Difference between revisions of "Part:BBa K3044027"

Revision as of 02:38, 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.

The fluorescence pictures of the E.coli WT, the target bacterium expressing gfp and the target bacteria that have received either the sgRNA or the Cas9/sgRNA system (figure 1A and figur 2A). The wildtype (WT) E.coli K12 Top10 is not fluorescent and the E.coli harboring the gfp gene, for both expression (gfp' 0.33 and gfp 0.86). The sgRNA is included as a control ensuring that sgRNA itself is not downregulates the fluorescent signal. In contrast, the fluorescence in the bacteria with our Cas9/sgRNA system is knocked out. The microscopy image of Cas9/sgRNA system only one bacteria is fluorescent for gfp 0.86, and no green colonies with gfp 033, illustrating a high efficiency of our system. Furthermore, the FACS histograms support the microscopy data. Thereby showing that our system can be used to knockout gfp. This biobrick can potentially be modified to target any other gene.

Sequence and Features