CN117664941A - Method for detecting editing efficiency of adenine base editor and nucleic acid composition - Google Patents
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Abstract
The present application provides a method and nucleic acid composition for detecting the editing efficiency of an adenine base editor. The detection method comprises the following steps: co-transfecting the target cells with an inactivated fluorescent protein expression system, an sgRNA expression system, and an adenine base editor; the sgRNA expression system targets the inactivated fluorescent protein expression system; culturing the transfected target cells, detecting the number of cells expressing the fluorescent protein and the number of cells not expressing the fluorescent protein, and calculating the ratio of the cells expressing the fluorescent protein; and determining the editing efficiency of the adenine base editor according to the cell ratio of the fluorescent protein. The method can realize simultaneous detection of a plurality of samples, and has the advantages of low cost, short period and the like.
Description
Technical Field
The present application relates to the biotechnology field, and in particular to a method and a nucleic acid composition for detecting the editing efficiency of an adenine base editor.
Background
The CRISPR/Cas9 technology utilizes the guide sequences (tracrRNA and crRNA) in RNA double chains to form base pairs with DNA target sequences, DNA double-chain breaks (Double Strand Breaks, DSB) are generated at the target points, and homologous recombination (Homology Directed Repair, HDR) and Non-homologous end joining (Non-Homologous End Joining, NHEJ) repair pathways in cells are induced, so that modification such as site-directed knockout, replacement, insertion and the like of genomic DNA is realized. However, DSB-initiated DNA repair is difficult to achieve with efficient and stable single base mutations; NHEJ is easy to cause random insertion and deletion, and causes frame shift mutation, thereby affecting the function of target genes; HDR, although more accurate than NHEJ, is less efficient in repairing homologous recombination in cells. Among the most recent known human pathogenic mutations, the largest class is the point mutation, also known as Single-nucleotide variation (SNV), and about 2/3 of human diseases occur in association with SNV.
The base editing technology is a novel target gene modification technology developed based on a CRISPR/Cas system, can realize the site-directed mutation of single nucleotide without cutting off a nucleic acid skeleton, and can directly and chemically modify target bases in genome and transcriptome editing processes. Gene editing tools developed by combining CRISPR/Cas systems with a single deaminase can precisely make single base substitutions in DNA or RNA without creating DNA Double Strand Breaks (DSBs) or requiring donor DNA templates in living cells. Compared with other traditional artificial nuclease systems such as CRISPR/Cas9, the base editor is more accurate and safer. Thus, base editors have found wide application in the biomedical field, including gene function studies, directed protein evolution, genetic lineage tracing, disease modeling, and gene therapy. Common base editors include cytosine base editors (Cytosine Base Editor, CBEs) and adenine base editors (Adenine Base editor, ABEs).
Currently, methods for detecting ABEs editing efficiency include first generation sequencing and second generation sequencing. One generation of sequencing can directly see whether base substitution occurs in a target sequence, but only one sample can be tested at a time, and when a large number of samples need to be detected, the adoption of one generation of sequencing is unfavorable for the optimization of a base editor ABEs. The second generation sequencing can test a plurality of samples simultaneously, but has long on-machine time, complex sequence analysis and poor rapid screening effect on ABEs. In addition, the ABEs editing efficiency can be judged by directly establishing a tyrosine albino mouse according to the fur color (whether albino occurs) of the mouse, but the mouse strain has long establishment period and complex operation.
Accordingly, the conventional technology has yet to be improved.
Disclosure of Invention
Based on this, one or more embodiments of the present application provide a method and nucleic acid composition for detecting the editing efficiency of an adenine base editor. The technical proposal comprises:
according to an aspect of the embodiments of the present application, there is provided a method of detecting editing efficiency of an adenine base editor, comprising the steps of:
co-transfecting the target cells with an inactivated fluorescent protein expression system, an sgRNA expression system, and an adenine base editor; the sgRNA expression system targets the inactivated fluorescent protein expression system;
culturing the transfected target cells, detecting the number of cells expressing the fluorescent protein and the number of cells not expressing the fluorescent protein, and calculating the ratio of the cells expressing the fluorescent protein; and
determining the editing efficiency of the adenine base editor according to the cell ratio of the fluorescent protein.
In one embodiment, the inactivated fluorescent protein expression system includes a PAM fragment, a fluorescent protein encoding fragment, and a coding fragment for a stop codon.
In one embodiment, the inactivated fluorescent protein expression system further comprises one or more tryptophan encoding fragments.
In one embodiment, the coding segment of the stop codon is 3n bases apart from the PAM segment, wherein 0.ltoreq.n.ltoreq.6.
In one embodiment, the adenine base editor comprises one or more of pHAGE-To-TadA, -TnpB, pHAGE-To-Nt.bspD6I-TnpB-TadA, and NG-ABE8 e.
In one embodiment, the PAM fragment has a nucleotide sequence of TTGAT, NGG, NG, TTTR, NRN or NYN.
In one embodiment, the inactivated fluorescent protein expression system comprises one or more of an inactivated enhanced green fluorescent protein expression system, an inactivated enhanced yellow fluorescent protein expression system, an inactivated enhanced blue fluorescent protein expression system, an inactivated enhanced cyan fluorescent protein expression system, an inactivated green fluorescent protein expression system, an inactivated red fluorescent protein expression system, an inactivated orange fluorescent protein expression system, an inactivated yellow fluorescent protein expression system, an inactivated blue fluorescent protein expression system, and an inactivated cyan fluorescent protein expression system.
In one embodiment, the primer pair for constructing the inactivated enhanced green fluorescent protein expression system comprises:
a forward primer with a nucleotide sequence shown as SEQ ID NO. 1, and a reverse primer with a nucleotide sequence shown as one or more of SEQ ID NO. 14-SEQ ID NO. 27.
In one embodiment, the primer pair for constructing the sgRNA expression system comprises:
the nucleotide sequences are one or more pairs of primer pairs shown as SEQ ID NO 28-29, SEQ ID NO 30-31, SEQ ID NO 32-33, SEQ ID NO 34-35, SEQ ID NO 6-37, SEQ ID NO 38-39, SEQ ID NO 40-41, SEQ ID NO 42-43, SEQ ID NO 44-45, SEQ ID NO 46-47, SEQ ID NO 48-49, SEQ ID NO 50-51, SEQ ID NO 52-53 and SEQ ID NO 54-55.
