Pin-point™ Synthetic sgRNA Validated Control

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Gene editing
Pin-point™ Reagents
Pin-point™ Synthetic sgRNA Validated Control

Pin-point™ Synthetic sgRNA Validated Controls

Synthetic sgRNA controls to verify optimal parameters for precise gene editing without inducing DNA double-strand breaks.

Each control sgRNA is chemically synthesized, HPLC purified, and biologically validated for optimal use with either Adenine base editor (ABE) or Cytosine base editor (CBE) Pin-point base editing mRNAs.

Pin-point synthetic sgRNA are 128-nucleotide chimera fusions of the crRNA, tracrRNA, and a proprietary aptameric sequence. They are chemically modified for nuclease resistance on both 5’ and 3’ ends of the molecule.

The sgRNA controls are recommended as positive controls for base editing experiments utilizing Pin-point ABE or Pin-point CBE mRNAs.

Gene-specific positive controls and kits are designed and validated for assessment of base editing and phenotypic protein knockout where applicable. They are configured as follows:

Individual Pin-point synthetic sgRNA ABE or CBE validated control
Synthetic sgRNA control kits containing:

Individual Pin-point synthetic sgRNA ABE or CBE validated control
Forward and reverse detection primers come in the 10 nmol size

Need custom guides?

Design and order custom CBE or ABE Pin-point sgRNA 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 that only cuts or “nicks” a single strand of DNA. For cytosine base editing (CBE), the nCas9 is fused to a sequence of uracil glycosylase (UGI) inhibitors (Komor, 2016). For adenine base editing (ABE), the nCas9 is not fused to any UGI.
[2] a cytidine deaminase (rAPOBEC) or an adenine deaminase (ABE-flex or ABE-exact) fused to an aptamer binding protein. The rAPOBEC 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). ABE-flex converts A-T base pairs to G-C base pairs if the base pairs are within positions 4 to 8 of the base editing window while ABE-exact converts A-T base pairs to G-C base pairs if the base pairs are within positions 5 to 6 of the base editing window.
[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 the base editing 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. See our Pin-point base editing sgRNA design tool to design your own sgRNAs for gene knockout.

Pinpoint abe global product page

Illustration of Pin-point base editing system.

The utilization of nCas9 (light gray) with or without UGIs (red) 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 = cytidine deaminase, turquoise = adenine deaminase) via aptamer binding protein (orange) to perform base editing.

Table 1: 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*
	T cells (250,000 cells per well in a 24 well plate)		iPSCs (100,000 cells per well in a 12 well plate)		HSPCs (50,000 cells per well in a 96 well plate)		HEK 293T cells (50,000 cells per well in a 96 well plate)
	CBE	ABE	CBE	ABE	CBE	ABE	CBE
	20 pmol/well	20 pmol/well	40 pmol/well	40 pmol/well	62.5 pmol/well	62.5 pmol/well	60 pmol/well
2 nmol	100	100	50	50	32	32	33
5 nmol	250	250	125	125	80	80	83
10 nmol	500	500	250	250	160	160	167

Table 2: How much Pin-point ABE 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*
	T cells (250,000 cells per well in a 24 well plate)			iPSCs (100,000 cells per well in a 12 well plate)			HSPCs (50,000 cells per well in a 96 well plate)
	ABE nCas9 mRNA	ABE-exact deaminase mRNA	ABE-flex deaminase mRNA	ABE nCas9 mRNA	ABE-exact deaminase mRNA	ABE-flex deaminase mRNA	ABE nCas9 mRNA	ABE-exact deaminase mRNA	ABE-flex deaminase mRNA
	1.5 μg/well	1 μg/well	0.1 μg/well	1 μg/well	1 μg/well	0.1 μg/well	1.5 μg/well	1 μg/well	0.4 μg/well
10 μg (2 μg/μL)	-	10	100	-	10	100	-	10	25
20 μg (2 μg/μL)	13	20	200	20	20	200	13	20	50
100 μg (2 μg/μL)	67	100	1000	100	100	1000	67	100	250
500 μg (2 μg/μL)	333	-		500	-	-	333	-	-

