CN115148281B - Automatic design method and system for gene editing point mutation scheme - Google Patents
Automatic design method and system for gene editing point mutation scheme Download PDFInfo
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Abstract
The application relates to the technical field of gene editing, in particular to an automatic design method and system for a point mutation scheme of gene editing, wherein the method comprises the following steps: determining mutation types and mutation sites according to a positioning method; and (B) step (B): judging whether the mutation site is suitable for point mutation gene Editing by a Base modifying method, if so, executing the step C: designing a Base edition scheme according to mutation types, and screening gRNA sequences; if not, executing the step D: designing a CRISPR and Cas 9-based RNP scheme according to mutation types, screening a gRNA sequence, selecting an oligo sequence and introducing mutation sites; step E: carrying out sequence complexity analysis and GC content calculation on the gene sequences within each range of 500bp before and after the mutation site, and storing analysis results; step F: and generating and outputting a gRNA targeting region schematic diagram and a scheme report according to the analysis result. The method can automatically design the gene editing scheme, realize the point mutation gene editing on the gene and improve the efficiency and the accuracy.
Description
Technical Field
The invention relates to the technical field of gene editing, in particular to an automatic design method and system for a point mutation scheme of gene editing.
Background
In the prior art, CRISPR/Cas9 directs Cas9 endonuclease to cleave PAM upstream of a target sequence by designing a specific guide RNA recognition target sequence, causing target site DNA double strand breaks; after double strand break of DNA, repair is carried out on the cutting site by utilizing a non-homologous end joining (NHEJ) or homologous recombination (HDR) mode of cells, so that gene knockout, knock-in or point mutation at the DNA level is realized. CRISPR/Cas9 is one of the most hot techniques in the field of life science due to the advantages of simple operation, low cost, editing efficiency and the like, and has been widely applied to gene editing related function research of various types of organisms, such as mammals (rats, mice, pigs, rabbits, monkeys and the like), zebra fish, stem cells, tumor cell lines, bacterial fungi and the like.
The design of a CRISPR/Cas 9-based point mutation gene editing scheme seems to be not difficult, but for a gene editing beginner, a few days are required, and the effect is not necessarily ideal; for researchers with genetic editing experience, it may be difficult to obtain an optimal solution because of incomplete information sources and limited human knowledge. Therefore, the manual design is complicated and time-consuming, and the research of a computer intelligent method for automatically designing the gene editing scheme is now a problem to be solved.
Disclosure of Invention
Aiming at the defects, the invention aims to provide an automatic design method and an automatic design system for a point mutation scheme of gene editing, which can comprehensively analyze sequences and gRNA near a target mutation site and automatically design the method for the gene editing scheme, so that the point mutation gene editing on the gene is realized, and the efficiency and the accuracy of a selection scheme are improved.
To achieve the purpose, the invention adopts the following technical scheme:
an automatic design method for a gene editing point mutation scheme comprises the following steps:
step A: determining mutation types and mutation sites according to a localization method, wherein the localization method comprises an Amino Acid method, a SNP method, a Chromosomal Location method and a Nucleotide Sequence method;
and (B) step (B): judging whether the mutation site is suitable for the Base modifying method to carry out point mutation gene Editing, if yes, executing the step C, and if no, executing the step D;
step C: designing a Base modifying scheme according to mutation types, and screening a gRNA sequence, wherein the gRNA sequence needs to contain mutation sites;
step D: designing a CRISPR and Cas 9-based RNP scheme according to mutation types, screening a gRNA sequence, selecting an oligo sequence and introducing mutation sites, wherein the gRNA sequence needs to contain the mutation sites;
step E: carrying out sequence complexity analysis and GC content calculation on the gene sequences within the range of 500bp before and after the mutation site, and storing analysis results;
step F: and generating and outputting a gRNA targeting region schematic diagram and a scheme report according to the analysis result.
Preferably, in step a, the Amino Acid method includes the following steps: selecting a species, inputting a gene, querying an NCBI database and an Ensembl database and acquiring transcripts, gene sequences and coding sequence data of the gene; selecting one transcript and one mutation type, and inputting related parameters to obtain a final mutation site;
the SNP method comprises the following steps: inputting an SNP site number, inquiring an NCBI database to obtain the data of the gene where the site is located, the position of the site on a chromosome and the allele of the SNP site, and calculating the position of the SNP site on the gene through the positions of the gene and the SNP site on the chromosome; selecting one of the allelic bases, and automatically calculating mutation types according to the bases and the allelic bases of the original sequence;
the Chromosomal Location method comprises the following steps: selecting species and genome versions, selecting chromosome numbers, and inquiring a custom database to obtain the gene sequence of the chromosome; selecting one mutation type, inputting parameters of a start position and an end position, inquiring an Ensembl database to obtain a base corresponding to the position segment and a gene containing the position segment, and calculating to obtain the position of the mutation position segment on the gene;
the Nucleotide Sequence method comprises the following steps: selecting a species, inputting a gene, querying an NCBI database and an Ensembl database and acquiring transcripts, gene sequences and coding sequence data of the gene; one of the transcripts is selected, and parameters of a wild type sequence and a mutated sequence are input, and mutation types and mutation sites are automatically calculated according to the difference between the two sequences and the wild type sequence at the positions on the gene.
