Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

Base editors create specific point mutations in the genome, but they’re inefficient compared to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due to this inefficiency it is crucial for scientists to not only easily identify base editing events in real-time but also enrich for base-edited cells in their experiments. In the past few years, scientists have created an array of base editing reporters that can help you do just that.

Monitoring APOBEC and Cas9-mediated editing in real time

The Harris lab created ACE, a reporter that monitors APOBEC (a family of cytidine deaminase proteins) and Cas9-mediated editing in real time (St. Martin et al., 2018). The ACE reporter is a bicistronic construct that consists of a mutated mCherry and a downstream constitutively active eGFP. To create the inactive mCherry, the lab introduced a 43 base-pair insertion that disrupts fluorescence due to a frameshift. Restoration of fluorescence can only occur through editing by a APOBEC-Cas9n-UGI complex. So how exactly does this work?

Figure 1: APOBEC- and Cas9-mediated editing (ACE) reporter in action. Image from St. Martin et al., 2018.

APOBEC-Cas9n-UGI targets its preferred trinucleotide motifs (5’-TCA-to-TUA) found within the 43 base-pair mCherry insertion. Editing of these motifs generates lesions that are subject to excision and ssDNA cleavage by canonical base excision repair enzymes. At the same time Cas9n cleaves the opposing DNA strain which results in two DSBs that can be repaired by NHEJ restoring mCherry expression. Cells can then be sorted using Fluorescence-activated cell sorting (FACS) and the editing efficiency can be easily determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry. ACE was used to identify cells that have been successfully edited with APOBEC3A and APOBEC3B (St. Martin et al., 2018).

TLDR: The ACE reporter from the Harris Lab relies on the restoration of a frame-shift in mCherry that restores fluorescence to monitor APOBEC-Cas-9 mediated editing.

eGFP reporters for single base editing by APOBEC-Cas9

Though the ACE reporter quantifies APOBEC-Cas9 efficiency in real-time, it relies on DSBs which is not ideal for monitoring single base edits (St. Martin et al., 2018).

To circumvent sequencing and the need for a DSB in their previous reporter, the Harris Lab created a panel of eGFP reporters to quantify the on-target DNA editing efficiency of APOBEC-Cas9 editisome complexes in real-time (St. Martin et al., 2019). To create the eGFP reporters the Harris lab individually mutated three codons in eGFP to eliminate fluorescence. This created three inactivated eGFP reporters- eGFP L202, eGFP L138, and eGFP L93.

The modified eGFPs were placed downstream of wild-type mCherry and a T2A site and expressed in cells. mCherry is constitutively expressed to identify successfully transfected cells. C-to-T editing by the APOBEC-Cas9 editosome would restore eGFP fluorescence, making it easy to quantify single base editing efficiency by dividing the number of eGFP and mCherry positive cells by the number of only mCherry positive cells (Martin et al., 2019).

Figure 2: Editing efficiencies using base editing reporters. Image from St. Martin et al., 2019.

The Harris lab used these eGFP reporters to analyze the base editing capability of the seven human APOBEC3 proteins, finding that APOBEC3A and APOBEC3bctd are the most efficient (Martin et al., 2019).

TLDR: The eGFP reporters from the Harris Lab circumvents the need for DSBs in monitoring on-target DNA editing efficiency of APOBEC-Cas9 editisome complexes. The eGFP reporters rely on the correction of point mutations in eGFP that results in the restoration of fluorescence.

Transient reporter for editing enrichment (TREE)

The Wang and Brafman lab developed a transient reporter for editing enrichment (TREE) to purify single base edited cells without the need for single cell isolation and downstream sequencing (Standage-Beier et al. 2019). TREE is a real-time, fluorescent based identification system for the isolation of base-edited cell populations.

To develop this method the lab was inspired by work that used an integrated GFP reporter that is converted into BFP (blue fluorescent protein) upon CRISPR/Cas9 homology directed repair (HDR) (Glasser et al., 2016). For TREE they engineered a BFP variant that undergoes conversion to GFP after being targeted by a cytidine deaminase base DNA editor. Specifically the BFP mutant (BFPH66) contains a histidine at amino acid 66. This histidine is encoded by a ‘CAC’ codon that is converted to a ‘TAC’ or ‘TAT’ after a C-to-T base editing event. This edit changes the histidine to a tyrosine generating a GFP variant (GFPY66) that has a shifted emission spectra. Thus a successful base editing event would result in GFP fluorescence that can be visualized and sorted via flow cytometry.

