阅读说明：本技术 一种新型基因组编辑工具（Lb2Cas12a-RVR）及其构建方法和应用方法 (Novel genome editing tool (Lb2Cas12a-RVR) and construction method and application method thereof ) 是由 谷峰 刘潇宇 林莉 于 2020-01-15 设计创作，主要内容包括：本发明公开了一种新型基因组编辑工具(Lb2Cas12a-RVR)及其构建方法和应用方法,其技术方案要点是包括包括编码表达Lb2Cas12a-RVR突变型蛋白编码质粒pcDNA3.1-hLb2Cas12a-RVR,编码蛋白的DNA序列如SEQ ID NO.1所示,该新型改造的基因组编辑工具及其构建方法和应用方法,以解决在精确位点特异性基因编辑的低活性和低保真性的问题,有助于临床肿瘤治疗靶标的筛选,药物开发,致病突变纠正等进行深入研究,其在人类细胞中具有基因组编辑活性,并且具有更好的的PAM灵活性、高保真度和特殊的切割方式。(The invention discloses a novel genome editing tool (Lb2Cas12a-RVR) and a construction method and an application method thereof, and the technical scheme is characterized by comprising a mutant protein coding plasmid pcDNA3.1-hLb2Cas12a-RVR for coding and expressing Lb2Cas12a-RVR, wherein the DNA sequence of the coding protein is shown as SEQ ID NO. 1.)

1. A novel genome editing tool (Lb2Cas12a-RVR), characterized by: comprises encoding and expressing Lb2Cas12a-RVR mutant protein encoding plasmid pcDNA3.1-hLb2Cas12a-RVR, and the DNA sequence of the encoding protein is shown as SEQ ID NO. 1.

2. A method of construction of the gene editing tool (Lb2Cas12a-RVR) according to claim 1, characterized in that: the method comprises the following steps:

step 21: comprises a primer, wherein the primer is provided with corresponding mutation, and the sequence of the primer is shown as SEQ ID NO. 7;

step 22: amplifying a fragment 1 with a sequence shown as SEQ ID NO.2 and an amplified fragment 2 with a sequence shown as SEQ ID NO.3 from a pY011(pcDNA3.1-hLb2Cpf1) vector;

step 23: the amplified fragment and the backbone were passed through Clon ExpressIIOne Step Cloning Kit to obtain the mutated vector pcDNA3.1-hLb2Cas12 a-RVR.

3. A method of application of a novel genome editing tool (Lb2Cas12a-RVR) according to claim 1, characterized in that: the method comprises the following steps:

step 33: comprises the steps of constructing crRNA aiming at a DNA target point of a certain pathogenic gene, and introducing the crRNA and an Lb2Cas12a-RVR expression vector in a eukaryotic cell together to repair the target site in situ;

step 34: constructing a GFP (green fluorescent protein) reporting system for evaluating the editing activity, efficiency and fidelity of Lb2Cas12a-RVR, wherein the sequence of a GFP gene is SEQ ID NO. 4;

step 35: constructing an Lb2Cas12a-RVR gene repair template RS1-GFP cell line, wherein the sequence of the utilized RS1-GFP gene is SEQ ID NO. 5;

step 36: extracting the processed eukaryotic cell genome DNA, performing PCR amplification by using a specific primer aiming at a target spot, constructing a high-throughput sequencing DNA library by using a PCR product of the genome DNA, and performing high-throughput sequencing.

Technical Field

The invention relates to a novel genome editing tool (Lb2Cas12a-RVR) and a construction method and an application method thereof.

Background

As a novel genome editing tool, the new member of CRISPR family Cas12a has received extensive attention in recent years and has exposed its corners in the field of gene therapy of human cell and animal and plant models. Cas12 a-mediated targeted genome engineering techniques have achieved a wide range of research and medical applications. However, existing studies mostly focus on FnCas12a, assas 12a and LbCas12a, and limited to this, mammalian-related genome editing studies also mostly spread around assas 12a and LbCas12a, however, for broad application in precise site-specific human genome editing, the limited target selection spectrum and low activity, low fidelity would still be bottlenecks in its mediated application. Therefore, there is an urgent need to develop new Cas12a nucleases with improved characteristics for manipulating the human genome.