Preferably, in the Amino Acid method, one mutation type is selected, and relevant parameters are input to obtain final mutation sites, which correspond to the following steps:
when the mutation type is selected as "mutation": inputting parameters of 'amino acid sequence number' and 'mutated amino acid', and reversely calculating according to the amino acid sequence number to obtain the accurate position of the mutation site on the gene;
when the mutation type is selected as "delete": inputting parameters of a start position and an end position, acquiring a sequence to be deleted from a coding sequence according to the two positions, and calculating the accurate position of the deleted sequence fragment on the gene according to the distribution position of the coding region on the gene;
when the mutation type is selected as "insert": inputting parameters of a start position and an inserted sequence, and calculating the position of the inserted sequence on the gene according to the distribution position of the coding region on the gene;
when the mutation type is selected as "delete+insert": the parameters "start position", "end position" and "inserted sequence" are input, the sequence fragment to be deleted is obtained from the coding sequence according to the start position and the end position, and the accurate position of the deleted sequence fragment on the gene is calculated.
Preferably, the step C: judging whether the mutation site is suitable for point mutation gene Editing by a Base modifying method or not, wherein the judgment conditions comprise: a single base is mutated and the base is changed from C to T or G to a.
Preferably, the step D: designing a CRISPR and Cas 9-based RNP scheme according to mutation types, screening gRNA, selecting an oligo sequence and introducing mutation sites, and specifically comprising the following steps:
step D1: screening the gRNA sequence according to mutation types, and grading the screened gRNA;
step D2: selecting gRNA1 and gRNA2 with the highest specificity fraction in the gRNA sequence;
step D3: designing Oligo sequences for gRNA1 and gRNA2, respectively;
step D4: a mutation site is introduced in the Oligo sequence.
Preferably, the step D1: screening the gRNA sequence according to mutation types, and grading the screened gRNA; the mutation types comprise four types of mutation, insertion, deletion and insertion, and the corresponding operation steps are carried out for different mutation types;
when the mutation type is "mutation", the method specifically comprises the following steps:
step a1: screening all gRNA sequences within 50bp ranges at the upstream and downstream of the mutation site, and acquiring information such as specificity score, off-target condition, cutting score and the like of the gRNA sequences through CRISPOR online software;
step a2: screening out gRNA with the specificity score of more than 80, the cutting efficiency of more than 0.5, no off-target and the GC content of 40-60% according to the conditions such as the specificity score, the cutting score, the off-target condition, the GC content and the like of the gRNA;
step a3: grnas were ranked according to distance between mutation site and gRNA cleavage site:
a: n is less than or equal to 5, B:5<N is less than or equal to 10, C:10< N is less than or equal to 23, D: n >23 (A is highest);
step a4: firstly selecting a gRNA with the minimum relative position N from high to low according to the grade as gRNA1, selecting the gRNA with the highest specificity score as gRNA2 if two or more gRNA sequences exist in the same grade, otherwise selecting the gRNA with the minimum relative position N from the next grade as gRNA2;
step a5: designing Oligo sequences for the gRNA1 and the gRNA2 respectively, and taking the end of the cutting site and the end of the mutation site, which is farthest from the cutting site, as the start to extend outwards by 60bp respectively, wherein the sequences in the range are Oligo sequences;
step a6: introducing a mutation site into the Oligo;
step a7: analyzing the complexity and GC content of sequences within 500bp before and after the mutation site;
when the mutation type is "insert, delete, delete+insert", the method specifically comprises the following steps:
step b1: screening all gRNA sequences within 35bp of the upstream and downstream of the deletion/insertion site, and acquiring information such as specificity score, off-target condition, cutting score and the like of the gRNA sequences through online software;
step b2: screening out gRNA with specificity score greater than 80, cutting efficiency greater than 0.5, no off-target and GC content between 40% and 60% according to conditions such as gRNA specificity score, cutting score, off-target condition, GC content and the like;
step b3: grading the grnas according to the relative positions of the gRNA cleavage sites and the editing region:
a: n=0, wherein the cleavage site is inside the editing region, B: n is less than or equal to 5, C:5<N is less than or equal to 10, D:10< N <23;
step b4: if two or more gRNAs exist in the class A, selecting the two gRNAs with the highest specificity score as gRNA1 and gRNA2, otherwise, selecting the two gRNAs with the smallest relative position N from the next class as gRNA1 and gRNA2;
step b5: designing an Oligo: if the cutting position is in the editing region, respectively extending 60bp around the editing region as a center to serve as an Oligo sequence; if the cutting position is not in the editing area, respectively taking the cutting position and the farthest end of the editing area as the starting points to extend outwards by 60bp;
step b6: introducing a mutation site into the Oligo;
step b7: sequences within 500bp of each before and after the mutation site were analyzed for complexity and GC content.
