In Chlipid, we tend to create a biological system that can increase algae TAGs accumulation by knock-out
important genes in algal carbon metabolic pathways. We manage to build a genome editing system based on the
CRISPR-spCas9 system. The system is specially designed to target genes in Chlamydomonas reinhardtii
nucleus
genome. Here, based on what we have described in implementation, we present proof of concept with detailed
experiment results that mainly prove Chlipid’s foundational research system can work in a relevant context.
Stage 1: Preliminary test of the foundational research system
At the preliminary stage of testing the foundational research system, we based on an existing CRISPR/spCas9
plasmid designed to target photoreceptor genes in Chlamydomonas reinhardtii and perform codon
optimization to
construct pTX2038 and pTX2040.
Figure 1. Structure of pTX2038 and pTX2040.
We chose the commonly used electronic transformation to transform pTX2038 and pTX2040 without cloning specific
gRNAs but remaining gRNAs inserting site into algae.
Figure 2. Selection and genetic editing efficiency of thaumatin-resistant colonies after
transformation.
(A) Growth of Chlamydomonas reinhardtii in the plates after
electrotransformation (7
days). Negative control: no plasmid was added. All dishes shown in the figure contain TAP medium
supplemented
with 25 µg/mL of Hyg, except for the positive control of the wild-type Chlamydomonas reinhardtii
strain.
(B) Statistics of positive clones after transformation ang the frequency of
transformation.
Figure 3. Gel run of samples from colony PCR.
(A)
Sequence comparison of Cas9, HgR, and mCherry fragments in pTX2038.
(B) Sequence alignment of Cas9,
HgR, and StayGold fragments in pTX2040. M: 2000bp DNA marker; NC: wildtype; PC: Linear
plasmid. 1-3 indicate
the different transformants selected.
The brightness of PCR products of Hyg resistance gene, mCherry reporter gene, Cas9 protein and StayGold
reporter gene (Figure 3A and 3B) fragments were all consistent with the DNA bands of the positive control
group as well as being matched with the position of DNA Marker, which indicates that the fragmented PCR
products transferred into the vector were in a high concentration and normal expression state. In sum, the
effectiveness of transformation could be testified.
To further demonstrate the correct insertion of the vector at the cellular expression level, we performed
microscopy whose centers were the autofluorescent genes contained in the construct vectors: mCherry and
StayGold. And autofluorescence in red of Chlorophyll was used as a negative control.
Figure 4. Images of WT Chlamydomonas reinhardtii and positive clones after transformation at 20×
magnification. WT:wild type; DIC:bright field. 60ms exposure time; Chlorophyll: EX:Form
625nm to 650nm, 10ms exposure time; mCherry:EX:Form 515nm to 555nm, 300ms exposure time;
StayGold:EX:Form 465nm to 495nm, 300ms exposure time.
Compared with the wildtype, the cells of Chlamydomonas reinhardtii with fluorescent reporter gene
showed
different degrees of fluorescence and normal autofluorescence expression of chlorophyll, which further proved
that the vector could be expressed normally when transferred into Chlamydomonas reinhardtii
(Figure 4).
Stage 2: Further proof of the foundational research system
For the plasmids, we noticed that although we have successfully proved that Cas9 protein and the reporters
were transformed into algae, we only had concrete test results on reporters. Cas9 protein hasn't had the
chance to show its ability! We found that psy1 (phytoene synthase-1 gene, disruption of psy1 produces white
colonies that are easy to detect and count) is a valuable and mind-blowing reporter gene which is an ideal in
vivo target gene for us to attempt our first transformation towards
Chlamydomonas reinhardtii.
We used the tool called CRISPOR on the sgRNA design website, together with the
off-target prediction
model we constructed, to perform sgRNA design for psy1. The following are the test results on psy1
knock-out.
Figure 5. The phytoene synthase gene, PSY1, was chosen as a target gene. sgRNA transcription driven by
Chlamydomonas U6 promoters (CrU6) was assayed.
Comparison between wildtype Chlamydomonas reinharditii and transformed positive clone of PSY1 gene in bright
field (Figure 5).
Comparison between wildtype Chlamydomonas reinharditii and transformed positive clone of PSY1 gene in bright
field (Figure 5).
Here, we provide a scheme to verify the successful introduction and proper operation of the CRISPR/Cas system
by targeting the endogenous PSY1 gene for vector construction and transformation. Moreover, by microscopic
bright-field comparison of the wild type with the PSY1 mutant, if the field of view turns white, the system
could prove to be feasible.