According to another aspect of embodiments of the present application, there is provided a nucleic acid composition for detecting editing efficiency of an adenine base editor, comprising an inactivated enhanced green fluorescent protein expression system and an sgRNA expression system, the sgRNA expression system targeting the inactivated fluorescent protein expression system;
alternatively, comprising a primer pair for constructing the inactivated enhanced green fluorescent protein expression system and a primer pair for constructing the sgRNA expression system;
optionally, the primer pair for constructing the inactivated enhanced green fluorescent protein expression system comprises: a forward primer with a nucleotide sequence shown as SEQ ID NO. 1, and a reverse primer with a nucleotide sequence shown as one or more of SEQ ID NO. 14-SEQ ID NO. 27;
optionally, constructing the primer pair of the sgRNA expression system includes: the nucleotide sequences are one or more pairs of primer pairs shown as SEQ ID NO 28-29, SEQ ID NO 30-31, SEQ ID NO 32-33, SEQ ID NO 34-35, SEQ ID NO 6-37, SEQ ID NO 38-39, SEQ ID NO 40-41, SEQ ID NO 42-43, SEQ ID NO 44-45, SEQ ID NO 46-47, SEQ ID NO 48-49, SEQ ID NO 50-51, SEQ ID NO 52-53 and SEQ ID NO 54-55.
Compared with the prior art, the method has the following beneficial effects:
the application provides a rapid and efficient method for detecting the editing efficiency of a base editor. When the base editor edits, the inactivated fluorescent protein expression system can be edited to be re-expressed, and the editing efficiency of the base editor can be intuitively reflected by detecting the cell proportion of the expressed fluorescent protein. In addition, the method can realize simultaneous detection of a plurality of samples, and has the advantages of low cost, short period and the like.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow-through fluorescence statistics for EGFP expression systems with different numbers of tryptophan inserted therein;
FIG. 2 is a flow chart of fluorescence after insertion of PAM sequences into EGFP expression systems;
FIG. 3 is a flow chart of fluorescence after insertion of 2 tryptophan at different positions of an EGFP expression system;
FIG. 4 is a flow-through fluorescence statistics for EGFP expression system with 2 tryptophan inserted at different locations;
FIG. 5 is a flow fluorescent statistical graph of an inactivated EGFP expression system;
FIG. 6 is a statistical graph of editing efficiency of pHAGE-To-TadA-TnpB editing enzyme;
FIG. 7 is a statistical graph of editing efficiency of pHAGE-To-Nt.BspD61-TnpB-TadA editing enzyme.
Detailed Description
The detailed description of the embodiments of the present application will be presented in order to make the above objects, features and advantages of the present application more obvious and understandable. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is, however, susceptible of embodiment in many other forms than those described herein and similar modifications can be made by those skilled in the art without departing from the spirit of the application, and therefore the application is not to be limited to the specific embodiments disclosed below.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, and the like used in the present application are commercially available or may be prepared by existing methods.
Some embodiments of the present application provide a method of detecting editing efficiency of an adenine base editor, comprising steps S10 to S30.
Step S10: co-transfecting the target cells with an inactivated fluorescent protein expression system, an sgRNA expression system, and an adenine base editor; the sgRNA expression system targets the inactivated fluorescent protein expression system;
step S20: culturing the transfected target cells, detecting the number of cells expressing the fluorescent protein and the number of cells not expressing the fluorescent protein, and calculating the ratio of the cells expressing the fluorescent protein; and
step S30: determining the editing efficiency of the adenine base editor according to the cell ratio of the fluorescent protein.
Understandably, when the normal fluorescent protein expression system transfects the target, the fluorescent protein expressing cells account for 100% or about 100%; in the case of an inactivated fluorescent protein expression system, the fluorescent protein-expressing cell ratio is 0 or about 0; after the base editor edits the inactivated fluorescent protein expression system, the cell ratio expressing the fluorescent protein is the editing efficiency of the base editor. Therefore, the editing efficiency of the base editor can be intuitively determined according to the ratio of the fluorescent protein.
In some embodiments, before step S10, the method further includes the following steps:
culturing the target cells to a logarithmic growth phase; and digesting the target cells with pancreatin.
In some of these embodiments, in step S10, the target cell is selected from HEK-293T cells.
In some embodiments, in step S20, the culturing time is 48 to 50 hours.
In some of these embodiments, in step S20, the number of cells expressing the fluorescent protein is detected by one or more of the following methods: flow cytometry, fluorescence microscopy, and confocal microscopy.
In some of these embodiments, the adenine base editor comprises one or more of pHAGE-To-TadA-TnpB, pHAGE-To-Nt.bspD6I-TnpB-TadA, and NG-ABE8 e.
In some of these embodiments, the inactivated fluorescent protein expression system comprises a PAM (protospacer adjacent motif) fragment, a fluorescent protein encoding fragment, and a coding fragment for a stop codon.
Understandably, the coding fragment of tryptophan is TGG and the coding fragment of the stop codon is TAG or TGA. Under the action of the sgRNA expression system and the base editor, A is encoded into G, so that the encoded fragment of the stop codon is changed into the encoded fragment of tryptophan, and the inactivated fluorescent protein expression system is activated to re-express fluorescence.
In some specific examples thereof, the inactivated fluorescent protein expression system further comprises one or more tryptophan encoding fragments.
Optionally, the inactivated fluorescent protein expression system further comprises a tryptophan encoding fragment. The addition of a tryptophan coding segment increases the number of editable sites in the expression system, and does not affect the expression level of the fluorescent protein.
In some specific examples, the PAM fragment has a nucleotide sequence of TTGAT, NGG, NG, TTTR, NRN or NYN.
Understandably, when the adenine base editor is pHAGE-To-TadA-TnpB or pHAGE-To-Nt.bspD6I-TnpB-TadA, the PAM fragment corresponds To TTGAT.
In some of these embodiments, the coding segment of the stop codon is 3n bases apart from the PAM segment, where 0.ltoreq.n.ltoreq.6. Specifically, n may be any positive integer between 0 and 6, and may be, for example, 0, 1, 2, 3, 4, 5, or 6. That is, the fragments of the stop codon may be directly linked to the PAM fragment or may be distributed at intervals.