Further describing, the step D4: introducing mutation sites into an Oligo sequence, which specifically comprises the following steps:
step D41: inputting an Oligo sequence, judging whether the PAM structure of the gRNA on the Oligo sequence is changed, if so, outputting the Oligo sequence, and if not, executing the step D42;
step D42: judging whether the gRNA is in the gene editing region, if so, executing a step D43; if not, introducing random mutations in the gRNA: ngg→ngc, and then step D46 is performed;
step D43: introducing synonymous mutation from 3 'end to 5' end of gRNA in turn, judging whether the gRNA is on a PAM structure NGG, if so, executing a step D44, and if not, executing a step D45;
step D44: judging whether the PAM structure is changed, if not, returning to the step D43; if yes, go to step D46;
step D45: judging whether the target mutation point is overlapped with the target mutation point, if so, executing the step D46, otherwise, returning to the step D43; the method comprises the steps of carrying out a first treatment on the surface of the
Step D46: the Oligo sequence was modified and output.
The automatic design system for the gene editing point mutation scheme adopts the steps of the automatic design method for the gene editing point mutation scheme, and comprises a mutation site interpretation module, a judgment module, a first scheme design module, a second scheme design module, a calculation module and a result generation module;
the mutation site reading module is used for determining mutation types and mutation sites according to a positioning method;
the judging module is used for judging whether the mutation site is suitable for point mutation gene Editing by a Base modifying method;
the first scheme design module is used for designing a Base edition scheme and carrying out evaluation and screening of gRNA;
the second scheme design module is used for designing a CRISPR and Cas 9-based RNP scheme, screening gRNA, oligo sequence selection and mutation site introduction;
the calculation module is used for carrying out sequence complexity analysis and GC content calculation on sequences within the range of 500bp before and after the mutation site, and storing analysis results;
the result generation module is used for generating and outputting a gRNA targeting region schematic diagram and a scheme report according to the analysis result.
An electronic device comprising a memory, a processor and a computer program stored on the memory and executable on the processor, the processor implementing the steps of a method for automatically designing a point mutation scheme for gene editing as described above when the program is executed by the processor.
A non-transitory computer readable storage medium having stored thereon a computer program which, when executed by a processor, implements the steps of a method of automatically designing a point mutation scheme for gene editing as described above.
One of the above technical solutions has the following beneficial effects: aiming at the problems of complicated and time-consuming manual design scheme, higher dependence on experience of experiment technicians and the like, the method for comprehensively analyzing sequences and gRNA near a target mutation site and automatically designing a gene editing scheme is provided, so that point mutation gene editing on genes is realized: the method comprises the steps of selecting one of Amino Acid, SNP (single nucleotide polymorphism site), chromosomal Location (chromosome position) and Nucleotide Sequence (nucleotide sequence) of a mutation site, determining mutation types and mutation sites according to different positioning methods, comprehensively analyzing sequences before and after the mutation sites, and designing two types of point mutation schemes which are Base edition and RNP point mutation schemes respectively, wherein the Base edition scheme comprises gRNA evaluation and screening, and the RNP point mutation scheme comprises gRNA screening, oligo selection and mutation site introduction. The point mutation scheme is designed on line to replace a complicated and time-consuming manual scheme, and only parameters of site information are selected, and the optimal scheme can be obtained by gRNA screening and additionally introducing mutation on Oligo, so that a great amount of time is saved for scientific research users.
Drawings
FIG. 1 is a method flow diagram of one embodiment of the present invention;
FIG. 2 is a schematic flow chart of the introduction of mutation sites in Oligo according to the present invention;
FIG. 3 is a graph of output results for one embodiment of the invention;
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.
The following describes a method for automatically designing a point mutation scheme for gene editing according to an embodiment of the present invention with reference to fig. 1 to 2, comprising the steps of:
step A: determining mutation types and mutation sites according to a localization method, wherein the localization method comprises an Amino Acid method, a SNP method, a Chromosomal Location method and a Nucleotide Sequence method;
and (B) step (B): judging whether the mutation site is suitable for the Base modifying method to carry out point mutation gene Editing, if yes, executing the step C, and if no, executing the step D;
step C: designing a Base modifying scheme according to mutation types, and screening a gRNA sequence, wherein the gRNA sequence needs to contain mutation sites;
step D: designing a CRISPR and Cas 9-based RNP scheme according to mutation types, screening a gRNA sequence, selecting an oligo sequence and introducing mutation sites, wherein the gRNA sequence needs to contain the mutation sites;
step E: carrying out sequence complexity analysis and GC content calculation on the gene sequences within the range of 500bp before and after the mutation site, and storing analysis results;
step F: and generating and outputting a gRNA targeting region schematic diagram and a scheme report according to the analysis result.