In addition, the genome of a cell is affected by various endogenous or exogenous factors during the self-replication of the chromosome, and thus various forms of damage exist, thereby initiating a series of cytological reactions. The types of DNA damage are many, with Double Strand Break (DSB) being the most severe. And repair of DNA DSBs is more difficult than other types of DNA damage, with unrepaired repair potentially leading to chromosome breakage and cell death, and mis-repair potentially leading to chromosome deletions, rearrangements, translocations, inversions, and the like. Incomplete repair of DNA damage can lead to genomic instability, and in order to combat damage, body cells evolve multiple repair systems to ensure genomic integrity, with homologous recombination repair (HDR) being the primary means of DNA DSB damage repair. HDR occurs primarily at the S phase of mitosis and at the G2 checkpoint, where DNA DSBs cause broken DNA ends to bind to sister chromatids and repair.

Based on applicants ' previous studies (Tu, M.et al.A ' new release of life ': FnCpf1 stress DNAs cleavage activity for genome editing in human cells.nucleic acids research.2017; 45(19): 11295-. Therefore, the Lb2Cas12a mutant is developed into a more efficient human genome editing tool, the application of an applicant to a gene editor can be greatly expanded, the Lb2Cas12a mutant has extensive application in basic research, clinical treatment, microbial breeding, plant breeding and the like, and a powerful research tool is particularly provided for clinical disease treatment.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a novel modified genome editing tool and a construction method and an application method thereof, and the novel modified genome editing tool and the construction method and the application method thereof are used for solving the problems of low activity and low fidelity of specific gene editing at an accurate site, and are beneficial to the screening of clinical tumor treatment targets, the development of medicines, the correction of pathogenic mutation and the like for deep research. It has genome editing activity in human cells, and has better PAM flexibility, high fidelity, and a special cleavage pattern.

In order to achieve the purpose, the invention provides the following technical scheme: a novel modified genome editing tool comprises a coding expression Lb2Cas12a-RVR mutant protein coding plasmid pcDNA3.1-hLb2Cas12a-RVR, and the DNA sequence of the coding protein is shown as SEQ ID NO. 1.

A method for constructing a gene editing tool comprises the following steps:

step 21: comprises a primer, wherein the primer is provided with corresponding mutation, and the sequence of the primer is shown as SEQ ID NO. 7;

step 22: amplifying a fragment 1 with a sequence shown as SEQ ID NO.2 and an amplified fragment 2 with a sequence shown as SEQ ID NO.3 from a pY011(pcDNA3.1-hLb2Cpf1) vector;

step 23: the amplified fragment and the backbone were subjected to Clonexpress II One Step Cloning Kit to obtain the mutated vector pcDNA3.1-hLb2Cas12 a-RVR.

An application method of an Lb2Cas12a-RVR gene editing tool comprises the following steps:

step 33: comprises the steps of constructing crRNA aiming at a DNA target point of a certain pathogenic gene, and introducing the crRNA and an Lb2Cas12a-RVR expression vector in a eukaryotic cell together to repair the target site in situ;

step 34: constructing a GFP (green fluorescent protein) reporting system for evaluating the editing activity, efficiency and fidelity of Lb2Cas12a-RVR, wherein the sequence of a GFP gene is SEQ ID NO. 4;

step 35: constructing an Lb2Cas12a-RVR gene repair template RS1-GFP cell line, wherein the sequence of the utilized RS1-GFP gene is SEQ ID NO. 5;

step 36: extracting the processed eukaryotic cell genome DNA, performing PCR amplification by using a specific primer aiming at a target spot, constructing a high-throughput sequencing DNA library by using a PCR product of the genome DNA, and performing high-throughput sequencing.

The invention has the beneficial effects that: by passing

Drawings

FIG. 1 is a schematic diagram of pcDNA3.1-hLb2Cas12a-RVR plasmid;

FIG. 2 is a schematic diagram of the pSin-GFP plasmid;

FIG. 3 is a schematic diagram of the pSin-RS1-GFP plasmid;

FIG. 4 shows the result of HDR repair of pcDNA3.1-hLb2Cas12a-RVR in RS1 gene.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. In which like parts are designated by like reference numerals. It should be noted that the terms "front," "back," "left," "right," "upper" and "lower" used in the following description refer to directions in the drawings, and the terms "bottom" and "top," "inner" and "outer" refer to directions toward and away from, respectively, the geometric center of a particular component.

