Difference between revisions of "Part:BBa K3044027"

Revision as of 03:24, 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.

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.

Time experiment measuring Cas9/sgRNA efficiency

To identify in which phase Cas9 is most efficient, a time experiment measuring percentage fluorescence in the bacterial population was performed using FACS. OD_600 and FACS values were measured with one hour intervals for 5 hours. To interpret the FACS data, it was assumed that all populations were distributed normally based on a cell size plot. The data is plotted with percentage fluorescence over time and OD when comparing the WT and Cas9/sgRNA system with gfp 0.86 as illustrated in Figure 3. Fluorescence is seen for gfp 0.86 (Figure 3A) at all time intervals and OD_600 values. This is expected as this strain is the positive fluorescence control. Minor fluorescence is observed for the WT (Figure 3B) at the beginning and end of the experiment. This is caused by the division of the GFP-absorbance with cell size. In the lag- and stationary phase the cell size of the bacteria is smaller, giving rise to minimal fluorescence observation, caused by uncertainty in the FACS measurements. Through the remaining time intervals and OD600 values, no fluorescence is observed as expected, as the cell size of the bacteria increases through the exponential phase. Cas9/sgRNA1 (Figure 3C) shows no fluorescence through the plot, except in the first measurement. This is also caused by a smaller cell size in the lag phase compared to the later phases. By comparing these three plots, it is observed that knockout of gfp 0.86 gene expression takes place instantaneously which probably means the moment after the Cas9/sgRNA is transformed into the chassis.

This proves that the Cas9/sgRNA system is able to fast and efficiently knockout the target gene, which in this case is gfp.

Sequence and Features