In the prior art, CRISPR/Cas9 directs Cas9 endonuclease to cleave PAM upstream of a target sequence by designing a specific guide RNA recognition target sequence, causing target site DNA double strand breaks; after double strand break of DNA, repair is carried out on the cutting site by utilizing a non-homologous end joining (NHEJ) or homologous recombination (HDR) mode of cells, so that gene knockout, knock-in or point mutation at the DNA level is realized. CRISPR/Cas9 is one of the most hot techniques in the field of life science due to the advantages of simple operation, low cost, editing efficiency and the like, and has been widely applied to gene editing related function research of various types of organisms, such as mammals (rats, mice, pigs, rabbits, monkeys and the like), zebra fish, stem cells, tumor cell lines, bacterial fungi and the like.
The design of a CRISPR/Cas 9-based point mutation gene editing scheme seems to be not difficult, but for a gene editing beginner, a few days are required, and the effect is not necessarily ideal; for researchers with genetic editing experience, optimal solutions may not be selected because the information sources are not comprehensive.
In the embodiment of the invention, aiming at the problems that the manual design scheme is complicated and time-consuming, the experience of experimental technicians is high, and the like, the invention provides a method capable of comprehensively analyzing sequences and gRNA near a target mutation site and automatically designing a gene editing scheme, so as to realize point mutation gene editing on genes: the method comprises the steps of selecting one of Amino Acid, SNP (single nucleotide polymorphism site), chromosomal Location (chromosome position) and Nucleotide Sequence (nucleotide sequence) of a mutation site, determining mutation types and mutation sites according to different positioning methods, comprehensively analyzing sequences before and after the mutation sites, and designing two types of point mutation schemes which are Base edition and RNP point mutation schemes respectively, wherein the Base edition scheme comprises gRNA evaluation and screening, and the RNP point mutation scheme comprises gRNA screening, oligo selection and mutation site introduction. The point mutation scheme is designed on line to replace a complicated and time-consuming manual scheme, and only parameters of site information are selected, and the optimal scheme can be obtained by gRNA screening and additionally introducing mutation on Oligo, so that a great amount of time is saved for scientific research users.
Still further illustratively, in step A, the Amino Acid method comprises the steps of: selecting a species, inputting a gene, querying an NCBI database and an Ensembl database and acquiring transcripts, gene sequences and coding sequence data of the gene; selecting one transcript and one mutation type, and inputting related parameters to obtain a final mutation site;
the SNP method comprises the following steps: inputting an SNP site number, inquiring an NCBI database to obtain the data of the gene where the site is located, the position of the site on a chromosome and the allele of the SNP site, and calculating the position of the SNP site on the gene through the positions of the gene and the SNP site on the chromosome; selecting one of the allelic bases, and automatically calculating mutation types according to the bases and the allelic bases of the original sequence;
the Chromosomal Location method comprises the following steps: selecting species and genome versions, selecting chromosome numbers, and inquiring a custom database to obtain the gene sequence of the chromosome; selecting one mutation type, inputting parameters of a start position and an end position, inquiring an Ensembl database to obtain a base corresponding to the position segment and a gene containing the position segment, and calculating to obtain the position of the mutation position segment on the gene; it should be noted that, the gene sequence of the chromosome is derived from the NCBI database, and the sequence of one chromosome has hundreds of millions of character strings, so that the sequence is simplified after being processed and stored in a custom database on a server, and the query speed is improved.
The Nucleotide Sequence method comprises the following steps: selecting a species, inputting a gene, querying an NCBI database and an Ensembl database and acquiring transcripts, gene sequences and coding sequence data of the gene; one of the transcripts is selected, and parameters of a wild type sequence and a mutated sequence are input, and mutation types and mutation sites are automatically calculated according to the difference between the two sequences and the wild type sequence at the positions on the gene.