Table 3: How much Pin-point CBE 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*
	T cells (250,000 cells per well in a 24 well plate)		iPSCs (100,000 cells per well in a 12 well plate)		HSPCs (50,000 cells per well in a 96 well plate)		HEK 293T cells (50,000 cells per well in a 96 well plate)
	CBE nCas9 mRNA	CBE rAPOBEC deaminase mRNA	CBE nCas9 mRNA	CBE rAPOBEC deaminase mRNA	CBE nCas9 mRNA	CBE rAPOBEC deaminase mRNA	CBE nCas9 mRNA	CBE rAPOBEC deaminase mRNA
	1.56 μg/well	0.22 μg/well	2.56 μg/well	0.4 μg/well	2.8 μg/well	2 μg/well	1 μg/well	0.1 μg/well
20 μg (2 μg/μL)	12	90	8	50	7	10	20	200
100 μg (2 μg/μL)	64	450	39	250	36	50	100	1000
500 μg (2 μg/μL)	320	2252	195	1250	179	250	500	5000

For additional Supporting Data please see our Pin-point Base Editing mRNA, Custom Pin-point Synthetic sgRNA and Pin-point Synthetic sgRNA Non-targeting Controls pages.

pinpoint control sg rna global product page

Figure 1: Base editing efficiencies with the Pin-point platform using Pin-point mRNAs and control synthetic sgRNA reagents in various cell types.

(A) Pin-point CBE nCas9 mRNA, CBE rAPOBEC deaminase mRNA, and synthetic sgRNA CBE validated controls were delivered to activated human T cells, induced pluripotent stem cells (iPSCs) and hematopoietic stem and progenitor cells (HSPCs) via electroporation. (B) Pin-point ABE nCas9 mRNA, ABE-exact deaminase mRNA, and synthetic sgRNA ABE validated controls were delivered to T cells, iPSCs and HSPCs via electroporation. (C) Pin-point ABE nCas9 mRNA, ABE-flex deaminase mRNA, and synthetic sgRNA ABE validated controls were delivered to T cells, iPSCs and HSPCs via electroporation. Base editing efficiencies were measured at 3 days post electroporation by Sanger sequencing the edited loci using Pin-point control PCR primers. N=2-4 technical replicates. Bars – average ± SD.

graph-Pinpoint-ABE-flex-multiplex-Tcells-global-product-page-new

Figure 2: Efficient multiplex base editing with the Pin-point ABE platform in human T cells.

Activated human T cells were electroporated with Pin-point ABE nCas9 mRNA, ABE-flex deaminase mRNA, and synthetic sgRNA ABE validated controls targeting CD52, TRAC and PDCD1. (A) Base editing levels were assessed by Sanger sequencing of PCR products using Pin-point analysis primers at 3 days post electroporation. (B) Percent protein knockout was evaluated by flow cytometry at 5-6 days post electroporation. To induce the expression of PD1, T cells were cultured in the presence of phorbol12- myristate 13-acetate (PMA) and ionomycin for 48 h prior. Expression of CD52 and TCRa/b were analyzed on non-stimulated cells. Percentage of negative cells for each marker in the edited and un-edited (negative control, neg ctrl) reported as percentage of live cells. N=2 technical replicates. Bars – average ± SD.

graph-Pinpoint-CBE-multiplex-Tcells-global-product-page.jpg

Figure 3: Efficient multiplex base editing with the Pin-point CBE platform in human T cells.
Activated human T cells were electroporated with Pin-point CBE mRNAs and Pin-point synthetic sgRNA CBE validated controls for the 3 targets (CD52, PDCD1 and TRAC), or non-targeting control (NTC #1) sgRNA. Mock-electroporated (EP) cells were used as negative controls. (A) 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. (B) Cells were 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 analyzed 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.

Figure 4: Triple knockout (KO) with the Pin-point CBE platform.
(A) Percentage of individual cells that are negative for all three markers simultaneously (CD52, PD1 and TCRa/b) in the samples in Figure 3 (edited with the Pin-point CBE 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. (B) 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 panel A.