaved. This was in-fact observed for the entire construct, and controlled for using a construct that was missing Cas9 (Figure 8).

Figure 8. Colony counts of RFP and decoy plasmid containing clones (red and white, respectively): Cas9/repeat-spacer/tracrRNA-sense (CSS) and repeat-spacer/tracrRNA-sense (SS). While Cas9 was under constitutive expression, repeat-spacer and tracrRNA were under arabinose inducible promoters. Therefore, the addition of 10 ug/ml of arabinose added during transformation recovery was compared.

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