Further describing, in the Amino Acid method, one mutation type is selected, and relevant parameters are input to obtain final mutation sites, which correspond to the following steps:
when the mutation type is selected as "mutation": inputting parameters of 'amino acid sequence number' and 'mutated amino acid', and reversely calculating according to the amino acid sequence number to obtain the accurate position of the mutation site on the gene; for example: the amino acid sequence number is 50, the original amino acid is Ser (sequence AGC) and is mutated into Gly (sequence GGC), namely A-G, and the accurate position of the mutation of A-G on the gene can be obtained according to the position of the 50 th amino acid on the gene;
when the mutation type is selected as "delete": inputting parameters of a start position and an end position, acquiring a sequence to be deleted from a coding sequence according to the two positions, and calculating the accurate position of the deleted sequence fragment on the gene according to the distribution position of the coding region on the gene;
when the mutation type is selected as "insert": inputting parameters of a start position and an inserted sequence, and calculating the position of the inserted sequence on the gene according to the distribution position of the coding region on the gene;
when the mutation type is selected as "delete+insert": the parameters "start position", "end position" and "inserted sequence" are input, the sequence fragment to be deleted is obtained from the coding sequence according to the start position and the end position, and the accurate position of the deleted sequence fragment on the gene is calculated.
Further illustratively, the step C: judging whether the mutation site is suitable for point mutation gene Editing by a Base modifying method or not, wherein the judgment conditions comprise: a single base is mutated and the base is changed from C to T or G to a.
Further illustratively, the step D: designing a CRISPR and Cas 9-based RNP scheme according to mutation types, screening gRNA, selecting an oligo sequence and introducing mutation sites, and specifically comprising the following steps:
step D1: screening the gRNA sequence according to mutation types, and grading the screened gRNA;
step D2: selecting gRNA1 and gRNA2 with the highest specificity fraction in the gRNA sequence;
step D3: designing Oligo sequences for gRNA1 and gRNA2, respectively;
step D4: a mutation site is introduced in the Oligo sequence.
Further describing, the step D1: screening the gRNA sequence according to mutation types, and grading the screened gRNA; the mutation types comprise four types of mutation, insertion, deletion and insertion, and the corresponding operation steps are carried out for different mutation types;
when the mutation type is "mutation", the method specifically comprises the following steps:
step a1: screening all gRNA sequences within 50bp ranges at the upstream and downstream of the mutation site, and acquiring information such as specificity score, off-target condition, cutting score and the like of the gRNA sequences through CRISPOR online software; it should be noted that CRISPOR online software is an existing biological database and online tool;
step a2: screening out gRNA with the specificity score of more than 80, the cutting efficiency of more than 0.5, no off-target and the GC content of 40-60% according to the conditions such as the specificity score, the cutting score, the off-target condition, the GC content and the like of the gRNA;
step a3: grnas were ranked according to distance between mutation site and gRNA cleavage site:
a: n is less than or equal to 5, B:5<N is less than or equal to 10, C:10< N is less than or equal to 23, D: n >23 (A is highest);
step a4: firstly selecting a gRNA with the minimum relative position N from high to low according to the grade as gRNA1, selecting the gRNA with the highest specificity score as gRNA2 if two or more gRNA sequences exist in the same grade, otherwise selecting the gRNA with the minimum relative position N from the next grade as gRNA2;
step a5: designing Oligo sequences for the gRNA1 and the gRNA2 respectively, and taking the end of the cutting site and the end of the mutation site, which is farthest from the cutting site, as the start to extend outwards by 60bp respectively, wherein the sequences in the range are Oligo sequences;
step a6: introducing a mutation site into the Oligo;
step a7: analyzing the complexity and GC content of sequences within 500bp before and after the mutation site;
when the mutation type is "insert, delete, delete+insert", the method specifically comprises the following steps:
step b1: screening all gRNA sequences in the range of 35bp at the upstream and downstream of the deletion/insertion site, and acquiring information such as specificity score, off-target condition, cutting score and the like of the gRNA sequences through online software;
step b2: screening out gRNA with specificity score greater than 80, cutting efficiency greater than 0.5, no off-target and GC content between 40% and 60% according to conditions such as gRNA specificity score, cutting score, off-target condition, GC content and the like;
step b3: grading the grnas according to the relative positions of the gRNA cleavage sites and the editing region:
a: n=0, wherein the cleavage site is inside the editing region, B: n is less than or equal to 5, C:5<N is less than or equal to 10, D:10< N <23;
step b4: if two or more gRNAs exist in the class A, selecting the two gRNAs with the highest specificity score as gRNA1 and gRNA2, otherwise, selecting the two gRNAs with the smallest relative position N from the next class as gRNA1 and gRNA2;
step b5: designing an Oligo: if the cutting position is in the editing region, respectively extending 60bp around the editing region as a center to serve as an Oligo sequence; if the cutting position is not in the editing area, respectively taking the cutting position and the farthest end of the editing area as the starting points to extend outwards by 60bp;
step b6: introducing a mutation site into the Oligo;
step b7: sequences within 500bp of each before and after the mutation site were analyzed for complexity and GC content.
