Difference between revisions of "Part:BBa K3044027"

Revision as of 02:44, 22 October 2019

sgRNA/Cas9 for gfp knockout

This part is an assembly of a sgRNA-Cas9 system that can be used for knockout of the ‘’gfp’’ gene (BBa_E0040). This system has been tested in ‘’E. coli’’ K12 Top10

The Cas9 protein

Cas9 is the active protein in the CRISPR/Cas9 II system original discovered in Streptococcus pyogenes. Cas9 has endonuclease activity and induces a double stranded break in the target DNA sequence using its catalytic domains RuvC and HNH [1]. The protein forms a complex with the single guide RNA (sgRNA) which is responsible for target DNA identification. The sgRNA is designed according to the PAM sequence, a three nucleotide sequence in the target DNA, which is crucial to the Cas9 activity. The Cas9/sgRNA-system can be used for knockout of specific target genes. In this part the Cas9 protein is expressed with an IPTG inducible promoter (BBa_R0011) to enable control of Cas9 expression. The Cas9 protein is furthermore codon optimized for use in ‘’E. coli’’. dCas9 is an inactive form of the Cas9 protein which sterically inhibits gene transcription instead of performing a knockout. For more information on dCas9 read BBa_K3044008.

The sgRNA design

The sgRNA is composed of the sgRNA handle [2] and the 20 nucleotide spacer sequence that targets the gene [3]. This sgRNA BBa_K3044013 has the sequence 5’-CAAATTTTCTGTCAGTGGAG-3’ and is designed to target position 75-95 on the template strand of gfp (BBa_E0040). The sgRNA handle binds the Cas9 protein and the 20 nucleotides base pairs to the gene sequence right before the PAM sequence. The sgRNA were expressed with a 1.00 constitutive active Anderson promotor (BBa_J23100) ensuring high expression of the sgRNA. The Cas9/sgRNA part was tested in ‘’E. coli’’ K12 Top10 that expressed a low copy number plasmid pSB4C5 containing ‘’gfp’’ with either a 0.86 constitutive Anderson promoter (BBa_K3044006) or with a 0.33 constitutive Anderson promoter (BBa_K3044002). The Cas9/sgRNA system was transformed into these ‘’E. coli’’ expressing GFP and our expectations was that the Cas9/sgRNA system would downregulate the expression of GFP. We investigated this hypothesis by using Fluorescence-activated cell sorting (FACS) and fluorescence microscopy. The bacteria containing both the ‘’gfp’’ plasmid and the Cas9/sgRNA plasmid were grown overnight with IPTG to fully induce the expression of Cas9.

Figure 1: A: Microscopy image of the Cas9/sgRNA-system and the sgRNA alone transformed into a target bacterium expressing gfp (BBa_K3044002) from a weak (strength 0.33) promoter. Upper row: phase contrast microscopy images (20 ms exposure time). Middle row: fluorescence microscopy images (50 ms exposure time). Lower row: merged images of phase contrast and fluorescence. The different columns represent the E.coli WT, the target bacterium (gfp with promoter strength 0.33), the sgRNA on its own and the Cas9/sgRNA system transformed into the target bacterium. B: FACS histograms of GFP/area signal vs cell count of the mean of the three biological replicates. The histograms compare the E.coli WT (grey) and the target bacterium expressing gfp (green) which has received either sgRNA or a Cas9/sgRNA-system.

On figure 1A and 2A, the fluorescence images of the ‘’E. coli’’ wild type (WT), the chassis expressing only ‘’gfp’’ and the chassis that have received either the sgRNA or the Cas9/sgRNA system is shown. The WT is non fluorescent while the chassis is fluorescent, for both promoter strengths (gfp 0.33 and gfp 0.86). The sgRNA is included as a control ensuring that sgRNA in itself could not downregulate ‘’gfp’’ expression. In the fluorescence images, the chassis with our Cas9/sgRNA system are non fluorescent. This is as expected because the Cas9/sgRNA system induces a knockout of the ‘’gfp’’ gene. This is evident for the fluorescent images for both figure 1A and figure 2A. Figure 1B and 2B shows FACS histograms obtained from the different constructs which support the microscopy data. The sgRNA control has the same fluorescence as the chassis, while the Cas9/sgRNA system is non fluorescent like the WT. On figure 2B it could seem like the sgRNA has an inhibitory effect on the fluorescent expression. However, after performing an ANOVA analysis on the FACS data, the analysis showed that the only significant difference in fluorescence repression is between Cas9/sgRNA and the chassis (figure 3 and figure 4). This shows that the Cas9/sgRNA can efficiently knockout the ‘’gfp’’ gene. The data also indicates that the Cas9/sgRNA system can knockout a gene independent of the promoter strength of the gene. Additionally, this biobrick can potentially be modified to target any other gene by designing a new sgRNA.

Figure 2: A: Microscopy image of the Cas9/sgRNA-system and the sgRNA alone transformed into a target bacterium expressing gfp (BBa_K3044029) from a strong (strength 0.86) promoter. Upper row: phase contrast microscopy images (20 ms exposure time). Middle row: fluorescence microscopy images (50 ms exposure time). Lower row: merged images of phase contrast and fluorescence. The different columns represent the E.coli WT, the target bacterium (gfp with promoter strength 0.86), the sgRNA on its own and the Cas9/sgRNA system transformed into the target bacterium. B: Histograms of GFP/area signal vs cell count of the mean of the three biological replicates FACS. The histograms compare the E.coli WT (grey) and the target bacterium expressing gfp (green) which has received either sgRNA or a Cas9/sgRNA-system.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 478
Illegal BglII site found at 1552
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 27: / Line 27: @@
 On figure 1A and 2A, the fluorescence images of the ‘’E. coli’’ wild type (WT), the chassis expressing only ‘’gfp’’ and the chassis that have received either the sgRNA or the Cas9/sgRNA system is shown. The WT is non fluorescent while the chassis is fluorescent, for both promoter strengths (gfp 0.33 and gfp 0.86). The sgRNA is included as a control ensuring that sgRNA in itself could not downregulate ‘’gfp’’ expression. In the fluorescence images, the chassis with our Cas9/sgRNA system are non fluorescent. This is as expected because the Cas9/sgRNA system induces a knockout of the ‘’gfp’’ gene. This is evident for the fluorescent images for both figure 1A and figure 2A.
 Figure 1B and 2B shows FACS histograms obtained from the different constructs which support the microscopy data. The sgRNA control has the same fluorescence as the chassis, while the Cas9/sgRNA system is non fluorescent like the WT. On figure 2B it could seem like the sgRNA has an inhibitory effect on the fluorescent expression. However, after performing an ANOVA analysis on the FACS data, the analysis showed that the only significant difference in fluorescence repression is between Cas9/sgRNA and the chassis (figure 3 and figure 4).
-This shows that the Cas9/sgRNA can efficiently knockout the ‘’gfp’’ gene. The data also indicates that the Cas9/sgRNA system can knockout a gene independent of the promoter strength of the gene.
+This shows that the Cas9/sgRNA can efficiently knockout the ‘’gfp’’ gene. The data also indicates that the Cas9/sgRNA system can knockout a gene independent of the promoter strength of the gene. Additionally, this biobrick can potentially be modified to target any other gene by designing a new sgRNA.