Team:NTU-Singapore/Proof

Let it work...let it work...

With the hypothesis outlined and aims clearly defined, we started on simple experiments to see if our ideas worked! :)

Project Truncation

Project Evaluation

Project Evolution

Project truncation

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

NUC and REC2 Domain Truncations

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.

Left: Mean Fluorescence Intensity of ZsGreen activation. Right: Microscope image of HEK293FT cells 24 hours after transfection.

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.

Truncations of RuvCIII subdomains

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

Microscope image of HEK293FT cells 24 hours after transfection.

Truncations of the REC1 domain

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

Left: Mean Fluorescence Intensity of ZsGreen activation. Right: Microscope image 24 hours after transfection.

PI Domain Truncation

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.

Conclusion

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

MFI for truncations mutants that worked.

Evaluation

The overall flow of doing a T7E experiment is as such:

We cloned plasmids containing various guide RNA sequences into the Cas9/Cpf1 vector with a -P2A-OFP reporter. We transfected Human Embryonic Kidney 293 cells (HEK293 cells) with the plasmids and sort for transfected cells(OFP+) via FACS. We then extracted DNAs from these cells and PCR amplify the targeted genomic locus.

As DNA cleavage efficiency can be reflected from indel% due NHEJ’s dominance in DSB repair, we performed T7E assay on amplified products for the detection and quantification of indels.For the measurement of HDR efficiency in terms of HDR, the amplified product is then digested with the Restriction Enzyme corresponding to the RE site on the ssODN.

Workflow for T7E assay

In order to have a fair comparison among all endonucleases, we cloned them onto the same plasmid under the expression of two different promoters, CAG and EF1a. We did one round of T7E assay for every constructs on genomic locus with known editing efficiency to check if our cloning works.

Constructs used for Project Evaluation

T7E1 results for each constructs. a. SpCas9 b. SaCas9 c. NmCas9 d. AsCpf1 e. LbCpf1

We then check the expression level of the endonucleases under the two promoters via RT-qPCR to ensure our results are promoter independent. We found that the proteins expressed under the EF1a promoter has 1.5-fold higher expression. However, even with differing expression levels, the measurement of NHEJ efficiency using the two promoter correlates well with an EF1a:CAG ratio of 0.9621(R2 = 0.7025).

a. Expression of endonuclease under CAG and EF1a promoter b. Plot of indel% between two promoters

With all conditions for a fair measurement checked, we proceed on to measure the NHEJ efficiency on the 5 genes with increasing expression levels. The endonuclease were targeted to regions with 17bp to 24 bp spacers. For a fair comparison, the target sequence for Cpf1 and Cas9 are the same. As the PAM sequence for SaCas9 and NmCas9 is impossible to be unified under the same PAM binding site, we searched for two sets of targets, set A for SaCas9 and others, set B for NmCas9 and others.

a. Target sites for each locus with PAM sites for Cas9 and Cpf1. Indel% on b. ALK, c. EGFR d. NF1 e. KDM6A f. STAG2 locus.

In general, we are able to derive that:
1.SpCas9 is able to work with efficiently even with a minimum 17bp spacer but not as well with 23nt spacers.
2.NmCas9 is unable to perform as well for most of the time.
3.SaCas9, LbCpf1 and AsCpf1 works well with 19-13nt spacer but not reliable with 17-18nt spacers.

Due to the scarcity of target sites with PAM binding site for both Cas9 and Cpf1, most target sites are within intronic regions of the gene. We then searched for other Cas9 and Cpf1 compatible target sites that are situated within protein coding regions. The results on protein coding regions parallels results we obtained from intronic regions.

Indel% for protein coding regions on a. APC b. ATM c. KDM5C

Although the spacer sequence is the same, the PAM-proximal sites of Cas9 and Cpf1 still differs. Hence, we shortened the distance of the sequence between PAM sites of Cas9s and Cpf1s to 7bp, so that every Cas9 and Cpf1 have the same seed sequence. Results confirm our conclusion that SpCas9 works well with 17nt spacer while others require longer spacers for efficient cleavage editing.

Indel% with the same seed on a. CACNA1D protein-coding region b. PPP1R12C protein-coding region

We then went on to compare how efficient is each enzyme in promoting HDR with a 100nt ssODN donor, with an Xba1 cutsite, using the same target sites as with the same seed region. Interestingly, LbCpf1 and AsCpf1 works efficiently to induce HDR for targeted genome editing.

HDR efficiency with the same seed on a. CACNA1D protein-coding region b. PPP1R12C protein-coding region