Further describing, the step D4: introducing mutation sites into an Oligo sequence, which specifically comprises the following steps:
step D41: inputting an Oligo sequence, judging whether the PAM structure of the gRNA on the Oligo sequence is changed, if so, outputting the Oligo sequence, and if not, executing the step D42;
step D42: judging whether the gRNA is in the gene editing region, if so, executing a step D43; if not, introducing random mutations in the gRNA: ngg→ngc, and then step D46 is performed;
step D43: introducing synonymous mutation from 3 'end to 5' end of gRNA in turn, judging whether the gRNA is on a PAM structure NGG, if so, executing a step D44, and if not, executing a step D45;
step D44: judging whether the PAM structure is changed, if not, returning to the step D43; if yes, go to step D46;
step D45: judging whether the target mutation point is overlapped with the target mutation point, if so, executing the step D46, otherwise, returning to the step D43; the method comprises the steps of carrying out a first treatment on the surface of the
Step D46: the Oligo sequence was modified and output.
The embodiment also discloses an automatic design system of the gene editing point mutation scheme, which adopts the steps of the automatic design method of the gene editing point mutation scheme, and comprises a mutation site interpretation module, a judgment module, a first scheme design module, a second scheme design module, a calculation module and a result generation module;
the mutation site reading module is used for determining mutation types and mutation sites according to a positioning method;
the judging module is used for judging whether the mutation site is suitable for point mutation gene Editing by a Base modifying method;
the first scheme design module is used for designing a Base edition scheme and carrying out evaluation and screening of gRNA;
the second scheme design module is used for designing a CRISPR and Cas 9-based RNP scheme, screening gRNA, oligo sequence selection and mutation site introduction;
the calculation module is used for carrying out sequence complexity analysis and GC content calculation on sequences within the range of 500bp before and after the mutation site, and storing analysis results;
the result generation module is used for generating and outputting a gRNA targeting region schematic diagram and a scheme report according to the analysis result.
The embodiment also discloses an electronic device, which comprises a memory, a processor and a computer program stored on the memory and capable of running on the processor, wherein the processor realizes the steps of the automatic design method of the gene editing point mutation scheme when executing the program.
The present embodiment also discloses a non-transitory computer readable storage medium having stored thereon a computer program which, when executed by a processor, implements the steps of a method for automatically designing a point mutation scheme for gene editing as described above.
Other configurations, etc. and operations of a method and system for automatically designing a point mutation scheme for gene editing according to an embodiment of the present invention are known to those skilled in the art, and will not be described in detail herein.
All or part of each module in the automatic design system of the gene editing point mutation scheme can be realized by software, hardware and a combination thereof. The above modules may be embedded in hardware or independent of a processor in the electronic device, or may be stored in software in a memory of the electronic device, so that the processor may call and execute operations corresponding to the above modules.
Those skilled in the art will appreciate that implementing all or part of the above-described methods may be accomplished by way of a computer program, which may be stored on a non-transitory computer readable storage medium and which, when executed, may comprise the steps of the above-described embodiments of the methods. Any reference to memory, storage, database, or other medium used in the various embodiments provided herein may include non-volatile and/or volatile memory. The nonvolatile memory can include Read Only Memory (ROM), programmable ROM (PROM), electrically Programmable ROM (EPROM), electrically Erasable Programmable ROM (EEPROM), or flash memory. Volatile memory can include Random Access Memory (RAM) or external cache memory. By way of illustration and not limitation, RAM is available in a variety of forms such as Static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), double Data Rate SDRAM (DDRSDRAM), enhanced SDRAM (ESDRAM), synchronous Link DRAM (SLDRAM), memory bus direct RAM (RDRAM), direct memory bus dynamic RAM (DRDRAM), and memory bus dynamic RAM (RDRAM), among others.
It will be apparent to those skilled in the art that, for convenience and brevity of description, only the above-described division of the functional units and modules is illustrated, and in practical application, the above-described functional distribution may be performed by different functional units and modules according to needs, i.e. the internal structure of the apparatus is divided into different functional units or modules to perform all or part of the above-described functions.
The above description of the specific embodiments of the present invention is merely for illustrating the technical route and features of the present invention, and is intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, but the present invention is not limited to the above-described specific embodiments. All changes or modifications that come within the scope of the appended claims are intended to be embraced therein.
