US-20260126430-A1 - TRANSIENT REPORTERS AND METHODS FOR BASE EDITING ENRICHMENT

Abstract

Provided herein are compositions and methods for real-time identification and isolation of base-edited cell populations. Also provided herein are methods for producing enriched isogenic lines of genetically modified cells, including base-edited human pluripotent stem cells. In particular, provided herein are methods utilizing transient expression of reporter proteins, the detectable signal of which is altered following base editing. Using the transient reporter with a base editor permits enrichment of isogenic populations of base-edited cells.

Inventors

• David Brafman
• Xiao Wang
• Nicholas Brookhouser
• Stefan Tekel
• Kylie Standage-Beier

Assignees

• ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY

Dates

Publication Date

20260507

Application Date

20250723

Claims (11)

1. 1 . A method for selecting a base edited cell, the method comprising (a) introducing into a cell a first nucleic acid sequence encoding one or more reporter proteins, a second nucleic acid sequence encoding a first sgRNA adjacent to a protospacer adjacent motif (PAM), wherein the first sgRNA comprises a protospacer sequence and is complementary to a portion of the nucleic acid sequence encoding one or more reporter proteins; a third nucleic acid encoding a second sgRNA adjacent to a protospacer adjacent motif (PAM), wherein the second sgRNA comprises a protospacer adjacent sequence and is complementary to a portion of a gene of interest to be base edited; and a fourth nucleic acid sequence encoding a base editor, wherein the first nucleic acid includes a PAM site adjacent to a base that when edited causes a change in a function or characteristic of the one or more reporter proteins and wherein the change in function or characteristic results in a detectable signal; (b) culturing the cell of step (a) for about 48 hours to about 72 hours under conditions sufficient for expression of proteins encoded by the first, second, third and fourth nucleic acid sequences; (e) sorting cells based on the presence or absence of a detectable signal, wherein a change in the detectable signal indicates that the base editor caused a base-to-base conversion or other genetic modification in the first nucleic acid sequence; and (f) selecting cells exhibiting the changed detectable signal from the sorted cells, thereby selecting base edited cells.
2. 2 . The method of claim 1 , wherein the base editor is selected from a cytidine deaminase base editor, an adenine base editor, Cas9-mediated adenosine base editor, and a prime editor.
3. 3 . The method of claim 1 , wherein one or more of the first, second, third, and fourth nucleic acids is provided in a vector.
4. 4 . The method of claim 3 , wherein the vector is an episomal vector.
5. 5 . The method of claim 1 , wherein the reporter protein is a fluorescent protein.
6. 6 . The method of claim 5 , wherein the fluorescent protein is a green fluorescent protein (GFP), a blue fluorescent protein (BFP), red fluorescent protein (RFP), luciferase, mCherry, or a variant or combination thereof.
7. 7 . The method of claim 6 , wherein the fluorescent protein is a BFP variant comprising a histidine at amino acid position 66 (numbered relative to SEQ ID NO:1) or a fusion protein of two fluorescent proteins linked via a linker including at least one stop codon and a PAM site.
8. 8 . The method of claim 1 , wherein the cell is a human cell.
9. 9 . The method of claim 8 , wherein the human cell is a human pluripotent stem cell.
10. 10 . The method of claim 9 , wherein the human pluripotent stem cell is a human induced pluripotent stem cell obtained from a somatic cell of a human subject having a disease-associated single nucleotide polymorphism.
11. 11 . The method of claim 1 , wherein the selecting is performed using a fluorescence activated cell sorter (FACS).

Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS The present application is a divisional application of U.S. application Ser. No. 17/347,360, filed Jun. 14, 2021, which claims the benefit of priority to U.S. Provisional Application No. 63/038,220, filed on Jun. 12, 2020, the content of which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under R01 GM106081, R01 GM121698, R01 GM131405 and R21 AG056706 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on Jan. 19, 2026, is named “112624_01526.xml” and is 477,294 bytes in size. BACKGROUND The rapid advancement of CRISPR Cas-based technologies has allowed for the modification (i.e., deletion, mutation and insertion) of human cells at precise genomic locations. For applications in which precise editing of a single nucleotide is desired, the CRISPR Cas machinery can be used to introduce site-specific double-stranded breaks (DSB) followed by homology-directed repair (HDR) using an exogenous DNA template. However, HDR is inefficient in mammalian cells, especially in recalcitrant cells such as human pluripotent stem cells (hPSCs), and repair of DSB is predominantly achieved through non-homologous end joining (NHEJ). In addition, NHEJ results in insertion or deletion of nucleotides (indels), resulting in undesired disruption (e.g. frameshift mutations, premature stop codons, deletion) of the targeted genes. As an alternative to standard gene editing approaches that require a DSB, several groups have reported the development of deaminase base editors that do not rely on HDR to introduce single nucleotide genomic changes. Broadly speaking, these base editors consist of a fusion of three components—a D10A nickase Cas endonuclease, cytidine deaminase (APOBEC1), and a DNA uracil glycosylase inhibitor (UGI). This complex is capable of converting cytosine to thymine (or adenine to guanine on the complementary strand) without the need for a DSB and homology repair template. Overall, genome modification through the use of base editors has been shown to result in formation of fewer indels when compared to HDR-based methods. Despite the advantages that deaminase base editors offer, identification and isolation of cell populations that have been successfully edited remains challenging. Specifically, there is no readily detectable phenotype to distinguish edited from unedited cells. In turn, isolation of edited cell populations requires single cell isolation followed by downstream sequencing verification. Some progress has been made to help enrich for edited cells, such as co-transfecting plasmids with a fluorescent reporter and using flow cytometry to isolate reporter-positive cells. Similarly, base editors fused to fluorescent proteins have been used to enrich for edited cell populations. However, these techniques are only reporters of transfection (RoT) and do not report on base editing activity within a cell population. Accordingly, there remains a need in the art for materials and efficient methods for selecting and enriching for base-edited human cells. There also remains a need in the art for efficient methods for producing isogenic populations of base edited human cells, particularly human pluripotent stem cell populations having targeted genetic modifications. BRIEF SUMMARY OF THE DISCLOSURE In a first aspect, a polynucleotide encoding one or more reporter polypeptides, the polynucleotide including a PAM site adjacent to a base that when the base is edited a change in a function or characteristic of the one or more reporter polypeptides occurs. The polynucleotide may encode at least one reporter polypeptide with at least 90% sequence identity to SEQ ID NO: 2, wherein the polynucleotide encodes histidine at amino acid at position number 66 relative to SEQ ID NO: 1, and encodes glycine at amino acid position number 72 relative to SEQ ID NO: 1. Alternatively, the polynucleotide may encode a reporter polypeptide with at least 90% sequence identity to one of SEQ ID NO: 316 or 318. In one alternative, the polynucleotide comprises a polynucleotide selected from the group consisting of SEQ ID NO: 258, 259 and 260. In a second aspect, provided herein is a kit. The kit can comprise or consist essentially of a first nucleic acid sequence encoding one or more reporter proteins, wherein the first nucleic acid includes a PAM site adjacent to a base that when edited causes a change in a function or characteristic of the one or more reporter proteins; a second nucleic acid sequence encoding a first sgRNA adjacent to a protospacer adjacent motif (PAM), wherein the first sgRNA comprises a protospacer sequence and is complementary to a portion