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CN-115386623-B - Method and kit for detecting editing site of base editor

CN115386623BCN 115386623 BCN115386623 BCN 115386623BCN-115386623-B

Abstract

The present application relates to a method for detecting the site of editing nucleic acid by a base editor, and a kit for carrying out said method. The application also relates to a method for detecting the editing efficiency or off-target effect of a base editor editing nucleic acid.

Inventors

  • YI CHENGQI
  • LEI ZHIXIN
  • MENG HAOWEI
  • LV ZHICONG

Assignees

  • 北京大学

Dates

Publication Date
20260512
Application Date
20220520
Priority Date
20210520

Claims (20)

  1. 1. A method for detecting editing sites, editing efficiencies, or off-target effects of a base editor for non-diagnostic purposes editing a target nucleic acid, comprising the steps of: (1) Providing an editing product of a base editor editing a target nucleic acid, comprising a base editing intermediate comprising a first nucleic acid strand and a second nucleic acid strand, wherein the first nucleic acid strand comprises editing bases generated as a result of the base editor editing the target nucleic acid; (2) Creating a single-stranded break nick within a segment comprising the editing base using an endonuclease in the first nucleic acid strand; (3) Introducing nucleotides labeled with a first labeling molecule and nucleotides labeled with a second labeling molecule at or downstream of the single strand break with a nucleic acid polymerase having strand displacement activity to produce a labeled product containing the first labeling molecule and the second labeling molecule; (4) Isolating or enriching the labeled product using a first binding molecule capable of specifically recognizing and binding to the first label molecule; (5) Determining the sequence of the labeled product; Thereby, determining an editing site, editing efficiency, or off-target effect of the base editor editing the target nucleic acid; Wherein the nucleotide labeled with the second labeling molecule is a 5-aldehyde cytosine deoxyribonucleotide, and the method further comprises: protecting the nucleotide marked with the second marker molecule which may be present in the editing product before the step (3), and, After step (3), prior to determining the sequence of the tagged product, treating the tagged product with a compound selected from the group consisting of malononitrile or borane-based compounds to alter the base complementary pairing ability of the nucleotides comprising the second tagged molecule.
  2. 2. The method of claim 1, wherein in step (2), a single strand break nick is created in the first nucleic acid strand within a segment of 10nt upstream to 10nt downstream of the editing base.
  3. 3. The method of claim 1, wherein the base editor is a single base editor or a double base editor.
  4. 4. The method of claim 1, wherein the base editor is a cytosine base editor, an adenine base editor, or an adenine and cytosine double base editor.
  5. 5. The method of claim 1, wherein the target nucleic acid is genomic nucleic acid or mitochondrial nucleic acid.
  6. 6. The method of claim 1, wherein the editing product is a product of the base editor editing the target nucleic acid extracellular or intracellular.
  7. 7. The method of claim 1, wherein the editing product is a product of the base editor editing a target nucleic acid in an organelle.
  8. 8. The method of claim 1, wherein the method further comprises, prior to step (1), the step of contacting the base editor with the target nucleic acid under conditions that allow the base editor to edit the target nucleic acid, thereby producing an edited product.
  9. 9. The method of claim 1, wherein the method further comprises, prior to step (1), the step of contacting the base editor with the target nucleic acid, either extracellular or intracellular, under conditions that allow the base editor to edit the target nucleic acid, thereby producing an edited product.
  10. 10. The method of claim 1, wherein the method further comprises, prior to step (1), the step of contacting the base editor with the target nucleic acid in an organelle under conditions that allow the base editor to edit the target nucleic acid, thereby producing an edited product.
  11. 11. The method of claim 1, wherein the method further comprises, prior to step (1), the step of introducing the base editor into the cell such that the base editor contacts and base edits the target nucleic acid in the cell to produce an edited product, or introducing a nucleic acid molecule encoding the base editor into the cell and expressing the base editor, the base editor contacting and base editing the target nucleic acid in the cell to produce an edited product.
  12. 12. The method of claim 1, wherein the method further comprises, prior to step (1), the step of introducing the base editor into the cell so that the base editor contacts and base edits the target nucleic acid in the cell to produce an edited product, or introducing a nucleic acid molecule encoding the base editor into the cell so that it expresses the base editor, the base editor contacting and base editing the target nucleic acid in the cell to produce an edited product.
  13. 13. The method of claim 11, wherein in step (1), the base-edited target nucleic acid is extracted or isolated from the cell, and optionally fragmented, thereby obtaining the edited product.
  14. 14. The method of claim 12, wherein in step (1), the base-edited target nucleic acid is extracted or isolated from within the organelle, and optionally fragmented, thereby obtaining the edited product.
  15. 15. The method of claim 11, wherein in step (1), a base-edited target nucleic acid is extracted or isolated from the cell, and nucleic acid fragmentation and end repair are performed, thereby obtaining the edited product.
  16. 16. The method of claim 12, wherein in step (1), a base-edited target nucleic acid is extracted or isolated from within the organelle, and nucleic acid fragmentation and end repair are performed, thereby obtaining the edited product.
  17. 17. The method of claim 15 or 16, wherein the terminal repair is a filling of the 5 'terminal overhang and/or excision of the 3' terminal overhang.
  18. 18. The method of claim 1, wherein the second nucleic acid strand does not undergo base editing or does not contain editing bases.
  19. 19. The method of claim 1, wherein the editing base is selected from uracil or hypoxanthine.
  20. 20. The method of claim 1, wherein in step (2), a single strand break cut is made at or upstream or downstream of the position of the editing base.