Claims (10)
1. An automatic design method for a gene editing point mutation scheme is characterized by comprising the following steps: the method comprises the following steps:
step A: determining mutation types and mutation sites according to a positioning method, wherein the positioning method comprises an Amino Acid method, a SNP method, a chromosomal positioning method and a NucleotideSequence method;
and (B) step (B): judging whether the mutation site is suitable for the Baseedition method to edit the point mutation gene, if so, executing the step C, and if not, executing the step D;
step C: designing a Baseedition scheme according to mutation types, and screening a gRNA sequence, wherein the gRNA sequence needs to contain mutation sites;
step D: designing a CRISPR and Cas 9-based RNP scheme according to mutation types, screening a gRNA sequence, selecting an oligo sequence and introducing mutation sites, wherein the gRNA sequence needs to contain the mutation sites;
step E: carrying out sequence complexity analysis and GC content calculation on the gene sequences within the range of 500bp before and after the mutation site, and storing analysis results;
step F: and generating and outputting a gRNA targeting region schematic diagram and a scheme report according to the analysis result.
2. The method for automatically designing a point mutation scheme for gene editing according to claim 1, wherein: in step a, the amioacid method includes the steps of: selecting a species, inputting a gene, querying an NCBI database and an Ensembl database and acquiring transcripts, gene sequences and coding sequence data of the gene; selecting one transcript and one mutation type, and inputting related parameters to obtain a final mutation site;
the SNP method comprises the following steps: inputting an SNP site number, inquiring an NCBI database to obtain the data of the gene where the site is located, the position of the site on a chromosome and the allele of the SNP site, and calculating the position of the SNP site on the gene through the positions of the gene and the SNP site on the chromosome; selecting one of the allelic bases, and automatically calculating mutation types according to the bases and the allelic bases of the original sequence;
the ChromosomalLocation method comprises the following steps: selecting species and genome versions, selecting chromosome numbers, and inquiring a custom database to obtain the gene sequence of the chromosome; selecting one mutation type, inputting parameters of a start position and an end position, inquiring an Ensembl database to obtain a base corresponding to the position segment and a gene containing the position segment, and calculating to obtain the position of the mutation position segment on the gene;
the NucleotedSequence method comprises the following steps: selecting a species, inputting a gene, querying an NCBI database and an Ensembl database and acquiring transcripts, gene sequences and coding sequence data of the gene; one of the transcripts is selected, and parameters of a wild type sequence and a mutated sequence are input, and mutation types and mutation sites are automatically calculated according to the difference between the two sequences and the wild type sequence at the positions on the gene.
3. The method for automatically designing a point mutation scheme for gene editing according to claim 2, wherein: in the AminoAcid method, one mutation type is selected, and relevant parameters are input to obtain final mutation sites, which correspond to the following steps:
when the mutation type is selected as "mutation": inputting parameters of 'amino acid sequence number' and 'mutated amino acid', and reversely calculating according to the amino acid sequence number to obtain the accurate position of the mutation site on the gene;
when the mutation type is selected as "delete": inputting parameters of a start position and an end position, acquiring a sequence to be deleted from a coding sequence according to the two positions, and calculating the accurate position of the deleted sequence fragment on the gene according to the distribution position of the coding region on the gene;
when the mutation type is selected as "insert": inputting parameters of a start position and an inserted sequence, and calculating the position of the inserted sequence on the gene according to the distribution position of the coding region on the gene;
when the mutation type is selected as "delete+insert": the parameters "start position", "end position" and "inserted sequence" are input, the sequence fragment to be deleted is obtained from the coding sequence according to the start position and the end position, and the accurate position of the deleted sequence fragment on the gene is calculated.
4. The method for automatically designing a point mutation scheme for gene editing according to claim 1, wherein: the step C: judging whether the mutation site is suitable for point mutation gene editing by a BaseEditing method or not, wherein the judgment conditions comprise: a single base is mutated and the base is changed from C to T or G to a.
5. The method for automatically designing a point mutation scheme for gene editing according to claim 1, wherein: the step D: designing a CRISPR and Cas 9-based RNP scheme according to mutation types, screening gRNA, selecting an oligo sequence and introducing mutation sites, and specifically comprising the following steps:
step D1: screening the gRNA sequence according to mutation types, and grading the screened gRNA;
step D2: selecting gRNA1 and gRNA2 with the highest specificity fraction in the gRNA sequence;
step D3: designing Oligo sequences for gRNA1 and gRNA2, respectively;
step D4: a mutation site is introduced in the Oligo sequence.
