In order to assay the binding affinity of dCas9 to it’s target, we used the VP64-p65-Rta(VPR) transcription activation domain fused dCas9, to activate a GFP reporter construct co-transfected into HEK293FT cells. Another plasmids is used for gRNA expression and OFP marker for transfected cells. The cells are then passed through the flow cytometry to measure the Mean Fluorescent Intensity(MFI) of transfected cells. Gates were set on OFP+ cells and histogram of GFP intensity is shown for OFP+ cells.

Left: Plasmids used for reporter assay. Right: flow cytometry plot and MFI measurement

As the focus of dCas9 in on it's DNA binding capability, we first delete the domains of the nuclease(NUC) lobe which are the three RuvC domains and HNH domain. On Recognition(REC) lobe, the REC2 domain has been shown to be deletable, despite with a reduced expression. Regions that we started on are as shown:

Schematic depicting the truncations of individual whole domains of SpCas9 that were evaluated.

Activation of ZsGreen reporter gene using NUC and REC truncations

As shown above, the ∆HNH mutant is still able to bind to it’s target while other NUC truncations failed to activate our reporter. We were very excited when we found out that the HNH domain can be deleted. The HNH and RuvC domains are known for their function as a helicase. Studies for specificity enhancement have shown that excess energy is provided for the separation of the non-targeted strand. We could only hypothesize that with HNH domain deleted, strand separation can still be aided by the RuvC domains.

Next, we asked whether we might be able to delete smaller pieces of RuvCIII and still be able to activate gene expression using dSpCas9-VPR. It has been showed that the Cas9-RNA-DNA ternary complex exists in two different states, before and after the non-targeted strand(NTS) cleavage.

Conformations of RuvCIII in state A, before NTS cleavage and state B, after NTS cleavage

As shown above, when Cas9 transits from state A to state B, the loop connected to the HNH domain switches from an unstructures loop to an alpha helix(red) and is pulled towards the blue-white helix(in RuvCIII-2). This forces the white region within the blue helix to bend in order to accommodate the red helix. Also, the loop regions hightlighted in purple were not resolved in state B meaning that they may be flexible and not essential for binding interactions. As we know that the HNH can be deleted, this region involved in the movement of HNH might not be essential as well.

On the other hand, the cyan-highlighted(RuvCIII-1) helixes also has minimal interaction with non-targeted strand of DNA. Hence, we chose these two regions within RuvCIII as candidates for truncation.

Schematic of RuvCIII sub-domain deletions

We tried the RuvCIII deletions as well as with HNH deletions in combination to see if our deletions can be combined.

Activation of a zsGreen reporter gene using the various RuvCIII truncations

it has been shown that sub-regions of the REC1 domain in the vicinity of REC2 abolishes the binding of the Cas9 to the DNA-RNA heteroduplex. From the crystal structures, we saw that these regions were closed to the PAM-proximal “seed” region of the heteroduplex.

As specificity studies have shown that mismatches at the PAM-distal sites, 3' end of target sequence, can be tolerated(refer below), we hypothesized that protein-DNA interaction is weaker at PAM-distal regions and hence, may be redundant for binding.

Tolerance of PAM-distal mismatches. Source: DNA targeting specificity of RNA-guided Cas9 nucleases

We chose three regions of the REC1 domain which are close to the PAM-distal site(10bp range) and found three regions where the distance of both ends are close to each other and can be replaced by the GGGS linker. We named them REC1-1, REC1-2 and REC1-3.

Sub-domians within REC1 at PAM-distal end

Schematic of different truncations of the REC1 domain tested

Truncations made within REC1

PI domain plays an essential role in binding to the target sequence at the PAM sequence and also gRNA binding. As a result, we realized it may not be easily achievable by deleting the whole PI region. Yet we believed PI domain has potential to be truncated because that is where the main difference is found between SpCas9 (370aa) and the much more compact SaCas9 (144aa). Thus, we have made a structural comparison of the PI domain between SpCas9 and SaCas9. SpCas9 was found to have extra 5 alpha helices from 1225E – 1318G. This identified sequence was named as PI1-all for the labeling of the subsequent experiments.

Alignment of SpCas9 and SaCas9. (Domain for PI1-all deletion is highlighted by red stroke.)

