Part:BBa_K2643010

gRNA expression cassette for CRISPR-targeting lacZ

gRNA_lacZ

Usage and Biology

Usage

This part is designed to allow in vivo CRISPR-mediated targeting of the lacZ gene at position 1190 bp (relative to the start of the ORF), which can be useful for evaluation of efficiency/effectivity of CRISPR-mediated editing approaches through blue-white screening. For sole plasmid propagation, there are no specific host strain requirements, other than being compatible with the antibiotic marker and replication origin. For actual testing of the functionality of the gRNA, the host strain should host at least one fully functional copy of the lacZ gene (either genomic or episomal).

Biology

lacZ is a bacterial gene that encodes for beta-galactosidase, of which one of the catalytic abilities is hydrolyzation of lactose . Widely, it is known for cleaving an analogue compound named X-gal, resulting in formation of a blue colored product. This chromogenic activity is commonly used to verify cloning attempts . This composite part holds a sequence that codes for a gRNA that targets lacZ. The gRNA is constitutively expressed when brought inside a bacterial host. Our in vivo screen methodology relies on lacZ disruption through targeted integration. To this extent, the assay makes use of blue-white screening, which happens on medium complemented with X-gal, IPTG and relevant antibiotics. IPTG induces expression of beta-galactosidase, the lacZ gene product, which has chromogenic activity when hydrolyzing X-gal.

Characterization

Introduction

In our project, we aim to targetedly alter a local DNA sequence of interest. Apart from in vitro testing, we have designed and experimented with an in vivo approach. This approach would enable higher throughput screens of the functionality of our methodology and quicker selection of better constructs, based on blue/white screenings and antibiotic selections. The approach taken in the in vivo screen involves single-step targeted genome editing in E.coli, in order to demonstrate the activity of our fusion construct. Putatively, this single-step genome editing can be used by itself as a tool in synthetic biology.

Strain construction

This part was constructed by means of EcoRI HF & PstI HF restriction-ligation cloning. After chemical transformation of the ligate, formed colonies on the LB+Cam plate were subjected to diagnostic PCR using internal primers IV003 and IV004, resulting in a 314 bp amplicon (figure 1).

Figure 1.Gel electrophoresis results from colony PCR on construction of this part. Colony 2 was used for subsequent plasmid isolation and sequence verification.

Procedure

To visualize in vivo whether a donor DNA sequence is indeed integrated close to a target sequence, we built a strain that harbors the dxCas9-Tn5 fusion protein, as well as a lacZ gRNA expression cassette, both on the same episomal vector. IPTG-Induction of protein expression enables immediate loading of the fusion construct with the gRNA. An additional requirement for activity is a donor DNA sequence flanked by MEs, which can be recognized by the Tn5 transposase domain for cut and paste integration near the gRNA target. This disrupts the lacZ gene and renders catalytically inactive beta-galactosidase, which can be visualized by blue/white screening with X-gal (figure 2).

Figure 2.Overview of the single-step genome editing mechanism in the in vivo assay. (A) The fusion construct is loaded with a gRNA that targets lacZ and recognizes ME flanked kanamycin resistance cassette for (B) picking it up. (C) CRISPR-mediation drives the loaded complex to the genomic lacZ locus, where the transposase domain (D) integrates the donor DNA. (E) The host strain contains an integrated copy of kanamycin resistance, while the lacZ gene is disrupted.

In our laboratory experiments, the donor DNA sequence was a kanamycin resistance expression cassette, flanked with MEs. Integration of this linear fragment into the lacZ locus would thereby confer the ability to grow on medium containing kanamycin, while still making use of the blue/white screen (note the selection medium contains kanamycin, chloramphenicol, IPTG and X-gal). In presence of kanamycin, cells should only be able to grow if the supplied donor DNA sequence is integrated into the genome. For non-targeted integration, intact lacZ confers chromogenic catalytic functionality when growing on X-gal, resulting in a blue phenotype. For targeted integration, lacZ is disrupted, resulting in loss of chromogenic capability and thus in a white phenotype. This composite part purposely contains both RFP and kanamycin, in order to distinguish surviving colonies based on whether genomic integration occurred, or whether the plasmid is simply propagated. The latter is a false positive, expected to confer a red color. In combination with blue product formation by intact LacZ, this is expected to result in a purple phenotype. Table 1 gives an overview of the expected possibilities.

Table 1. Overview of expected phenotypes in the in vivo assay.

Genomic Integration	Template with RFP cotransformed?	Growth on LB+Cam+Kan?	lacZ disrupted?	Colony phenotype*
On-target	no	yes	yes	white
On-target	yes	yes	yes	red
Off-target	no	yes	no	blue
Off-target	no	no	no	purple
None	no	no	no	n/a
None	yes	yes	no	purple

*phenotype when growing on LB+Cam+Kan+IPTG+X-gal

Results & Conclusion

This part was cloned next to the dxCas9-Tn5 fusion construct in the pACYC vector using Gibson Assembly. As a negative control, Gibson Assembly was performed without gRNA insert. This negative control showed colony growth on LB+Cam medium plates, but notably less compared to the plate with insert. This indicates that there is residual template pACYC plasmid left in the sample used for Gibson Assembly. Colony PCR using primers FP018 and IV002 showed that this part was indeed cloned into the vector (figure 3). Subsequent sequence verification identified the desired construct in one of the subjected colonies (colony 1).

Figure 3.Gel electrophoresis results from colony PCR on construction of pACYC vector containing this part and BBa_K2643000. Colonies 1 and 2 were used for subsequent plasmid isolation and sequence verification.

The constructed pACYC vector contains all elements needed for producing and loading the lacZ gRNA onto the dxCas9-domain of the fusion protein. We have not shown in vivo activity of the dxCas9 protein being loaded with this gRNA, but we did an in vitro study on a different gRNA (in which only 20bp target sequence is substituted by another 20bp target), which did show that the gRNA scaffold. is recognized and loaded by the dxCas9 protein. We have not been able to demonstrate that this gRNA is targeting the lacZ gene in vivo. Note that this characterisation is fully dependent on the activity of the Tn5 transposase domain, which is supposed to disrupt the lacZ locus through insertion of a donor sequence. In our setup, we used part BBa_K2643011 as donor sequence.

Source

This sequence was designed in silico and ordered as a Gblock from IDT. The Gblock was designed to be flanked with biobrick prefix and suffix.

Safety

This part does not pose any danger for safe work in BSL-1 laboratory work spaces. The expression of the fusion construct is regulated through IPTG induction, and the Tn5 activity is controlled by supplying a Mosaic End flanked donor DNA sequence. In our case, this is a kanamycin resistance marker, which is a common sequence used in BSL-1 lab spaces. The gRNA targets the lacZ gene at position 1190bp (relative to the start of the ORF). Since our experimental approach makes use of T7 polymerase expression systems, we needed a lacZ-containing strain that also harbors T7 expression machinery, driving us to work with E. coli BL21(DE3). The strain’s complete genome sequence was used as a template to choose a convenient gRNA target site in lacZ, and for excluding presence of other Mosaic Ends in the genome.