A simplified base editing library guide-RNA gene editing tool
By designing guide-RNAs tools that simplify base editing libraries, the problem of difficulty in screening multiple PAM sequences in existing technologies has been solved, achieving efficient screening and simplification of target sequences, and improving the efficiency and accuracy of gene editing.
Patent Information
- Application Number
- CN202310867155.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-14
- Publication Date
- 2025-10-31
- Estimated Expiration
- 2043-07-14
AI Technical Summary
In existing technologies, designing base editing libraries containing multiple protospacer adjacent mode sequences is difficult to simplify and efficient for screening target sequences.
This invention provides a simplified guide-RNA gene editing tool for base editing libraries, which includes an interactive module, a target sequence simplification module, and a display module. Target sequences can be screened by setting priorities and preset simplification rules, and it supports flexible design and user definition of multiple PAM types.
It achieves efficient simplification of base editing libraries, reduces target sequence repetition by approximately 30%, supports the use of multiple gene editors, and improves the efficiency and accuracy of gene editing.
Smart Images
Figure CN116825209B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to gene editing, and more specifically to a guide-RNA gene editing tool that simplifies the base editing library. Background Technology
[0002] Gene editing is a form of genetic engineering that involves replacing, inserting, modifying, or deleting DNA in various genomes; targeted manipulation is a key characteristic of gene editing. Before the advent of common nuclease-based gene editing technology platforms, gene editing technology, which emerged in the 1990s, was limited by its low efficiency. Because precise editing of genes or genomes plays a crucial role in biologists' understanding of gene function within organisms and in advancing modern gene therapy, scientists worldwide have been continuously exploring more advanced gene editing technologies.
[0003] CRISPR / Cas base editors can accurately convert single nucleotides without causing double-strand breaks (DSBs), avoiding genomic damage during gene editing. The development of base editing technology has brought new breakthroughs to gene editing, and gene editing technology is now widely used in gene therapy, gene function analysis, and other fields.
[0004] One of the key challenges in base editing technology is the selection of guide RNA (gRNA). While some base editing tools have been developed to design gRNAs, designing a simplified base editing library containing multiple protospacer adjacent (PAM) sequences for gRNA screening remains a challenge. Summary of the Invention
[0005] (a) Technical problems to be solved
[0006] In view of the above-mentioned shortcomings of the existing technology, the present invention provides a guide-RNA gene editing tool for simplifying base editing libraries, which can effectively overcome the deficiency of the existing technology in that it cannot effectively simplify base editing libraries containing multiple protospacer adjacent motif sequences.
[0007] (II) Technical Solution
[0008] To achieve the above objectives, the present invention provides the following technical solution:
[0009] A simplified base editing library guide-RNA gene editing tool includes an interaction module, a target sequence simplification module, and a display module;
[0010] The interactive module allows users to simultaneously search for target sequences with multiple PAMs and set priorities as needed;
[0011] The target sequence simplification module simplifies the target sequence based on preset simplification rules to reduce the reproducibility of genetic sites in the target sequence;
[0012] The display module shows all the target sequences that meet the requirements on the page, allowing users to select the appropriate target sequence.
[0013] Preferably, the target sequence simplification module simplifies the target sequence based on preset simplification rules, including:
[0014] For a single PAM sequence:
[0015] If multiple target sequences have the same mutation site in the gene sequence, one of the target sequences can be randomly selected and retained in the base editing library;
[0016] If a mutation site in one target sequence contains a mutation site in another target sequence, the target sequence with more mutation sites is selected and retained in the base editing library.
[0017] For multiple PAM sequences:
[0018] If multiple target sequences have the same mutation site in the gene sequence but different PAM types, the target sequence with the highest PAM type priority is selected and retained in the base editing library;
[0019] If a mutation site in one target sequence contains a mutation site in another target sequence, but the PAM types are different, then the PAM type priority is ignored, and the target sequence with more mutation sites is selected and retained in the base editing library.
[0020] Preferably, the interactive module further includes the following functions:
[0021] Users can manually define the editing window according to their selected gene editor type and enter the sequence of the target gene or fragment in the text area according to the selected target genome.
[0022] Preferably, the display module displays all eligible target sequences on the page for the user to select a suitable target sequence, including:
[0023] All eligible target sequences are displayed on the page, and users can select the appropriate target sequence based on GC content, prediction of potential off-target sites, and annotation of codon changes.
[0024] (III) Beneficial Effects
[0025] Compared with the prior art, the simplified base editing library guide-RNAs gene editing tool provided by this invention is a user-friendly tool that can construct efficient and comprehensive gene editing libraries. This tool can simultaneously screen multiple PAM target sequences according to different base editing methods and extract information of all target sequences within the coding region.
[0026] Furthermore, after in-depth comparison with other gene editing tools (including BE-target, CRISPR-BETS, beditor, designer, and PnB design), the technical solution of this application was found to have the following advantages:
[0027] 1) Supports user-defined gene editor types. By modifying the gene editor type and the parameters of the editing window, users can define whether to use one or multiple gene editors simultaneously.