It will be appreciated that the coding fragments of the stop codon, when ligated in different positions, can be used to test the efficiency of the base editor to edit different mutation sites. Since the sgRNA sequence is usually at most 21bp, the editable base site is at most 21bp away from the PAM, and the coding fragment of the corresponding stop codon is at most 18bp apart from the PAM fragment.
In some embodiments, the inactivated fluorescent protein expression system comprises one or more of an inactivated enhanced green fluorescent protein expression system, an inactivated enhanced yellow fluorescent protein expression system, an inactivated enhanced blue fluorescent protein expression system, an inactivated enhanced cyan fluorescent protein expression system, an inactivated green fluorescent protein expression system, an inactivated red fluorescent protein expression system, an inactivated orange fluorescent protein expression system, an inactivated yellow fluorescent protein expression system, an inactivated blue fluorescent protein expression system, and an inactivated cyan fluorescent protein expression system.
In some specific examples thereof, the primer pair for constructing the inactivated enhanced green fluorescent protein expression system comprises:
a forward primer with a nucleotide sequence shown as SEQ ID NO. 1, and a reverse primer with a nucleotide sequence shown as one or more of SEQ ID NO. 14-SEQ ID NO. 27.
In some of these embodiments, the primer pair for constructing the sgRNA expression system comprises:
the nucleotide sequences are one or more pairs of primer pairs shown as SEQ ID NO 28-29, SEQ ID NO 30-31, SEQ ID NO 32-33, SEQ ID NO 34-35, SEQ ID NO 6-37, SEQ ID NO 38-39, SEQ ID NO 40-41, SEQ ID NO 42-43, SEQ ID NO 44-45, SEQ ID NO 46-47, SEQ ID NO 48-49, SEQ ID NO 50-51, SEQ ID NO 52-53 and SEQ ID NO 54-55.
The method for detecting the editing efficiency of the adenine base editor has the advantages of being rapid, simple, visual and the like.
Some embodiments of the present application also provide a nucleic acid composition for detecting the editing efficiency of an adenine base editor, comprising an inactivated enhanced green fluorescent protein expression system and an sgRNA expression system, the sgRNA expression system targeting the inactivated fluorescent protein expression system;
alternatively, a primer pair for constructing the inactivated enhanced green fluorescent protein expression system and a primer pair for constructing the sgRNA expression system are included.
In some of these embodiments, the primer pair for constructing the inactivated enhanced green fluorescent protein expression system comprises: a forward primer with a nucleotide sequence shown as SEQ ID NO. 1, and a reverse primer with a nucleotide sequence shown as one or more of SEQ ID NO. 14-SEQ ID NO. 27.
In some of these embodiments, the primer pair for constructing the sgRNA expression system comprises: the nucleotide sequences are one or more pairs of primer pairs shown as SEQ ID NO 28-29, SEQ ID NO 30-31, SEQ ID NO 32-33, SEQ ID NO 34-35, SEQ ID NO 6-37, SEQ ID NO 38-39, SEQ ID NO 40-41, SEQ ID NO 42-43, SEQ ID NO 44-45, SEQ ID NO 46-47, SEQ ID NO 48-49, SEQ ID NO 50-51, SEQ ID NO 52-53 and SEQ ID NO 54-55.
The inactivated enhanced green fluorescent protein expression system and the sgRNA expression system can be used for simultaneously detecting the editing efficiency of a base editor for editing one or more different base substitution sites; it can also be used to detect edit windows of the ABE system.
The present application will be further described with reference to specific examples and comparative examples, which should not be construed as limiting the scope of the present application. The materials used in the following examples were all commercially available, unless otherwise specified, the equipment used, and the processes involved, unless otherwise specified, were all routinely selected by those skilled in the art.
Example 1:
(1) Effect of tryptophan insertion amount on EGFP expression level
In order to introduce PAM fragment (TTGAT) and tryptophan-encoding base fragment into the template plasmid, primers as shown in table 1 were designed for PCR amplification, wherein W is an abbreviation for tryptophan and the prefix number represents the number of encoded tryptophan.
TABLE 1
a) First round PCR amplification: pSin-EGFP-ires-puro plasmid (nucleotide sequence shown as SEQ ID NO: 57) was provided as a template, and 0W-F, 1W-F, 2W-F, 3W-F, 4W-F, 5W-F, 6W-F, 7W-F were used as upstream primers, and EGFP-R was used as downstream primer, and 0W-F+R fragment (779 bp), 1W-F+R fragment (782 bp), 2W-F+R fragment (785 bp), 3W-F+R fragment (788 bp), 4W-F+R fragment (791 bp), 5W-F+R fragment (794 bp), 6W-F+R fragment (797 bp) and 7W-F+R fragment (800 bp) were amplified, respectively.
The nucleotide sequence shown in SEQ ID NO. 57 is as follows:
cggaccgccactgccaattacctgtggtttcatttactctaaacctgtgattcctctgaattattttcattttaaagaaattgtatttgttaaatatgtactacaaacttagtagttggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagcagaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataaggtagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaagtgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatatcgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctgggcgggactggggagtggcgagccctcagatcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgccacaaatggcagtattcatccacaattttaaaagaaagggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccactttggtcgataagctttgcaaagatggataaagttttaaacagagaggaatctttgcagctaatggaccttctaggtcttgaaaggagtgggaattggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaggaattcgattaattcgattatcgccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtccggactcagatctcgataaactaatcactagttcgaaggatccgcatgcatctagggcggccaattccgcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgtgattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataagcttgccacaacccacaaggagacgaccttccatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtcccccgggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgacccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgataataggcggccgctcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcaggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggccttgacattataatagatttagcaggaattgaactaggagtggagcacacaggcaaagttctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctgggggcgcgcccctcgaggccgccatggtcatagctgtttgacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgc
the first round PCR amplification system (total 50. Mu.L) included: 1. Mu.L of template plasmid (template plasmid diluted to 100 ng/. Mu.L), 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 25. Mu.L of 2 Xmix (Takara:max DNA Polymerase, R045A) and 22. Mu.L of water.
