Pin-point™ Base Editing mRNA

The Pin-point™ base editing mRNA system allows for precisely directed point mutation edits without inducing double stranded breaks or the need for homology directed repair.

Pin-point™ base editing mRNAs harness the DNA targeting ability of a nickase Cas9, with the DNA editing activity of a cytidine deaminase to introduce targeted point mutations when used in combination with Pin-point synthetic sgRNA. The platform consists of an mRNA for translation of a mammalian codon-optimized nCas9 and an mRNA for translation of a mammalian codon-optimized deaminase fused to an aptamer binding protein.

Pin-point nCas9 mRNA

The Pin-point nCas9 mRNA contains a human codon-optimized version of the S. pyogenes nickase Cas9 (D10A) with a 5’ and 3’ nuclear localization signal (NLS), fused to UGI. The nCas9 mRNA is in vitro transcribed, capped using CleanCap AG, and polyadenylated for translation and nuclear localization of the protein.

Pin-point Rat APOBEC mRNA

The Pin-point Rat APOBEC mRNA contains a human codon-optimized version of an aptamer binding protein fused to a cytidine deaminase base editor enzyme with a 5’ NLS. The Rat APOBEC mRNA is in vitro transcribed, capped using CleanCap AG, and polyadenylated for translation and nuclear localization of the protein.

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128 nt Pin-point sgRNA are available now

Modified or unmodified, which one should I use?

Both Pin-point nCas9 mRNA and Pin-point Rat APOBEC mRNA come in either unmodified or fully substituted with 5-methoxy-uridine (5moU modified) formats for optimized usage in different cell types. Additionally, each experiment with the Pin-point base editing system requires usage of nCas9 and Rat APOBEC mRNAs in combination with Pin-point synthetic sgRNA. View the "Ordering information" tab to find a table of the suggested use of the Pin-point mRNA formulations by cell type. Read our application note that discusses specific use cases for selecting unmodified versus 5moU modified mRNAs.

Table 1: Should I select Pin-point unmodified or Pin-point 5moU modified mRNAs?

*Note: Suggested use is based on optimized functionality when delivering Pin-point mRNAs as outlined in the Technical Manual and Quick protocols found in the Resources tab. Use of these mRNAs in additional cell types and/or with alternative workflows could result in different results. Read our application note that discusses specific use cases for selecting unmodified versus 5moU modified mRNAs.
Suggested use of Pin-point mRNAs by cell type
HEK293T cells	unmodified
Primary human T cells	unmodified
Induced pluripotent stem cells (iPSCs)	unmodified
Hemopoietic Stem and Progenitor Cells (HSPCs)	5moU modified

Table 2: 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 3: 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.

3-days post electroporation

Figure 6
Representative editing and total cell count at 3 days post electroporation when delivering a Custom Pin-point Synthetic sgRNA targeting a chain of the major histocompatibility complex class 1 and either Pin-point unmodified or Pin-point 5moU modified mRNAs in T cells, iPSCs, and HSPCs.