[0028] 2) The selection of PAM is very flexible. Users can design target sequences for multiple PAMs at the same time and set the priority of different PAM types. The target sequence with the highest priority of PAM type can be selected and retained in the base editing library.
[0029] 3) To avoid repetition of the same mutation site on the target sequence, the target sequence designed based on one or more PAMs supports simplification of the target sequence with the same mutation nucleotide at the same site. Through relevant experiments and data analysis, the simplification effect of the target sequence of the base editing library can be achieved by about 30%. Attached Figure Description
[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.
[0031] Figure 1 This is a simplified example diagram of a single PAM sequence in this invention;
[0032] Figure 2 This is a simplified example diagram of multiple PAM sequences in this invention. Detailed Implementation
[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0034] A simplified base editing library guide-RNA gene editing tool includes an interaction module, a target sequence simplification module, and a display module;
[0035] The interactive module allows users to simultaneously search for target sequences with multiple PAMs and set priorities as needed;
[0036] The target sequence simplification module simplifies the target sequence based on preset simplification rules to reduce the reproducibility of genetic sites in the target sequence;
[0037] The display module shows all the target sequences that meet the requirements on the page, allowing users to select the appropriate target sequence.
[0038] ① The target sequence simplification module simplifies the target sequence based on preset simplification rules, including:
[0039] For a single PAM sequence, such as Figure 1 As shown:
[0040] If multiple target sequences have the same mutation site in the gene sequence, one of the target sequences can be randomly selected and retained in the base editing library;
[0041] If a mutation site in one target sequence contains a mutation site in another target sequence, the target sequence with more mutation sites is selected and retained in the base editing library.
[0042] For multiple PAM sequences, such as Figure 2 As shown:
[0043] If multiple target sequences have the same mutation site in the gene sequence but different PAM types, the target sequence with the highest PAM type priority is selected and retained in the base editing library;
[0044] If a mutation site in one target sequence contains a mutation site in another target sequence, but the PAM types are different, then the PAM type priority is ignored, and the target sequence with more mutation sites is selected and retained in the base editing library.
[0045] ②The interactive module also includes the following functions:
[0046] Users can manually define the editing window according to their selected gene editor type and enter the sequence of the target gene or fragment in the text area according to the selected target genome.
[0047] ③ The display module shows all eligible target sequences on the page, allowing users to select the appropriate target sequence, including:
[0048] All eligible target sequences are displayed on the page, and users can select the appropriate target sequence based on GC content, prediction of potential off-target sites, and annotation of codon changes.
[0049] After in-depth comparison with other gene editing tools (including BE-target, CRISPR-BETS, beditor, designer, and PnBdesign), the technical solution of this application has the following advantages:
[0050] 1) Supports user-defined gene editor types. By modifying the gene editor type and the parameters of the editing window, users can define whether to use one or multiple gene editors simultaneously.
[0051] 2) The selection of PAM is very flexible. Users can design target sequences for multiple PAMs at the same time and set the priority of different PAM types. The target sequence with the highest priority of PAM type can be selected and retained in the base editing library.
[0052] 3) To avoid repetition of the same mutation site on the target sequence, the target sequence designed based on one or more PAMs supports simplification of the target sequence with the same mutation nucleotide at the same site. Through relevant experiments and data analysis, the simplification effect of the target sequence of the base editing library can be achieved by about 30%.
[0053] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A simplified base editing library guide-RNA gene editing tool, characterized in that: It includes an interaction module, a target sequence simplification module, and a display module; The interactive module allows users to simultaneously search for target sequences with multiple PAMs and set priorities as needed; The target sequence simplification module simplifies the target sequence based on preset simplification rules to reduce the reproducibility of genetic sites in the target sequence; The display module shows all the target sequences that meet the requirements on the page, allowing users to select the appropriate target sequence. The target sequence simplification module simplifies the target sequence based on preset simplification rules, including: For a single PAM sequence: If multiple target sequences have the same mutation site in the gene sequence, one of the target sequences can be randomly selected and retained in the base editing library; If a mutation site in one target sequence contains a mutation site in another target sequence, the target sequence with more mutation sites is selected and retained in the base editing library. For multiple PAM sequences: If multiple target sequences have the same mutation site in the gene sequence but different PAM types, the target sequence with the highest PAM type priority is selected and retained in the base editing library; If a mutation site in one target sequence contains a mutation site in another target sequence, but the PAM types are different, then the PAM type priority is ignored, and the target sequence with more mutation sites is selected and retained in the base editing library.
2. The guide-RNA gene editing tool for a simplified base editing library according to claim 1, characterized in that: The interactive module also includes the following functions: Users can manually define the editing window according to their selected gene editor type and enter the sequence of the target gene or fragment in the text area according to the selected target genome.
3. The guide-RNA gene editing tool for a simplified base editing library according to claim 2, characterized in that: The display module displays all eligible target sequences on the page, allowing the user to select the appropriate target sequence, including: All eligible target sequences are displayed on the page, and users can select the appropriate target sequence based on GC content, prediction of potential off-target sites, and annotation of codon changes.