The reaction procedure for the first round of PCR amplification included: pre-denaturation at 98 ℃ for 5min; denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 15s, and circulation for 34 times; extending at 72 ℃ for 5min, and preserving at 12 ℃.
b) Second round PCR amplification: the products of the first round of PCR amplification are used as templates, GFP1-F is used as an upstream primer, EGFP-R is used as a downstream primer, and GFP1-0W, GFP1-1W, GFP1-2W, GFP1-3W, GFP1-4W, GFP1-5W, GFP1-6W and GFP1-7W fragments with EcoRI enzyme cleavage sites are obtained through amplification respectively.
The second round PCR amplification system (total 50. Mu.L) included: 2. Mu.L of the first round PCR product as a template (the first round product concentration was recovered at 50 ng/. Mu.L), 1. Mu.L of the upstream primer, 1. Mu.L of the downstream primer, 25. Mu.L of 2 Xmix (Takara:max DNA Polymerase, R045A) and 21. Mu.L of water.
The reaction procedure for the second round of PCR amplification included: pre-denaturation at 98 ℃ for 5min; denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 15s, and circulation for 34 times; extending at 72 ℃ for 5min, and preserving at 12 ℃.
c) And (3) enzyme cutting: the products of the second round of PCR amplification and pSin-EGFP-ires-puro vector were digested with EcoRI-HF (NEB: R3101V) and BamHI-HF (NEB: R0136), and the bands were separated by agarose gel electrophoresis, and the target band was recovered using a kit (Omega: D2500-01).
The enzyme digestion system comprises: 2. Mu.L of pSin-EGFP-ires-puro plasmid (500 ng/. Mu.L), 5. Mu.L of 10 XrCutSmart buffer, 1. Mu.L of BamHI, 1. Mu.L of LEcoRI and 41. Mu.L of dH 2 O。
d) Ligation transformation: pSin-EGFP-ires-puro and GFP1-0W to GFP1-7W, transformed, plated (Amp-resistant) and incubated overnight at 37℃after ligation with T4 DNA ligase (NEB: M0202V). The next day, the monoclonal strains are respectively selected for sequencing, plasmids with correct sequencing are amplified and cultured, and internal toxicity is removed, so that pSin-EGFP-ires-puro-0W (hereinafter referred to as 0W) to pSin-EGFP-ires-puro-7W (hereinafter referred to as 7W) are obtained.
The ligation transformation system comprises: mu.L of 10 XT 4 DNA Ligase buffer, 50ng Vector DNA (7.5 kb), 50ng Insert DNA (780 bp), 1. Mu. L T4 DNA Ligase, no nucleic acid water make up to 20. Mu.L.
e) Detecting fluorescence value: HEK-293T cells were cultured to logarithmic growth phase, after pancreatin digestion for 30s, complete medium was added to terminate digestion, followed by uniform blowing with a gun, and cell counting was performed. Spread in 20 wells of 24-well plate 0.9X10 respectively 5 HEK-293T cells (Mock, 0W, 1W, 2W, 3W, 4W, 5W, 6W, 7W, pSin-EGFP-ires-puro as a group, two replicates).
Mock, 0W, 1W, 2W, 3W, 4W, 5W, 6W, 7W, pSin-EGFP-ires-puro were diluted to 100 ng/. Mu.L, respectively, to prepare 50. Mu.L transfection systems: 43.5. Mu.L opti-mem medium, 5. Mu.L plasmid and 1.5. Mu.L Turbolect (Thermo Scientific, R0531). 50 μl of transfection system was added to each well, mixed with HEK-293T cells with gentle shaking, and after 48h incubation, the fluorescence values were detected by flow cytometry, and the detection results are shown in FIG. 1.
As can be seen from FIG. 1, the fluorescence values expressed by the 1W and 2W plasmids are not greatly different, while the fluorescence expressed by the 3W to 7W plasmids is obviously reduced, and 2W is selected as a test template by comprehensively considering the fluorescence expression intensity and the number of editable sites. Wherein 0W is a PAM sequence inserted only, and the fluorescence value of the PAM sequence is basically equivalent to that of pSin-EGFP-ires-puro (EGFP in figure 1), which indicates that the insertion of the PAM sequence does not influence the expression of EGFP; FIG. 2 is a flow chart of 0W and pSin-EGFP-ires-puro plasmids.
(2) Influence of tryptophan insertion position on EGFP expression level
To introduce base fragments encoding 2 tryptophan at different positions of the template plasmid, primers as shown in Table 2 were designed for PCR amplification.
TABLE 2
| Numbering device | Primer name | Sequence (5 '-3') |
| SEQ ID NO:1 | EGFP-R | TGCATGCGGATCCTTCGAACTAG |
| SEQ ID NO:2 | GFP1-F | CGTGAGGGAATTCGATTATCGCCATGGTTGATTGG |
| SEQ ID NO:5 | 2W-F | CGCCATGGTTGATTGGTGGGTGAGCAAGGGCGAGGAG |
| SEQ ID NO:11 | 2W-34-F | CGCCATGTTGATGTGAGCTGGTGGGAGGAGCTG |
| SEQ ID NO:12 | 2W-56-F | CGCCATGTTGATGTGAGCAAGGGCTGGTGGCTGTTCACC |
| SEQ ID NO:13 | 2W-78-F | CGCCATGTTGATGTGAGCAAGGGCGAGGAGTGGTGGACCGGGGTG |
a) First round PCR amplification: the pSin-EGFP-ires-puro plasmid is used as a template, 2W-F, 2W-34-F, 2W-56-F and 2W-78-F are used as a primer, EGFP-R is used as a primer at the downstream, and fragments of 1 st to 2 th codons, 3 rd to 4 th codon positions, 5 th to 6 th codons and 7 th to 8 th codons, which are positioned immediately after the PAM sequence, are respectively amplified to obtain the base encoding 2 tryptophan.
b) Second round PCR amplification: and respectively amplifying the products amplified by the first round of PCR as templates, GFP1-F as an upstream primer and EGFP-R as a downstream primer to obtain fragments with EcoRI enzyme cutting sites.
c) The products of the second round PCR amplification were digested with pSin-EGFP-ires-puro vectors, respectively.
d) Ligation transformation: ligating the cleavage products of step c) to obtain pSin-EGFP-12Trp (base encoding 2 tryptophan at the 1 st and 2 nd codon positions immediately after the PAM sequence), pSin-EGFP-34Trp (base encoding 2 tryptophan at the 3 rd and 4 th codon positions immediately after the PAM sequence), pSin-EGFP-56Trp (base encoding 2 tryptophan at the 5 th and 6 th codon positions immediately after the PAM sequence), pSin-EGFP-78Trp (base encoding 2 tryptophan at the 7 th and 8 th codon positions immediately after the PAM sequence).
