Pin-point™ Base Editing mRNA

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Gene editing
Pin-point™ Reagents
Pin-point™ Base Editing mRNA

Pin-point™ Base Editing mRNA

Pin-point base editing mRNAs should be combined with Pin-point validated control sgRNAs, Pin-point Non-targeting control sgRNAs, or Pin-point custom sgRNAs to effectively base edit cells for targeted point mutation or gene knockout without inducing DNA double stranded breaks. Pin-point nCas9 and deaminase mRNAs are available in both Adenine base editor (ABE) and Cytosine base editor (CBE) configurations.

Pin-point™ base editing mRNAs harness the DNA targeting ability of a nickase Cas9, with the DNA editing activity of an adenine or cytidine deaminase to introduce targeted point mutations when used in combination with Pin-point synthetic sgRNA. All mRNAs are available with either 5-methoxy-uridine (5moU) base modifications or fully unmodified throughout.

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

Pin-point ABE-flex and ABE-exact deaminase mRNAs
The Pin-point ABE-flex and ABE-exact deaminase mRNAs are designed using Profluent's frontier AI models and biologically validated for optimal A to G editing. The mRNAs are in vitro transcribed, capped using CleanCap AG, and polyadenylated for translation and nuclear localization of the protein.

Pin-point CBE nCas9 mRNA
The Pin-point CBE 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 a sequence of uracil DNA glycosylase inhibitor (UGI). The nCas9 mRNA is in vitro transcribed, capped using CleanCap AG, and polyadenylated for translation and nuclear localization of the protein.

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

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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: 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
Hematopoietic Stem and Progenitor Cells (HSPCs)	5moU modified

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

Table 4: 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

For additional Supporting Data please see our Pin-point Synthetic sgRNA Validated Controls, Pin-point Synthetic sgRNA Non-targeting Controls, or Custom Pin-point Synthetic sgRNA pages

pinpoint abe flex titration tcells global product page

Figure 1: Efficient base editing in activated human T cells with the Pin-point ABE platform at CD52 locus.
(A) CD52 sgRNA spacer sequence. Numbers indicate positions within spacer. A6 is on-target for generating splice donor mutation and protein knockout; other A positions are bystanders in this example. (B-C) Base editing levels following mRNA titrations of Pin-point ABE-exact (B) and ABE-flex (C) at on-target A (6) and the bystander A (12). Amounts of Pin-point ABE nCas9 mRNA and synthetic sgRNA were kept constant. Percent A to G conversion was calculated from Sanger sequencing of the PCR products using our Pin-point analysis primers. N=2 technical replicates. Points – average ± SD.

Figure 2: Efficient base editing in hematopoietic stem and progenitor cells (HSPCs) with the Pin-point ABE platform at HBG1/2 promoter regions.
(A) HBG1/2 sgRNA spacer sequence. A5 and A8 fall within BCL11A binding site. Other A positions are bystander. (B) Base editing levels following editing with nCas9 ABE mRNA, ABE-exact or ABE-flex deaminase mRNAs, and HBG1/2 synthetic sgRNA control. Bars – average ± SD. N=2 technical replicates. (C) Base editing levels following titration of Pin-point ABE-flex mRNA in HSPCs. Amounts of Pin-point ABE nCas9 mRNA and synthetic sgRNA were kept constant. Percent A to G conversion was calculated from Sanger sequencing of the PCR products using our Pin-point analysis primers. N=2 technical replicates. Bars and points – average ± SD.

pinpoint abe unmod 5mo u global product page

Figure 3: Evaluating the effects of Pin-point ABE mRNA modification on base editing levels.
(A) Unmodified or 5moU-modified ABE nCas9 mRNA and ABE-exact deaminase mRNA were delivered to activated human T cells via electroporation, together with Pin-point CD52 synthetic sgRNA ABE validated control. (B) Unmodified or 5moU-modified ABE nCas9 mRNA and ABE-flex deaminase mRNA were co-delivered with Pin-point CD52 synthetic sgRNA ABE validated control to activated human T cells via electroporation. (C) Unmodified or 5moU-modified ABE nCas9 mRNA and ABE-flex deaminase mRNA were co-delivered with Pin-point HBG1/2 synthetic sgRNA ABE validated control to HSPCs via electroporation. Percent A to G conversion was calculated from Sanger sequencing of the PCR products using our Pin-point analysis primers. N=2 technical replicates. Bars – average ± SD.

pinpoint cbe unmod 5mo u global product page

Figure 4: Pin-point CBE base editing using unmodified or 5moU modified mRNAs in T cells, HSPCs, and induced pluripotent stem cells (iPSCs).
Representative editing(A) and total cell count at 3 days post electroporation (B) 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 CBE mRNAs in activated human T cells, iPSCs, and HSPCs.

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Figure 5: 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.