Referring to fig. 1 to 4, the novel genome editing tool for modification in this example includes a plasmid pcdna3.1-hLb2Cas12a-RVR encoding mutant protein expressing Lb2Cas12a-RVR, and the DNA sequence of the encoded protein is shown as seq id No. 1.

The improvement is as follows: SEQ ID NO.1 Lb2Cas12a-RVR gene sequence (artificially synthesized)

atgtactatgagtccctgaccaagcagtaccccgtgtctaagacaatccggaatgagctgatccctatcggcaagacactggataacatccgccagaacaatatcctggagagcgacgtgaagcggaagcagaactacgagcacgtgaagggcatcctggatgagtatcacaagcagctgatcaacgaggccctggacaattgcaccctgccatccctgaagatcgccgccgagatctacctgaagaatcagaaggaggtgtctgacagagaggatttcaacaagacacaggacctgctgaggaaggaggtggtggagaagctgaaggcccacgagaactttaccaagatcggcaagaaggacatcctggatctgctggagaagctgccttccatctctgaggacgattacaatgccctggagagcttccgcaacttttacacctatttcacatcctacaacaaggtgcgggagaatctgtattctgataaggagaagagctccacagtggcctacagactgatcaacgagaatttcccaaagtttctggacaatgtgaagagctataggtttgtgaaaaccgcaggcatcctggcagatggcctgggagaggaggagcaggactccctgttcatcgtggagacattcaacaagaccctgacacaggacggcatcgatacctacaattctcaagtgggcaagatcaactctagcatcaatctgtataaccagaagaatcagaaggccaatggcttcagaaagatccccaagatgaagatgctgtataagcagatcctgtccgatagggaggagtctttcatcgacgagtttcagagcgatgaggtgctgatcgacaacgtggagtcttatggcagcgtgctgatcgagtctctgaagtcctctaaggtgagcgccttctttgatgccctgagagagtctaagggcaagaacgtgtacgtgaagaatgacctggccaagacagccatgagcaacatcgtgttcgagaattggaggacctttgacgatctgctgaaccaggagtacgacctggccaacgagaacaagaagaaggacgataagtatttcgagaagcgccagaaggagctgaagaagaataagagctactccctggagcacctgtgcaacctgtccgaggattcttgtaacctgatcgagaattatatccaccagatctccgacgatatcgagaatatcatcatcaacaatgagacattcctgcgcatcgtgatcaatgagcacgacaggtcccgcaagctggccaagaaccggaaggccgtgaaggccatcaaggactttctggattctatcaaggtgctggagcgggagctgaagctgatcaacagctccggccaggagctggagaaggatctgatcgtgtactctgcccacgaggagctgctggtggagctgaagcaggtggacagcctgtataacatgaccagaaattatctgacaaagaagcctttctctaccgagaaggtgaagctgaactttaatcgcagcacactgctgCGcggctgggatcggaatGTggagacagacCGcctgggcgtgctgctgctgaaggacggcaagtactatctgggcatcatgaacacaagcgccaataaggccttcgtgaatccccctgtggccaagaccgagaaggtgtttaagaaggtggattacaagctgctgccagtgcccaaccagatgctgccaaaggtgttctttgccaagagcaatatcgacttctataacccctctagcgagatctactccaattataagaagggcacccacaagaagggcaatatgttttccctggaggattgtcacaacctgatcgacttctttaaggagtctatcagcaagcacgaggactggagcaagttcggctttaagttcagcgatacagcctcctacaacgacatctccgagttctatcgcgaggtggagaagcagggctacaagctgacctatacagacatcgatgagacatacatcaatgatctgatcgagcggaacgagctgtacctgttccagatctataataaggactttagcatgtactccaagggcaagctgaacctgcacacactgtatttcatgatgctgtttgatcagcgcaatatcgacgacgtggtgtataagctgaacggagaggcagaggtgttctataggccagcctccatctctgaggacgagctgatcatccacaaggccggcgaggagatcaagaacaagaatcctaaccgggccagaaccaaggagacaagcaccttcagctacgacatcgtgaaggataagcggtatagcaaggataagtttaccctgcacatccccatcacaatgaacttcggcgtggatgaggtgaagcggttcaacgacgccgtgaacagcgccatccggatcgatgagaatgtgaacgtgatcggcatcgaccggggcgagagaaatctgctgtacgtggtggtcatcgactctaagggcaacatcctggagcagatctccctgaactctatcatcaataaggagtacgacatcgagacagattatcacgcactgctggatgagagggagggcggcagagataaggcccggaaggactggaacaccgtggagaatatcagggacctgaaggccggctacctgagccaggtggtgaacgtggtggccaagctggtgctgaagtataatgccatcatctgcctggaggacctgaactttggcttcaagaggggccgccagaaggtggagaagcaggtgtaccagaagttcgagaagatgctgatcgataagctgaattacctggtcatcgacaagagccgcgagcagacatcccctaaggagctgggaggcgccctgaacgcactgcagctgacctctaagttcaagagctttaaggagctgggcaagcagtccggcgtgatctactatgtgcctgcctacctgacctctaagatcgatccaaccacaggcttcgccaatctgttttatatgaagtgtgagaacgtggagaagtccaagagattctttgacggctttgatttcatcaggttcaacgccctggagaacgtgttcgagttcggctttgactaccggagcttcacccagagggcctgcggcatcaattccaagtggaccgtgtgcaccaacggcgagcgcatcatcaagtatcggaatccagataagaacaatatgttcgacgagaaggtggtggtggtgaccgatgagatgaagaacctgtttgagcagtacaagatcccctatgaggatggcagaaatgtgaaggacatgatcatcagcaacgaggaggccgagttctaccggagactgtataggctgctgcagcagaccctgcagatgagaaacagcacctccgacggcacaagggattacatcatctcccctgtgaagaataagagagaggcctacttcaacagcgagctgtccgacggctctgtgccaaaggacgccgatgccaacggcgcctacaatatcgccagaaagggcctgtgggtgctggagcagatcaggcagaagagcgagggcgagaagatcaatctggccatgaccaacgccgagtggctggagtatgcccagacacacctgctgaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagggatcctacccatacgatgttccagattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgactatgcctaa

The method for constructing a gene editing tool of the embodiment comprises the following steps:

step 21: comprises a primer, wherein the primer is provided with corresponding mutation, and the sequence of the primer is shown as SEQ ID NO. 7;

step 22: amplifying a fragment 1 with a sequence shown as SEQ ID NO.2 and an amplified fragment 2 with a sequence shown as SEQ ID NO.3 from a pY011(pcDNA3.1-hLb2Cpf1) vector;

step 23: the amplified fragment and the backbone were subjected to Clonexpress II One Step Cloning Kit to obtain the mutated vector pcDNA3.1-hLb2Cas12 a-RVR.

The improvement is as follows: SEQ ID NO.2 amplified fragment 1 sequence (Artificial Synthesis)

atgtactatgagtccctgaccaagcagtaccccgtgtctaagacaatccggaatgagctgatccctatcggcaagacactggataacatccgccagaacaatatcctggagagcgacgtgaagcggaagcagaactacgagcacgtgaagggcatcctggatgagtatcacaagcagctgatcaacgaggccctggacaattgcaccctgccatccctgaagatcgccgccgagatctacctgaagaatcagaaggaggtgtctgacagagaggatttcaacaagacacaggacctgctgaggaaggaggtggtggagaagctgaaggcccacgagaactttaccaagatcggcaagaaggacatcctggatctgctggagaagctgccttccatctctgaggacgattacaatgccctggagagcttccgcaacttttacacctatttcacatcctacaacaaggtgcgggagaatctgtattctgataaggagaagagctccacagtggcctacagactgatcaacgagaatttcccaaagtttctggacaatgtgaagagctataggtttgtgaaaaccgcaggcatcctggcagatggcctgggagaggaggagcaggactccctgttcatcgtggagacattcaacaagaccctgacacaggacggcatcgatacctacaattctcaagtgggcaagatcaactctagcatcaatctgtataaccagaagaatcagaaggccaatggcttcagaaagatccccaagatgaagatgctgtataagcagatcctgtccgatagggaggagtctttcatcgacgagtttcagagcgatgaggtgctgatcgacaacgtggagtcttatggcagcgtgctgatcgagtctctgaagtcctctaaggtgagcgccttctttgatgccctgagagagtctaagggcaagaacgtgtacgtgaagaatgacctggccaagacagccatgagcaacatcgtgttcgagaattggaggacctttgacgatctgctgaaccaggagtacgacctggccaacgagaacaagaagaaggacgataagtatttcgagaagcgccagaaggagctgaagaagaataagagctactccctggagcacctgtgcaacctgtccgaggattcttgtaacctgatcgagaattatatccaccagatctccgacgatatcgagaatatcatcatcaacaatgagacattcctgcgcatcgtgatcaatgagcacgacaggtcccgcaagctggccaagaaccggaaggccgtgaaggccatcaaggactttctggattctatcaaggtgctggagcgggagctgaagctgatcaacagctccggccaggagctggagaaggatctgatcgtgtactctgcccacgaggagctgctggtggagctgaagcaggtggacagcctgtataacatgaccagaaattatctgacaaagaagcctttctctaccgagaaggtgaagctgaactttaatcgcagcacactgctgCGcggctgggatcggaatGTggagacagacCGcctg