6. The method for automatically designing a point mutation scheme for gene editing according to claim 5, wherein: the step D1: screening the gRNA sequence according to mutation types, and grading the screened gRNA; the mutation types comprise four types of mutation, insertion, deletion and insertion, and the corresponding operation steps are carried out for different mutation types;
when the mutation type is "mutation", the method specifically comprises the following steps:
step a1: screening all gRNA sequences within 50bp ranges at the upstream and downstream of the mutation site, and acquiring the specificity score, off-target condition and cutting score information of the gRNA sequences through CRISPOR online software;
step a2: screening out gRNA with the specificity score larger than 80, the cutting efficiency larger than 0.5 and no off-target and the GC content between 40% and 60% according to the specificity score, the cutting score, the off-target condition and the GC content condition of the gRNA;
step a3: grnas were ranked according to distance between mutation site and gRNA cleavage site:
a: n is less than or equal to 5, B:5<N is less than or equal to 10, C:10< N is less than or equal to 23, D: n >23, A being the highest level;
step a4: firstly selecting a gRNA with the minimum relative position N from high to low according to the grade as gRNA1, selecting the gRNA with the highest specificity score as gRNA2 if two or more gRNA sequences exist in the same grade, otherwise selecting the gRNA with the minimum relative position N from the next grade as gRNA2;
step a5: designing Oligo sequences for the gRNA1 and the gRNA2 respectively, and taking the end of the cutting site and the end of the mutation site, which is farthest from the cutting site, as the start to extend outwards by 60bp respectively, wherein the sequences in the range are Oligo sequences;
step a6: introducing a mutation site into the Oligo;
step a7: analyzing the complexity and GC content of sequences within 500bp before and after the mutation site;
when the mutation type is "insert, delete, delete+insert", the method specifically comprises the following steps:
step b1: screening all gRNA sequences in the range of 35bp at the upstream and downstream of the deletion/insertion site, and acquiring the specificity score, off-target condition and cutting score information of the gRNA sequences through online software;
step b2: screening out gRNA with the specificity score larger than 80, the cutting efficiency larger than 0.5 and no off-target and the GC content between 40% and 60% according to the gRNA specificity score, the cutting score, the off-target condition and the GC content condition;
step b3: grading the grnas according to the relative positions of the gRNA cleavage sites and the editing region:
a: n=0, wherein the cleavage site is inside the editing region, B: n is less than or equal to 5, C:5<N is less than or equal to 10, D:10< N <23;
step b4: if two or more gRNAs exist in the class A, selecting the two gRNAs with the highest specificity score as gRNA1 and gRNA2, otherwise, selecting the two gRNAs with the smallest relative position N from the next class as gRNA1 and gRNA2;
step b5: designing an Oligo: if the cutting position is in the editing region, respectively extending 60bp around the editing region as a center to serve as an Oligo sequence; if the cutting position is not in the editing area, respectively taking the cutting position and the farthest end of the editing area as the starting points to extend outwards by 60bp;
step b6: introducing a mutation site into the Oligo;
step b7: sequences within 500bp of each before and after the mutation site were analyzed for complexity and GC content.
7. The method for automatically designing a point mutation scheme for gene editing according to claim 5, wherein: the step D4 is as follows: introducing mutation sites into an Oligo sequence, which specifically comprises the following steps:
step D41: inputting an Oligo sequence, judging whether the PAM structure of the gRNA on the Oligo sequence is changed, if so, outputting the Oligo sequence, and if not, executing the step D42;
step D42: judging whether the gRNA is in the gene editing region, if so, executing a step D43; if not, introducing random mutations in the gRNA: ngg→ngc, and then step D46 is performed;
step D43: introducing synonymous mutation from 3 'end to 5' end of gRNA in turn, judging whether the gRNA is on a PAM structure NGG, if so, executing a step D44, and if not, executing a step D45;
step D44: judging whether the PAM structure is changed, if not, returning to the step D43; if yes, go to step D46;
step D45: judging whether the target mutation point is overlapped with the target mutation point, if not, executing the step D46, otherwise, returning to the step D43;
step D46: the Oligo sequence was modified and output.
8. An automatic design system for a gene editing point mutation scheme is characterized in that: the method for automatically designing a gene editing point mutation scheme according to any one of claims 1 to 7, comprising a mutation site interpretation module, a judgment module, a first scheme design module, a second scheme design module, a calculation module and a result generation module;
the mutation site reading module is used for determining mutation types and mutation sites according to a positioning method;
the judging module is used for judging whether the mutation site is suitable for point mutation gene editing by using a Baseedition method;
the first scheme design module is used for designing a Baseedition scheme and carrying out evaluation and screening of gRNA;
the second scheme design module is used for designing a CRISPR and Cas 9-based RNP scheme, screening gRNA, oligo sequence selection and mutation site introduction;
the calculation module is used for carrying out sequence complexity analysis and GC content calculation on sequences within a range of 500bp before and after the mutation site, and storing analysis results;
the result generation module is used for generating and outputting a gRNA targeting region schematic diagram and a scheme report according to the analysis result.
9. An electronic device comprising a memory, a processor and a computer program stored on the memory and executable on the processor, wherein the processor performs the steps of a method for automatically designing a point mutation scheme for gene editing according to any one of claims 1 to 7 when the program is executed.
10. A non-transitory computer readable storage medium having stored thereon a computer program, wherein the computer program when executed by a processor implements the steps of a method for automatically designing a point mutation scheme for gene editing according to any one of claims 1 to 7.
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