Meanwhile, we have also looked at the evolutionary homology pattern of the variants species of Cas9 and found that 1138T to 1199P is less conserved as compared to the rest of PI domain. We then labeled it as PI2-all for potential target to be truncated. Hence, we have identified two target regions PI1 and PI2 for the truncation test.

We also made smaller truncations within PI1-all(PI1-1 to PI1-3) and PI2-all(PI-4) as we worry that large truncation may affect the structural integrity of the protein, as in the case of the RuvCIII truncation.

Truncations made within PI domain

Mean Fluorescence Intensity of each truncated PI structure

From the plot above, we know that unfortunately all the truncated dCas9 structures at PI domain do not exhibit similarly high activity as what we have obtained from Rec 1 and HNH truncations.

We then made a comparison among all the truncation combinations to see how the truncations compare among each other.

Size deleted	Mutant	Size
	Single Truncation
145	∆ REC1-1	1223
48	∆ REC1-3	1320
128	∆ REC2	1240
134	∆ HNH	1234
72	∆ RuvCIII-2	1296
	Double Truncations
182	∆ REC1-3 ∆HNH	1186
262	∆REC2 ∆HNH	1106
206	∆RuvCIII-2 ∆HNH	1162
	Triple Truncations
334	∆REC2 ∆RUVCIII-2 ∆HNH	1034

Sub-domians within REC1 at PAM-distal end

Briefly, we cloned plasmids containing various guide RNA sequences into the Cas9/Cpf1 vector with a red fluorescence gene reporter. We transfected Human Embryonic Kidney 293 cells (HEK293 cells) with the plasmids and isolated the successfully transfected cells through fluorescence-activated cell sorting (FACS). We then extracted DNAs from the cells and performed T7 endonuclease I surveyor assay to quantify efficiency of Cas9/Cpf1 editing on each DNA locus.

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Competition assay was conducted to isolate more efficient SpCas9 variants. Both the EZ plasmids and wild type::M13F (SpCas9WT::M13F) plasmid were introduced into BL21(DE3) that carries the selection plasmid, BBa_K2130004. The M13F sequence was introduced into the plasmid backbone of SpCas9WT, which allows us to differentiate the EZ plasmids from the WT plasmid via colony PCR. The cleaving efficiency of SpCas9WT::M13F was compared with SpCas9 WT and the results showed that the insertion of M13F did not affect its function.

Two gRNAs (1A and 4B) were used and both gRNAs were designed to target the Ampicillin gene on BBa_K2130004. This selection plasmid encodes the toxin CcdB gene under the inducible BAD promoter. In the presence of the gRNA and appropriate inducers, the CcdB-Ampicillin plasmid will be cleaved and hence, the toxin gene CcdB will not be expressed, allowing the cell to survive. While on the other hand, cells that carry a non-functional EZ SpCas9 variant, will not the CcdB-Ampicillin plasmid. As a result, the toxin CcdB gene will be expressed and lead to the cell death.

To perform the competition assay, we introduced equal amount (50ng) of plasmids (EZ + WT) into BL21(DE3) + BBa_K2130004. Colony PCR was performed on 32 randomly selected colonies to determine the EZ::WT ratio. The colony PCR results showed approximately 50% of the colonies contained the EZ plasmid and the other 50% contained the SpCas9WT::M13F plasmid. As we perform iterative rounds of competition assays, the ratio of EZ plasmid to SpCas9WT::M13F plasmid gradually increased, and eventually, all the tested cells contained only the EZ plasmid and none of them contained the SpCas9WT::M13F plasmid. The theory is that if the EZ SpCas9 variants are more efficient than SpCas9WT::M13F, cells that carry the EZ SpCas9 variants will replicate faster and their plasmid concentrations will increase as the rounds of competition assays increase.

Upon completing the competition assay, a few colonies were sequenced to determine their Cas9 mutations. The recurring variants were selected as the potential candidates for more efficient SpCas9. These selected variants of SpCas9 were recreated using Gibson Assembly in order to examine their cleaving efficiency in human cells. An alternative method employed to recreate the mutants was QuikChange site-directed mutagenesis (Agilent Scientific).