SEQ ID NO.3 amplified fragment 2 sequence (Artificial Synthesis)

ggcgtgctgctgctgaaggacggcaagtactatctgggcatcatgaacacaagcgccaataaggccttcgtgaatccccctgtggccaagaccgagaaggtgtttaagaaggtggattacaagctgctgccagtgcccaaccagatgctgccaaaggtgttctttgccaagagcaatatcgacttctataacccctctagcgagatctactccaattataagaagggcacccacaagaagggcaatatgttttccctggaggattgtcacaacctgatcgacttctttaaggagtctatcagcaagcacgaggactggagcaagttcggctttaagttcagcgatacagcctcctacaacgacatctccgagttctatcgcgaggtggagaagcagggctacaagctgacctatacagacatcgatgagacatacatcaatgatctgatcgagcggaacgagctgtacctgttccagatctataataaggactttagcatgtactccaagggcaagctgaacctgcacacactgtatttcatgatgctgtttgatcagcgcaatatcgacgacgtggtgtataagctgaacggagaggcagaggtgttctataggccagcctccatctctgaggacgagctgatcatccacaaggccggcgaggagatcaagaacaagaatcctaaccgggccagaaccaaggagacaagcaccttcagctacgacatcgtgaaggataagcggtatagcaaggataagtttaccctgcacatccccatcacaatgaacttcggcgtggatgaggtgaagcggttcaacgacgccgtgaacagcgccatccggatcgatgagaatgtgaacgtgatcggcatcgaccggggcgagagaaatctgctgtacgtggtggtcatcgactctaagggcaacatcctggagcagatctccctgaactctatcatcaataaggagtacgacatcgagacagattatcacgcactgctggatgagagggagggcggcagagataaggcccggaaggactggaacaccgtggagaatatcagggacctgaaggccggctacctgagccaggtggtgaacgtggtggccaagctggtgctgaagtataatgccatcatctgcctggaggacctgaactttggcttcaagaggggccgccagaaggtggagaagcaggtgtaccagaagttcgagaagatgctgatcgataagctgaattacctggtcatcgacaagagccgcgagcagacatcccctaaggagctgggaggcgccctgaacgcactgcagctgacctctaagttcaagagctttaaggagctgggcaagcagtccggcgtgatctactatgtgcctgcctacctgacctctaagatcgatccaaccacaggcttcgccaatctgttttatatgaagtgtgagaacgtggagaagtccaagagattctttgacggctttgatttcatcaggttcaacgccctggagaacgtgttcgagttcggctttgactaccggagcttcacccagagggcctgcggcatcaattccaagtggaccgtgtgcaccaacggcgagcgcatcatcaagtatcggaatccagataagaacaatatgttcgacgagaaggtggtggtggtgaccgatgagatgaagaacctgtttgagcagtacaagatcccctatgaggatggcagaaatgtgaaggacatgatcatcagcaacgaggaggccgagttctaccggagactgtataggctgctgcagcagaccctgcagatgagaaacagcacctccgacggcacaagggattacatcatctcccctgtgaagaataagagagaggcctacttcaacagcgagctgtccgacggctctgtgccaaaggacgccgatgccaacggcgcctacaatatcgccagaaagggcctgtgggtgctggagcagatcaggcagaagagcgagggcgagaagatcaatctggccatgaccaacgccgagtggctggagtatgcccagacacacctgctgaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagggatcctacccatacgatgttccagattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgactatgcctaa

SEQ ID NO.6 crRNA sequence (Artificial Synthesis)

Primer sequence of SEQ ID NO.7 (Artificial Synthesis)