Figure 3: Targeting pEF-BFP with a cytidine deaminase base editor results in shift in emission spectra from BFP to GFP. Image from Standage-Beier et al., 2019.

The team put TREE to the test by infecting HEK-293 cells with the BFP variant, a BE4am-Cas9 base editor, and sgRNA vectors for the BFP-to-GFP conversion and to target specific loci of interest in the genome. The Wang and Brafman lab showed that GFP positive cells isolated with TREE have a significantly higher frequency of genomic base pair edits of interest compared to cells segregated solely by a reporter of transfection, which only report the efficiency of plasmid delivery to a cell. In contrast, TREE provides a read out for plasmid delivery and subsequent base editing efficiency.

TLDR: TREE from the Wang and Brafman lab uses an engineered BFP variant that converts to GFP after a base editing event. GFP positive cells can be sorted and have a significantly higher frequency of base edits of interest than solely transfected cells.

“Gene On” (GO)- a functional reporter system to identify and enrich for base-editing activity

The reporters mentioned above all rely on GFP fluorescence and cell sorting using FACS which could limit their use across different cell types and systems.

To introduce flexibility, the Dow lab created a base editing reporter that detects and enriches for base editing events in vivo without relying solely on GFP (Katti et al., 2020). They named this reporter “Gene On” or GO. GO works by affecting protein translation of different reporter proteins. Protein translation requires the start codon of AUG immediately downstream of a kozak sequence. The Dow lab hypothesized that if they created an ATG codon from an ACG codon using a base-editor (C>T conversion) they could induce the translation of any detectable protein, not just fluorescent proteins.

Figure 4: Reporters in Gene On include a mScarlet reporter, luciferase reporter, and neomycin resistance reporter. Image adapted from Katti et al., 2020.

As a proof of concept the Dow lab tested their hypothesis with GFP fluorescence. To do this they generated a silent GFP construct, that contains an ACG start codon mutation, and integrated it into human and mouse cells that expressed either Cas9 or an optimized cytidine base editor. The lab then introduced an sgRNA targeting the GFPACG to the cells. As expected, only the cells that contained the cytidine base editor exhibited robust induction of GFP fluorescence confirming the efficacy of GO as a base-editing reporter.

As the initiation of protein translation at the start codon ATG is universal, the Dow lab successfully used GO to induce the translation of an array of different reporters including mScarlet-I, luciferase, antibiotic resistance markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing reporter toolbox. The Dow lab also demonstrated that GO is an effective reporter for adenine base editors, that make A-to-G edits in the genome (Gaudelli et al., 2017). GO is thus a flexible and adaptable tool to identify and enrich for base-editing events in vivo.

TLDR: Gene On (GO) from the Dow lab is a flexible reporter system for base-editing events in vivo. As GO relies on the correction of a mutated start codon to initiate protein expression, it can be used for an array of detectable proteins including fluorescent proteins, antibiotic resistance, and luciferase.

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References

Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464–471 . https://doi.org/10.1038/nature24644

Glaser A, McColl B, Vadolas J (2016) GFP to BFP Conversion: A Versatile Assay for the Quantification of CRISPR/Cas9-mediated Genome Editing. Molecular Therapy - Nucleic Acids 5:e334 . https://doi.org/10.1038/mtna.2016.48

Katti A, Foronda M, Zimmerman J, Diaz B, Zafra MP, Goswami S, Dow LE (2020) GO: a functional reporter system to identify and enrich base editing activity. Nucleic Acids Research 48:2841–2852 . https://doi.org/10.1093/nar/gkaa124

Martin AS, Salamango DJ, Serebrenik AA, Shaban NM, Brown WL, Harris RS (2019) A panel of eGFP reporters for single base editing by APOBEC-Cas9 editosome complexes. Scientific Reports 9: . https://doi.org/10.1038/s41598-018-36739-9

St. Martin A, Salamango D, Serebrenik A, Shaban N, Brown WL, Donati F, Munagala U, Conticello SG, Harris RS (2018) A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells. Nucleic Acids Research 46:e84–e84 . https://doi.org/10.1093/nar/gky332

Standage-Beier K, Tekel SJ, Brookhouser N, Schwarz G, Nguyen T, Wang X, Brafman DA (2019) A transient reporter for editing enrichment (TREE) in human cells. Nucleic Acids Research 47:e120–e120 . https://doi.org/10.1093/nar/gkz713

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