Pin-point™ Synthetic sgRNA Non-targeting Control

Gene editing
Pin-point™ Base Editing Platform
Pin-point™ Reagents
Pin-point™ Synthetic sgRNA Non-targeting Control

Pin-point™ Synthetic sgRNA Non-targeting Control

Non-targeting synthetic controls for evaluation of Pin-point base editing. Bioinformatically designed not to target any gene in the human genome.

Pin-point™ synthetic sgRNA non-targeting controls are designed and recommended for use as negative controls for Pin-point base editing experiments. These non-targeting controls will engage the Pin-point base editing complex, but will not target any PAM-adjacent sites in the human genome. Changes in viability or gene expression levels in cells treated with these controls likely reflect a baseline cellular response that can be compared to the levels in cells treated with target-specific Pin-point synthetic sgRNAs.

Highlights

Proprietary alignment tools used to verify mismatches or gaps to any potential target in the human genome
Ideal for establishing baseline levels

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128 nt Pin-point sgRNA are available now

Base editing with the Pin-point platform induces specific nucleotide changes without the formation of DNA double-strand breaks or indels.

This system consists of three components:

[1] a nuclease-defective “nickase” nCas9 fused to a uracil glycosylase (UGI) inhibitor that only cuts or “nicks” a single strand of DNA (Komor, 2016).
[2] a cytidine deaminase base editor (Rat APOBEC) fused to an aptamer binding protein. The cytidine deaminase enzyme converts C-G base pairs to T-A base pairs if the base pairs are within the editing window, (positions 4 to 8 at the PAM-distal end of the protospacer) (Collantes, 2021).
[3] an aptameric single guide RNA (sgRNA) that recruits the nCas9 and the aptamer-deaminase fusion to a specific DNA target site.

Delivery of all three components into mammalian cells induces C-G to T-A base conversion at the targeted site. This system can be used to make gene knockouts (through the introduction of premature stop codons (Billon, 2017), the disruption of splice donor and splice acceptor sites (Webber, 2019)), or to introduce point mutations.

Illustration of Pin-point base editing system.
The utilization of nCas9 (light gray) ensures that no DNA double-strand breaks occur and DNA damage response pathway is not triggered. Pin-point sgRNA contains an aptamer (green) that is used to recruit deaminase (blue) via aptamer binding protein (orange) to perform base editing.

Table 1: How much Pin-point mRNA do I need?

*Note: Exact amounts of each reagent should be optimized for each: experimental set-up, cell type, cell number, and plate format in order to achieve optimal results.
	Number of reactions by well/format size*
	HEK 293T cells (50,000 cells per well in a 96 well plate)		T cells (250,000 cells per well in a 24 well plate)
	Pin-point nCas9 mRNA	Pin-point Rat APOBEC mRNA	Pin-point nCas9 mRNA	Pin-point Rat APOBEC mRNA
	1 µg/well	0.1 µg/well	1.56 µg/well	0.22 µg/well
20 µg (2 µg/µl)	20	200	12	90
100 µg (2 µg/µl)	100	1000	64	450
500 µg (2 µg/µl)	500	5000	320	2252

Table 2: How much Pin-point sgRNA do I need?

*Note: Exact amounts of each reagent should be optimized for each: experimental set-up, cell type, cell number, and plate format in order to achieve optimal results.
	Number of reactions by well/format size*
	HEK 293T cells (50,000 cells per well in a 96 well plate)	T cells (250,000 cells per well in a 24 well plate)
	60 pmol/well	Singleplex 20 pmol/well or Multiplex (3 guides) at 20 pmol/well each
2 nmol	33	100
5 nmol	83	250
10 nmol	166	500

Figure 1
Pin-point base editing platform is efficient and reproducible across many experiments and T cells donors. Activated T cells were electroporated with Pin-point mRNAs and Pin-point synthetic sgRNAs for the 3 targets (CD52, PDCD1 and TRAC), or non-targeting control (NTC #1) sgRNA. Mock-electroporated (EP) cells were used as negative controls. Cells were harvested 6-7 days post electroporation, and base editing levels were calculated from Sanger sequencing data of PCR products, using the Pin-point analysis primers. N = 8 T cell donors, 5 independent experiments with duplicate or triplicate technical replicates. Bars are averages ± SD.

Pin-point reagents supporting data Figure 2A

Figure 2
Pin-point base editing platform efficiently induces multiplex gene knockout in primary T cells. Activated T cells were electroporated with Pin-point mRNAs and Pin-point synthetic sgRNAs for the 3 targets (CD52, PDCD1 and TRAC) and analyzed by flow cytometry 7 days post electroporation. To induce the expression of PD1, T cells were cultured in the presence of phorbol 12- myristate 13-acetate and ionomycin for 48 h prior. Expression of CD52 and TCRa/b were analysed on non-stimulated cells. Percentage of negative cells for each marker in the edited and mock-electroporated (EP) reported as percentage of live cells. N = 2 T cell donors with duplicate or triplicate technical replicates. Bars are averages ± SD.

Pin-point reagents supporting data Figure 2B

Figure 3
Representative flow cytometry plots from edited and control samples for each of the three markers. Negative cells for each marker are gated on the live population.

Pin-point reagents supporting data Figure 2C

Figure 4
Percentage of individual cells that are negative for all three markers simultaneously (CD52, PD1 and TCRa/b) in the samples, edited with the Pin-point platform, and mock-electroporated (EP) controls, reported as percentage of live cells, with no additional enrichment. To induce the expression of PD1, T cells were cultured in the presence of phorbol 12- myristate 13-acetate and ionomycin for 48 h prior to analysis to enable simultaneous quantification of all three markers. N = 2 T cell donors with duplicate or triplicate technical replicates. Bars are averages ± SD.

Pin-point reagents supporting data fig 2D

Figure 5
Representative flow cytometry plots from an edited sample to show the gating strategy: CD52 negative population gated on live cells; TCRa/b negative population gated on CD52 negative population; and PD1 negative population gated on CD52 and TCRa/b double negative population. The count of cells from this gate are the total number of cells negative for the three markers. The counts of triple negative cells are normalized on the counts of live cells and the percentage reported in the figure above.