The reaction system and the procedure of the first round of PCR amplification, the second round of amplification, the enzyme digestion and the ligation transformation are the same as those of the step (1).
e) Detecting fluorescence value: HEK-293T cells were cultured until the logarithmic phase, after pancreatin digestion for 30s, complete medium was added to terminate digestion, and then the cells were counted by blowing with a gun to uniformity. Lay in 0.9X10 in 14 wells of a 24 well plate 5 HEK-293T cells (Mock, 12Trp, 34Trp, 56Trp, 78Trp, pSin-EGFP-ires-puro) were grouped in duplicate.
The plasmids were diluted to 100 ng/. Mu.L respectively, and a 50. Mu.L transfection system was prepared: 43.5. Mu.L opti-mem medium, 5. Mu.L plasmid and 1.5. Mu.L LTurofect (Thermo Scientific, R0531). 50 mu L of transfection system is added into each hole, the mixture is mixed with HEK-293T cells uniformly by slight shaking, fluorescence values are detected through flow cytometry after culturing for 48 hours, FIG. 3 is a flow type fluorescence graph after different plasmids are transfected, and FIG. 4 is a statistical graph of the proportion of cells expressing EGFP fluorescence after different plasmids are transfected.
As is clear from FIGS. 3 to 4, the insertion position of the base has no significant effect on the EGFP expression level. Thus, inactivated EGFP expression systems can be constructed for different sites for detecting editing efficiency of ABEs.
(3) Detecting editing efficiency of ABEs
a) Construction of an inactivated EGFP expression System
And (3) constructing four inactivated plasmids respectively comprising TAGTGG (A2), TGATGG (A3), TGGTAG (A5) and TGGTGA (A6) fragments by using the pSin-EGFP-12Trp plasmid constructed in the step (2) as a template and adopting a primer pair with a nucleotide sequence shown in a table 3. Similarly, the inactivation plasmids of fragments A9, A11, A12, A14, A15, A17, A18, A20 and A21, respectively comprising A8 (the mutation of the G base into the A base occurs at the 8 th base position after the PAM sequence, the same applies below), A9-EGFP-34 Trp, pSin-EGFP-56Trp and pSin-EGFP-78Trp constructed in the step (2), are constructed and obtained, and the inactivation plasmids are named A2-dEGFP, A3-dEGFP … … A21-dEGFP, respectively. Since the sgRNA sequence is at most 21bp, the inactivating template was constructed to position 21.
TABLE 3 Table 3
| Numbering device | Primer name | Sequence (5 '-3') |
| SEQ ID NO:1 | EGFP-R | TGCATGCGGATCCTTCGAACTAG |
| SEQ ID NO:2 | GFP1-F | CGTGAGGGAATTCGATTATCGCCATGGTTGATTGG |
| SEQ ID NO:14 | A2-2W-F | CGCCATGTTGATTAGTGGGTGAGCAAGGGC |
| SEQ ID NO:15 | A3-2W-F | CGCCATGTTGATTGATGGGTGAGCAAGGGC |
| SEQ ID NO:16 | A5-2W-F | CGCCATGTTGATTGGTAGGTGAGCAAGGGC |
| SEQ ID NO:17 | A6-2W-F | CGCCATGTTGATTGGTGAGTGAGCAAGGGC |
| SEQ ID NO:18 | A8-2W-F | TCGCCATGGTTGATGTGAGCTAGTGGGAGGAGCTGT |
| SEQ ID NO:19 | A9-2W-F | TCGCCATGGTTGATGTGAGCTGATGGGAGGAGCTGT |
| SEQ ID NO:20 | A11-2W-F | TCGCCATGGTTGATGTGAGCTGGTAGGAGGAGCTGT |
| SEQ ID NO:21 | A12-2W-F | TCGCCATGGTTGATGTGAGCTGGTGAGAGGAGCTGT |
| SEQ ID NO:22 | A14-2W-F | TCGCCATGGTTGATGTGAGCAAGGGCTAGTGGCTGTTCACCGG |
| SEQ ID NO:23 | A15-2W-F | TCGCCATGGTTGATGTGAGCAAGGGCTGATGGCTGTTCACCGG |
| SEQ ID NO:24 | A17-2W-F | TCGCCATGGTTGATGTGAGCAAGGGCTGGTAGCTGTTCACCGG |
| SEQ ID NO:25 | A18-2W-F | TCGCCATGGTTGATGTGAGCAAGGGCTGGTGACTGTTCACCGG |
| SEQ ID NO:26 | A20-2W-F | TCGCCATGGTTGATGTGAGCAAGGGCGAGGAGTAGTGGACCGGGGTG |
| SEQ ID NO:27 | A21-2W-F | TCGCCATGGTTGATGTGAGCAAGGGCGAGGAGTGATGGACCGGGGTG |
To determine whether EGFP expression was completely silenced after TGG was changed to TGA or TAG in EGFP coding sequence, HEK-293T cells were transfected with 21 inactivated plasmids constructed, respectively, and the results are shown in FIG. 5: and if part of sites are found to have background leakage, the editing efficiency of the sites is calculated later, and the background leakage data is removed.
b) Construction of sgRNA expression System
Designing primers for constructing corresponding sgRNA expression systems for the 14 EGFP expression systems constructed in the step a), wherein the sequences of the primers are shown in table 4.
The sgRNA F sequence and the sgRNA R sequence were mixed at a ratio of 1:1, placed in a PCR apparatus at 95℃for 5min, and cooled to room temperature at 0.1℃every 8 s. Then the ligation and transformation are carried out on the pDONOR5.1-TnpB-empty vector (the nucleotide sequence of which is shown as SEQ ID NO: 58) after BbsI-HF (NEB: R3539S) enzyme digestion. Monoclonal was picked and colony PCR was performed with U6-F as the upstream primer and sgRNA-R as the downstream primer, with brighter bands selected for sequencing. The sequencing results were all consistent with the expected sequence (500 bp), indicating that the construction of the sgRNA plasmid was successful at each site.
