Performing genome engineering in bacteria using CRISPR/Cas9 usually requires the presence of three components in a cell at the same time, namely: Cas9, gRNA and a donor template
(Wasels et al. 2017).
Therefore we aimed to design a convenient system that allows easy adaption of CRISPR/Cas9 to target various sequences.
The LVL1 plasmid with p15A origin, pTET promotor, Cas9 CDS and tretacyclin-resisstence is shown.
Preparation of Cas9
A Cas9 and dCas9 LVL0 part was created as a CDS part in our Marburg Collection. The cas9 sequence was PCR amplified from pCas9CR4
(Reisch and Prather 2017).
To make this sequence compatible with our toolbox and the iGEM registry, we removed a BsmBI and EcoRI recognition site in the parts sequence creating the Cas9 LVL0 part BBa_K2560047. For further experiments, a dCas9 part could prove useful and therefore we induced mutations the nuclease loop in the RuvC1 (D10A) & HNH (H840A) subunits creating BBa_K2560054.
To establish CRISPR/Cas9 in V. natriegens, we conceived a Cas9 plasmid where the expression is tightly controlled by an inducible promoter. We expect this to help during cloning and transformation steps, as the toxicity of Cas9 expression is reduced in the absence of inducer. We chose pTet for this purpose. This decision is based on the characterization of this part that showed tight control and a 40 fold induction of this promoter when induced with ATc (Link to Results) . Finally, we created LVL1-pTet-Cas9, a plasmid with the Cas9 CDS under control of pTet (figure xxx). We chose to built this plasmid with p15A as the ori and a kanamycin resistance cassette. This enables cotransformation of this plasmid together with a second plasmid harboring the gRNA cassette.
The gRNA entry vector contained the scaffold of the gRNA and a sfGFP drop out for insertion of spacer sequences.
Construction of a gRNA entry Vector
To enable simple cloning of gRNAs, we constructed BBa_K2560305 an entry vector for gRNA spacer. The spacer is defined as the 20 bp at the 5’ end of the gRNA that confers sequence specificity to the target sequence in the genome. This parts design is based on BBa_K2457002 a part which harbors a gRNA targeting LacZ of E. coli. To achieve superior cloning flexibility we incorporated the approach of using a sfGFP dropout similar to the part entry vector of the Marburg Collection BBa_K2560002. Analogous to the cloning of small LVL0 parts, new spacer sequences can easily be cloned by annealing of oligos and subsequent ligation into the gRNA entry vector using a golden-gate reaction. This drop out helps to distinguish correctly assembled plasmids from the religated entry plasmid. The succesfully cloned plasmid then contains a gRNA expression cassette as well as a pMB1 ori and the chloramphenicol resistance cassette.
The two plasmids LVL1-pTet-Cas9 and BBa_K2560305 after integration of a spacer are compatible and can be cotransformed. As a third component a donor template has to be added to the transformation.
Both the pTET-Cas9 LVL1 plasmid and the gRNA entry vector assembled in one LVL2 plasmid.
We realized that cotransformation of three components into V. natriegens might be challenging and therefore, we designed a plasmid that is a combination of the previously designed LVL1-pTet-Cas9 and the gRNA entry vector BBa_K2560305. Using our toolbox, we converted the LVL1 construct into a LVL2 plasmid to remove the BsmBI recognition sites that are present in the LVL1 plasmid. Subsequently, we integrated the gRNA transcription unit including the sfGFP dropout into the 5’ Connector via Gibson assembly. Finally, we created a plasmid that possesses a Cas9 CDS with pTet and the gRNA entry sequence to facilitate easy cloning.
In most bacterial CRISPR/Cas9 approaches, a two plasmid system is used separating Cas9 and gRNA on two different plasmids (Wasels et al 2017). The reason is that during the process of gRNA cloning, a functional Cas9 transcription unit is present in the same cell with the correctly cloned gRNA, resulting in double strand breaks (DSB) leading to the death of the cells.
Our approach to overcome this challenge is to use E. coli as cloning chassis for the construction of the Cas9-gRNA plasmid which is then transformed in V. natriegens together with a donor template to perform genome engineering. This is possible because the spacer sequence targeting the genome of V. natriegens should not match any sequence in the genome of E. coli and therefore we do not expect creation of DSB.
Designing of donor templates
Different approaches for the design of donor templates are described in literature. The mutation can be introduced either as dsDNA in a linear form or integrated into a plasmid or as ssDNA (Jiang et al 2015).
We decided to use ssDNA because they can be easily ordered from companies offering synthesis. As a prove of concept we decided to tackle the dns gene on chromosome 1 of V. natriegens, as this mutation has already been shown not to be lethal and to significantly increase DNA yields of plasmid preparations (Lee et al 2017). We used two ssDNA oligos each targeting the leading or lagging strand. The oligos consists of the desired mutation flanked by 50 bp long homolougous sequences flanking the position of the introduced DSB. The last three nucleotides at both ends are connected via phosphorothioate-bonds providing the stabilization of the donor oligonucleotides and protection against digestion (Reisch and Prather 2017).
The donor template was designed to exchange four basepairs at the beginning of the dns gene with a three basepair stop-codon. In this way, the knock-out is ensured through the premature translation termination as well as a frameshift in the sequence.
The spacer sequences targeting the genomic sequence was designed using the CRISPR design tool of Geneious which utilizes the algorithm developed by
Doensch et al. (2014).
The alligment shows that the the donor template differ in two nucleotides from the dns-CDS of V. natriegens. The first nucelotide leads to the conversion of an aminoacid to a stopcodon whereas the second, missing nucleotide leads to a frameshift.