Description

Method and kit for detecting editing site of base editor Technical Field The present application relates to the field of gene editing (particularly base editing) technology. In particular, the application relates to a method for detecting the site of editing of a nucleic acid by a base editor (e.g., a single base editor or a double base editor), and a kit for performing the method. The application also relates to methods for detecting the editing efficiency or off-target effect of a base editor (e.g., single base editor or double base editor) editing nucleic acids. Background The cytosine base editor (cytosine base editor, CBE) was developed by David Liu et al, 2016, which fused rAPOBEC1 from rats to nCas (D10A) protein based on the CRISPR/Cas9 system (Komor, et al Nature 533,420-424, doi:10.1038/nature17946 (2016)). The editing principle of the design is that nCas which loses part of nucleic acid cutting activity can still be guided by sgRNA to drive rAPOBEC1 connected with nCas to a target site, then, the sgRNA and a DNA sequence of a target gene form an R ring (R-loop) structure, so that non-sgRNA complementary strand DNA (non-TARGET STRAND) in a single-stranded state in the R ring can be combined by APOBEC1 to deaminate cytosine (C) in a certain range on the strand into uracil (U), and finally, the uracil can complete uracil-thymine conversion through a subsequent DNA replication process, and thus, the base conversion from C to T (C-to-T) is finally realized. Thereafter, various new CBE editing systems, such as YE1-BE, BE4max, etc., were developed successively, with various degrees of optimization in terms of editing efficiency, active editing window, editable sequence range, etc (Kim,Y.B.et al.Nature biotechnology 35,371-376,doi:10.1038/nbt.3803(2017);Suzuki,K.et al.Nature 540,144-149,doi:10.1038/nature20565(2016)). Furthermore, david Liu et al in 2020 reported a RNA-free mitochondrial cytosine base editor DdCBE (DddA-modified CBE) that enabled a major breakthrough in mitochondrial gene editing (Mok, B.Y. et al Nature 583,631- +, doi:10.1038/s41586-020-2477-4 (2020)). Heretofore, introducing sgrnas into mitochondria has still faced significant challenges due to the presence of mitochondrial bilayer membranes, severely limiting the application of CRISPR/Cas 9-based CBE tools in mitochondrial gene editing. Compared with a CBE tool based on CRISPR/Cas9, the main change of DdCBE comprises the following two steps of firstly using TALE protein to replace sgRNA, realizing the identification of a target DNA chain, avoiding the difficult problem that the sgRNA is difficult to enter mitochondria, secondly using newly discovered double-stranded DNA deaminase DddA to replace APOBEC, deaminizing dC on double-stranded DNA at a target position into dU, and finally realizing the base conversion from dC to dT. In summary, there are a variety of cytosine base editing systems directed to the nucleus or mitochondria, and there are also in constant abundance. The core principle is that cytosine (C) is deaminated into uracil (U) at a targeted editing site, and finally, the uracil can finish uracil (U) to thymine (T) through a subsequent DNA replication process, so that the base conversion from C to T (C-to-T) is finally realized. After the development of cytosine base editors (Komor et al., 2016) by David Liu in 2016, an adenine base editor (adenine base editor, ABE) in 2017 (Gaudelli et al., 2017) was also developed, the principle of the technology was that Cas9 reached the targeted editing site under the guidance of sgRNA, and opening the DNA duplex to form an R-loop structure, and then adenine deaminase fused with Cas9 deaminates adenine in the editing window to form hypoxanthine (inosine, I). During repair and replication, hypoxanthine will be read as G by DNA polymerase, so that the transition from adenine (a) to guanine (G) eventually occurs. Through development for several years, the current utilization rate is ABEmax, and the system is based on a series of improvements such as mutation screening, codon optimization, nuclear localization signal introduction and the like of the initial ABE version, so that the editing efficiency of the target site is continuously improved. In 2020, david Liu and Jennifer a.doudena have newly reported a version of ABE with higher activity and are named ABE8e (RICHTER ET al, 2020). Only one TadA element is reserved on the basis of ABEmax in the ABE8e, and a plurality of mutations are carried out, so that the in vitro activity of the enzyme is improved (LAPINAITE ET al, 2020), and the editing efficiency of a targeting site in a cell is also greatly improved. Also, similar to CBE editing systems, a variety of ABE editing systems have been developed, all of which have the core principle that adenine is deaminated to hypoxanthine at the targeted editing site, after which these hypoxanthines can be passed through subsequent DNA replication processes to complete the hypoxanthine to guanine ba