The nucleotide sequence shown in SEQ ID NO. 58 is as follows:
acggatcgggagatctaccggtacgcgttgacgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgattcaagaatcccgaagtgaagaatcttgccgtccgtacatggacttgcccgaactgtggggaaacccatgaccgagacgagaacgctgcgctgaacattcggcgtgaagcgttggtggctgcgggaatctcagacaccttaaacgctcatggaggctatgtcagacctgcttcggcgggcaatggtctgcgaagtgagaatcacgcgactttagtcgtgtgaggttcaacggtcttcgagagagggtgaagacccggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgcttcggcatggcgaatgggacttttttactcgcggccgcttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatccgccaccatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgccgctagcggtgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggtagtggaacgcgtatggtgtctaagggcgaagagctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtggacaaccatcacttcaagtgcacatccgagggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggtggtcgagggcggccctctccccttcgccttcgacatcctggctactagcttcctctacggcagcaagaccttcatcaaccacacccagggcatccccgacttcttcaagcagtccttccctgagggcttcacatgggagagagtcaccacatacgaagatgggggcgtgctgaccgctacccaggacaccagcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaacttcacatccaacggccctgtgatgcagaagaaaacactcggctgggaggccttcaccgagacgctgtaccccgctgacggcggcctggaaggcagaaacgacatggccctgaagctcgtgggcgggagccatctgatcgcaaacgccaagaccacatatagatccaagaaacccgctaagaacctcaagatgcctggcatctactatgtggactacagactggaaagaatcaaggaggccaacaacgaaacctacgtcgagcagcacgaggtggcagtggccagatactgcgacctccctagcaaactggggcacaagcttaatccaaagaaaaagcggaaagtgtgagtcgacctcgagggggggcccggtacctttaagaccaatgacttacaaggcagctgtagatcttagccacatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggaattcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc
TABLE 4 Table 4
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c) Transferring the inactivated EGFP expression system constructed in the step a), the sgRNA expression system constructed in the step b) and the editing enzyme corresponding to the EGFP expression system into HEK-293T cells, and synthesizing Adenine Base Editors (ABEs) in the cells. ABEs edit the A base in the inactivated EGFP expression system to G under the action of sgRNA. The EGFP protein can be expressed by twisting nonsense mutations by editing TAGTG as TGGTGG, TGATGG as TGGTGG, TGGTAG and editing as TGGTGG, TGGTGA as TGGTGG.
The pHAGE-To-TadA-TnpB editase (nucleotide sequence shown as SEQ ID NO: 59) was tested using the EGFP expression system designed for different sites and the sgRNA described above, and the flow type fluorescence statistics are shown in Table 5, and FIG. 6 is a flow type fluorescence statistics diagram after background data removal.
TABLE 5
| Expression system | First group of | Second group of | Expression system | First group of | Second group of |
| Mock | 5.2 | 3.4 | A11 | 6.4 | 7.5 |
| EGFP | 99.2 | 99.6 | A12 | 6.8 | 5.8 |
| A2 | 19.3 | 18.6 | A14 | 2.1 | 2.6 |
| A3 | 6.6 | 8 | A15 | 1 | 1.7 |
| A5 | 10.4 | 9.6 | A17 | 5.5 | 5.8 |
| A6 | 13.6 | 16.1 | A18 | 14.3 | 12.4 |
| A8 | 6.6 | 6 | A20 | 2.2 | 2.2 |
| A9 | 7.1 | 5.4 | A21 | 0.3 | 0.5 |
The pHAGE-To-Nt.BspD61-TnpB-TadA editorial enzyme (nucleotide sequence shown as SEQ ID NO: 60) was tested using the EGFP expression system described above and the sgRNA described above, and the flow fluorescence statistics are shown in Table 6, and FIG. 7 is a flow fluorescence statistics graph after background data removal.
TABLE 6
| Expression system | First group of | Second group of | Expression system | First group of | Second group of |
| Mock | 2.2 | 1.1 | A11 | 22.1 | 27.9 |
| EGFP | 99.2 | 99.8 | A12 | 27.2 | 26.6 |
| A2 | 33.5 | 26.5 | A14 | 7.3 | 9.6 |
| A3 | 27.8 | 24.1 | A15 | 11.9 | 10.9 |
| A5 | 32.4 | 40.7 | A17 | 17.9 | 16.2 |
| A6 | 34.3 | 44.8 | A18 | 25.2 | 30.6 |
| A8 | 28.8 | 33.6 | A20 | 3 | 2.7 |
| A9 | 32.2 | 34.4 | A21 | 3.5 | 3.6 |
The nucleotide sequence shown in SEQ ID NO. 59 is as follows:
tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagcagaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataaggtagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaagtgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatatcgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctgggcgggactggggagtggcgagccctcagatcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgccgaattgacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggactagcgatgctagctgatgcgggccctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctccctatcagtgatagagatctccctatcagtgatagagatcgtcgacgagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctaccggttaaggtgaacgcgtgcaggctggcgccaccatggcccctaaaaagaaacgcaaggtcgaattcataaggaataaggctttcgtggtcaggctgtacccaaatgcggctcagactgaactgattaaccgcacgctgggtagcgcaaggttcgtctacaaccacttccttgcccgtcgcattgcggcctacaaggaaagcgggaagggactgacctacgggcaaacgagtagcgaactgacccttctgaagcaggctgaagaaacctcctggctctcggaagtagataagtttgctttgcagaactcgctgaaaaaccttgagaccgcgtacaagaacttctttcggactgtgaagcagtccggtaaaaaggtaggattcccacgtttcagaaagaagcgcacgggagagtcctaccggactcaattcaccaacaacaacatccaaattggggaaggtaggctcaaacttcctaagctgggatgggtgaaaaccaagggccagcaggatattcaagggaagattctgaatgtcactgtgcgccgtattcacgaaggccattacgaagcgtccgttctctgtgaagtcgagattccctacctgcctgcggctcccaagtttgcagcgggtgtggCtgtcggcatcaaggattttgccatcgtgaccgatggcgtgaggtttaagcatgaacagaatccgaaatattaccgctccaccctgaaaagacttcgtaaagctcagcaaaccctgtccagacggaagaagggcagcgcacgttacgggaaagcgaaaaccaagctggctcggattcacaagcgcattgtcaataagcgtcaggatttccttcacaagctcaccacctccctggtgcgtgagtacgaaatcatcggaaccgaacaccttaaacccgacaacatgcggaaaaatcgccgccttgcactgagcatcagtgatgcgggctggggtgagttcatccggcagttggaatacaaggcagcgtggtacgggcgactggtatctaaagtcagcccctactttccatctagccagttgtgtcatgactgcggattcaagaatcccgaagtgaagaatcttgccgtccgtacatggacttgcccgaactgtggggaaacccatgaccgagacgagaacgctgcgctgaacattcggcgtgaagcgttggtggctgcgggaatctcagacaccttaaacgctcatggaggctatgtcagacctgcttcggcgggcaatggtctgcgaagtgagaatcacgcgactttagtcgtgactagtggctctcccaagaagaagaggaaggtatctgaggtggagttttcccacgagtactggatgagacatgccctgaccctggccaagagggcacgggatgagagggaggtgcctgtgggagccgtgctggtgctgaacaatagagtgatcggcgagggctggaacagagccatcggcctgcacgacccaacagcccatgccgaaattatggccctgagacagggcggcctggtcatgcagaactacagactgattgacgccaccctgtacgtgacattcgagccttgcgtgatgtgcgccggcgccatgatccactctaggatcggccgcgtggtgtttggcgtgaggaactcaaaaagaggcgccgcaggctccctgatgaacgtgctgaactaccccggcatgaatcaccgcgtcgaaattaccgagggaatcctggcagatgaatgtgccgccctgctgtgcgatttctatcggatgcctagacaggtgttcaatgctcagaagaaggcccagagctccatcaactaaggcggccgctctggcatcgatgggagcctcgagtagggtaatctagataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctgagatcctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggcccgggttaattaaggaaagggctagatcattcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagcaagctcatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctccccgtggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacatgattacgaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatcaactggataactcaagctaaccaaaatcatcccaaacttcccaccccataccctattaccactgccaattacctgtggtttcatttactctaaacctgtgattcctctgaattattttcattttaaagaaattgtatttgttaaatatgtactacaaacttagtagt
the nucleotide sequence shown in SEQ ID NO. 60 is as follows:
tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagcagaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataaggtagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaagtgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatatcgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctgggcgggactggggagtggcgagccctcagatcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgccgaattgacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggactagcgatgctagctgatgcgggccctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctccctatcagtgatagagatctccctatcagtgatagagatcgtcgacgagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctaccggttaaggtgaacgcgtgcaggctggcgccaccatggcccctaaaaagaaacgcaaggtccggcagctggaggaggtgattgatctgctggaggtgtaccatgagaagaagaacgtgatcgaggagaagatcaaggcccggtttatcgccaacaagaacaccgtgttcgagtggctgacatggaacggcttcattattctgggcaatgccctcgagtacaagaataatttcgtgatcgatgaagagctgcagcccgtgacacacgctgccggcaaccagcctgatatggagattatctacgaggactttattgtgctgggcgaagtgaccacatcaaagggggccacacagtttaagatggagtctgagccagtcactcgccactacctgaacaaaaagaaggagctggaaaaacagggcgtggagaaggaactgtattgcctgttcatcgcccctgaaatcaacaaaaacacctttgaggagtttatgaagtacaacatcgtgcagaacaccaggatcatccctctgtctctgaagcagttcaacatgctgctgatggtgcagaagaagctgatcgaaaaaggccgcagactgtccagctacgacattaagaacctgatggtgagcctgtaccggaccacaatcgagtgcgaaagaaagtacacccagattaaagccggcctggaggaaaccctgaataactgggtggtggacaaggaggtgagattttctggaggatctagcggaggatcctctggcagcgagacaccaggaacaagcgagtcagcaacaccagagagcagtggcggcagcagcggcggcagcgaattcataaggaataaggctttcgtggtcaggctgtacccaaatgcggctcagactgaactgattaaccgcacgctgggtagcgcaaggttcgtctacaaccacttccttgcccgtcgcattgcggcctacaaggaaagcgggaagggactgacctacgggcaaacgagtagcgaactgacccttctgaagcaggctgaagaaacctcctggctctcggaagtagataagtttgctttgcagaactcgctgaaaaaccttgagaccgcgtacaagaacttctttcggactgtgaagcagtccggtaaaaaggtaggattcccacgtttcagaaagaagcgcacgggagagtcctaccggactcaattcaccaacaacaacatccaaattggggaaggtaggctcaaacttcctaagctgggatgggtgaaaaccaagggccagcaggatattcaagggaagattctgaatgtcactgtgcgccgtattcacgaaggccattacgaagcgtccgttctctgtgaagtcgagattccctacctgcctgcggctcccaagtttgcagcgggtgtggctgtcggcatcaaggattttgccatcgtgaccgatggcgtgaggtttaagcatgaacagaatccgaaatattaccgctccaccctgaaaagacttcgtaaagctcagcaaaccctgtccagacggaagaagggcagcgcacgttacgggaaagcgaaaaccaagctggctcggattcacaagcgcattgtcaataagcgtcaggatttccttcacaagctcaccacctccctggtgcgtgagtacgaaatcatcggaaccgaacaccttaaacccgacaacatgcggaaaaatcgccgccttgcactgagcatcagtgatgcgggctggggtgagttcatccggcagttggaatacaaggcagcgtggtacgggcgactggtatctaaagtcagcccctactttccatctagccagttgtgtcatgactgcggattcaagaatcccgaagtgaagaatcttgccgtccgtacatggacttgcccgaactgtggggaaacccatgaccgagacgagaacgctgcgctgaacattcggcgtgaagcgttggtggctgcgggaatctcagacaccttaaacgctcatggaggctatgtcagacctgcttcggcgggcaatggtctgcgaagtgagaatcacgcgactttagtcgtgactagtggctctcccaagaagaagaggaaggtatctgaggtggagttttcccacgagtactggatgagacatgccctgaccctggccaagagggcacgggatgagagggaggtgcctgtgggagccgtgctggtgctgaacaatagagtgatcggcgagggctggaacagagccatcggcctgcacgacccaacagcccatgccgaaattatggccctgagacagggcggcctggtcatgcagaactacagactgattgacgccaccctgtacgtgacattcgagccttgcgtgatgtgcgccggcgccatgatccactctaggatcggccgcgtggtgtttggcgtgaggaactcaaaaagaggcgccgcaggctccctgatgaacgtgctgaactaccccggcatgaatcaccgcgtcgaaattaccgagggaatcctggcagatgaatgtgccgccctgctgtgcgatttctatcggatgcctagacaggtgttcaatgctcagaagaaggcccagagctccatcaactaaggcggccgctctggcatcgatgggagcctcgagtagggtaatctagataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctgagatcctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggcccgggttaattaaggaaagggctagatcattcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagcaagctcatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctccccgtggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacatgattacgaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatcaactggataactcaagctaaccaaaatcatcccaaacttcccaccccataccctattaccactgccaattacctgtggtttcatttactctaaacctgtgattcctctgaattattttcattttaaagaaattgtatttgttaaatatgtactacaaacttagtagt
the pHAGE-To-Nt.BspD61-TnpB-TadA editing enzyme is improved on the basis of pHAGE-To-TadA-TnpB editing enzyme, and the efficiency of the improved editing enzyme is obviously improved, so that the method can accurately reflect the editing efficiency of a base editor.
