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Novel single-base editing technology and application thereof

A technology for editing enzymes and genes, which can be used in applications, recombinant DNA technology, genetic engineering, etc., and can solve the problems of large gene editing windows and low DNA editing accuracy.

Pending Publication Date: 2020-12-08
HUIGENE THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, in the existing DNA base editing methods, the accuracy of DNA editing is not high, that is, the gene editing window is too large

Method used

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  • Novel single-base editing technology and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0256] Example 1: Off-target RNA SNV detection for various single base editing systems

[0257] In this example, in order to assess the off-target effect of gene editing at the RNA level, CBE, BE3 (APOBEC1-nCas9-UGI) or ABE, ABE7.10 (TadA-TadA*-nCas9), and GFP with or without Single guide RNA (sgRNA) was transfected into cultured 293T cells. After 72 hours of incubation, GFP-expressing cells were harvested by FACS and then analyzed by RNA-seq. The experimental results of each group were compared with wild-type (WT, non-transfected) samples, calling RNA SNVs in each transfection group ( figure 1 A).

[0258] The 9 groups of transfected cells include expressing GFP, APOBEC1, BE3, BE3 with "site 3" sgRNA, BE3 with "RNF2" sgRNA, TadA-TadA*, ABE7.10, ABE7 with "site 1" sgRNA. 10. ABE7.10 cells with "site 2" sgRNA ( Figure 5 ).

[0259] First, the high targeting efficiency of DNA editing of BE3 and ABE7.10 in these 293T cells was verified using targeted deep sequencing, as sho...

Embodiment 2

[0263] Example 2: Characterization of off-target RNA SNVs

[0264] In this example, off-target RNA SNVs were characterized for each single base editing system.

[0265] The result is as figure 2 with Figure 7-12 shown.

[0266] Notably, almost 100% of the RNA SNVs identified in BE3-treated cells were G to A or C to U mutations, significantly higher than in GFP-transfected cells (e.g. figure 2 A and 2C and Figure 7 ). This mutational bias is identical to APOBEC1 itself, suggesting that these mutations are not spontaneous but induced by BE3 or APOBEC1.

[0267] Correspondingly, 95% of ABE7.10-induced mutations were A to G or U to C, consistent with the effect of ABE7.10 (eg figure 2 B and 2C and Figure 7 ).

[0268] From the results, it can also be noticed that the GFP group also exhibits some bias for A to G and U to C mutations (eg figure 2 C), which may be due to an innate mutation preference.

[0269] In any two samples of BE3- or ABE7.10-transfection groups...

Embodiment 3

[0272] Example 3: Single-cell RNA SNV analysis of cells transfected with a single base editing system

[0273] In this example, single-cell RNA-seq sequencing was performed on four groups of cells (WT, GFP, BE3-site 3 and ABE7.10-site 1) to avoid random off-target signal loss due to population averaging .

[0274] The result is as image 3 with Figure 13-17 shown.

[0275] On average, 10,932 RefSeq genes were detected in each single cell by approximately 6.07 million sequencing reads, with results such as image 3 Shown in B. Cells with high expression levels of the indicated deaminases were selected for further analysis, and the results Figure 13 shown. Also, severe RNA off-targets and similar mutational patterns were observed in those cells expressing base edits (e.g. image 3 C to 3F and Figure 14 with 15 ).

[0276] Interestingly, the percentage of off-target sites shared by any BE3- or ABE7.10-edited cells (4.5+ / -1.0%) was much lower than that of the cell pop...

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Abstract

The invention provides a novel single-base editing technology and application thereof. Specifically, the invention provides a gene editing enzyme which is characterized in that the structure of the gene editing enzyme is shown as a formula I of Z1-L1-Z2-L2-Z3-Z4 (I), wherein Z1 is an amino acid sequence of cytosine deaminase APOBEC3A; Z2 is an amino acid sequence of spCas9 (n); and Z1 has a mutation A corresponding to the 128th R residue of the sequence as shown in SEQ ID NO: 1; Z3 is a coding sequence of a uracil DNA glycosylase inhibitor (UGI); L1 and L2 are each independently an optional linker peptide sequence; Z4 is a none or nuclear localization signal element (NLS); And each '-' is independently a peptide bond. The invention also provides a gene single-base fixed-point editing method. The method provided by the invention is high in DNA editing precision, and can significantly reduce the RNA off-target effect.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a novel single base editing technology and its application. Background technique [0002] Since 2013, the new generation of gene editing technology represented by CRISPR / Cas9 has entered various experiments in the field of biology and is changing the traditional means of gene manipulation. [0003] DNA base editing methods developed in recent years are able to directly generate precise point mutations in genomic DNA without double-strand breaks (DSBs). Two classes of base editors have been reported: cytosine base editors (CBE, C to T and G to A) and adenine base editors (ABE, A to G, T to C). However, there is still a key problem in its application, that is, off-target effects. [0004] Previous studies have mainly focused on assessing off-target mutations in genomic DNA. Recent findings have shown that CBEs, but not ABEs, induce a large number of off-target single-nucleot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N9/22C12N15/55C12N15/90C12N5/10
CPCC12N9/78C12Y305/04001C12N9/22C12N15/907C12N15/90C12N5/10C12N15/10C12N15/62C07K19/00
Inventor 杨辉周昌阳
Owner HUIGENE THERAPEUTICS CO LTD
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