The results show that the method can rapidly and efficiently detect the editing efficiency of the base editor and can detect a plurality of samples at the same time.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A method for detecting the editing efficiency of an adenine base editor, comprising the steps of:
co-transfecting the target cells with an inactivated fluorescent protein expression system, an sgRNA expression system, and an adenine base editor; the sgRNA expression system targets the inactivated fluorescent protein expression system;
culturing the transfected target cells, detecting the number of cells expressing the fluorescent protein and the number of cells not expressing the fluorescent protein, and calculating the ratio of the cells expressing the fluorescent protein; and
determining the editing efficiency of the adenine base editor according to the cell ratio of the fluorescent protein.
2. The method of claim 1, wherein the inactivated fluorescent protein expression system comprises PAM fragments, fluorescent protein encoding fragments, and encoding fragments of a stop codon.
3. The method of detecting the editing efficiency of an adenine base editor of claim 2 wherein the inactivated fluorescent protein expression system further comprises one or more coding fragments of tryptophan.
4. The method for detecting the editing efficiency of an adenine base editor according to claim 2, wherein the coding fragment of the stop codon is 3n bases apart from the PAM fragment, wherein 0.ltoreq.n.ltoreq.6.
5. The method of detecting the editing efficiency of an adenine base editor according To any one of claims 1 To 4, wherein the adenine base editor comprises one or more of pHAGE-To-TadA-TnpB, pHAGE-To-Nt.bspD6I-TnpB-TadA and NG-ABE8 e.
6. The method for detecting the editing efficiency of an adenine base editor according to any one of claims 2 to 4, wherein the nucleotide sequence of said PAM fragment is TTGAT, NGG, NG, TTTR, NRN or NYN.
7. The method of detecting editing efficiency of an adenine base editor of any one of claims 1-4, wherein the inactivated fluorescent protein expression system comprises one or more of an inactivated enhanced green fluorescent protein expression system, an inactivated enhanced yellow fluorescent protein expression system, an inactivated enhanced blue fluorescent protein expression system, an inactivated enhanced cyan fluorescent protein expression system, an inactivated green fluorescent protein expression system, an inactivated red fluorescent protein expression system, an inactivated orange fluorescent protein expression system, an inactivated yellow fluorescent protein expression system, an inactivated blue fluorescent protein expression system, and an inactivated cyan fluorescent protein expression system.
8. The method for detecting editing efficiency of an adenine base editor according to claim 7 wherein constructing the primer pair of the inactivated enhanced green fluorescent protein expression system comprises:
a forward primer with a nucleotide sequence shown as SEQ ID NO. 1, and a reverse primer with a nucleotide sequence shown as one or more of SEQ ID NO. 14-SEQ ID NO. 27.
9. The method for detecting editing efficiency of an adenine base editor according to claim 8, wherein constructing the primer pair of the sgRNA expression system comprises:
the nucleotide sequences are one or more pairs of primer pairs shown as SEQ ID NO 28-29, SEQ ID NO 30-31, SEQ ID NO 32-33, SEQ ID NO 34-35, SEQ ID NO 6-37, SEQ ID NO 38-39, SEQ ID NO 40-41, SEQ ID NO 42-43, SEQ ID NO 44-45, SEQ ID NO 46-47, SEQ ID NO 48-49, SEQ ID NO 50-51, SEQ ID NO 52-53 and SEQ ID NO 54-55.
10. A nucleic acid composition for detecting the editing efficiency of an adenine base editor comprising an inactivated enhanced green fluorescent protein expression system and a sgRNA expression system, the sgRNA expression system targeting the inactivated fluorescent protein expression system;
alternatively, comprising a primer pair for constructing the inactivated enhanced green fluorescent protein expression system and a primer pair for constructing the sgRNA expression system;
optionally, the primer pair for constructing the inactivated enhanced green fluorescent protein expression system comprises: a forward primer with a nucleotide sequence shown as SEQ ID NO. 1, and a reverse primer with a nucleotide sequence shown as one or more of SEQ ID NO. 14-SEQ ID NO. 27;
optionally, constructing the primer pair of the sgRNA expression system includes: the nucleotide sequences are one or more pairs of primer pairs shown as SEQ ID NO 28-29, SEQ ID NO 30-31, SEQ ID NO 32-33, SEQ ID NO 34-35, SEQ ID NO 6-37, SEQ ID NO 38-39, SEQ ID NO 40-41, SEQ ID NO 42-43, SEQ ID NO 44-45, SEQ ID NO 46-47, SEQ ID NO 48-49, SEQ ID NO 50-51, SEQ ID NO 52-53 and SEQ ID NO 54-55.
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