WO2024093962A1 - Use of compact editing tool ebcas12a in gene editing - Google Patents
Use of compact editing tool ebcas12a in gene editing Download PDFInfo
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- 238000010362 genome editing Methods 0.000 title claims abstract description 35
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 9
- 108091033409 CRISPR Proteins 0.000 claims description 21
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 claims description 14
- 238000000338 in vitro Methods 0.000 claims description 8
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- AWXGSYPUMWKTBR-UHFFFAOYSA-N 4-carbazol-9-yl-n,n-bis(4-carbazol-9-ylphenyl)aniline Chemical compound C12=CC=CC=C2C2=CC=CC=C2N1C1=CC=C(N(C=2C=CC(=CC=2)N2C3=CC=CC=C3C3=CC=CC=C32)C=2C=CC(=CC=2)N2C3=CC=CC=C3C3=CC=CC=C32)C=C1 AWXGSYPUMWKTBR-UHFFFAOYSA-N 0.000 claims description 3
- 101000837344 Homo sapiens T-cell leukemia translocation-altered gene protein Proteins 0.000 claims description 3
- 102100028692 T-cell leukemia translocation-altered gene protein Human genes 0.000 claims description 3
- 108700004991 Cas12a Proteins 0.000 description 18
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- 125000006850 spacer group Chemical group 0.000 description 4
- 238000010354 CRISPR gene editing Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
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- the present invention belongs to the field of biomedicine, and specifically relates to an application of a class II type V CRISPR protein Cas12a (EbCas12a) from Erysipelotrichia bacterium in gene editing.
- EbCas12a class II type V CRISPR protein Cas12a
- Cas12 also known as Cpf1
- Cpf1 CRISPR system with gene editing effects that has been discovered. It has greatly expanded the editable range of gene editing system targets.
- Cas12a has the function of processing precursor RNA, which provides it with a more convenient and efficient editing ability for mediating multi-gene editing than the Cas9 system.
- the guide RNA of Cas12a is simpler in composition and more convenient in design.
- Zhang Feng's team first discovered another new member with gene editing capabilities besides the Cas9 system, Cas12a, also known as Cpf1, and classified it into the CRISPR system type 2 type V.
- Cas12a also known as Cpf1
- the editing efficiency of Cas12a is comparable to that of Cas9, and is lower than that of Cas9 at some targets.
- the off-target rate of Cas12a is extremely low.
- Cas12a is a safe gene editing tool.
- Cas12a forms sticky ends after cutting, while Cas9 forms flat ends.
- Cas12a has obvious advantages. Only Cas12a itself can complete the processing of precursor RNA, while the Cas9 system requires RNaseIII processing, which greatly promotes the application of Cas12a in multi-gene editing. In terms of PAM recognition, Cas12a recognizes 5’-TTTN-3’ or 5’-KYTV-3’, while Cas9 recognizes 5’-NGG-3’.
- Cas12a As a new gene editing tool, Cas12a, together with the Cas9 system, provides a powerful tool for scientific research and disease treatment. Based on the existing research on Cas12a, it is of great significance to discover a smaller and more compact Cas12a system to cope with gene editing events in various situations in the future.
- the purpose of the present invention is to provide a compact Cas12a editing tool EbCas12a for use in gene editing in view of the deficiencies in the prior art.
- the sequence shown in SED ID NO.2 is the sequence used by EbCas12a in eukaryotic cells.
- the PAM sequence recognized by the above-mentioned EbCas12a is mainly TTTV, and it can also weakly recognize TCTA, TTCA or CTTA, and the V represents A, C, or G.
- the above-mentioned EbCas12a has the ability to cut DNA and can perform gene editing on in vitro DNA and in vivo genomes at a specific site.
- the above-mentioned application of EbCas12a in gene editing includes in vitro gene editing, prokaryotic gene editing, and eukaryotic gene editing.
- prokaryotic gene editing the amino acid sequence of EbCas12a is shown in SED ID NO.1; in eukaryotic gene editing, the amino acid sequence of EbCas12a is shown in SED ID NO.2.
- amino acid sequence of the protein EbCas12a of the present invention is as follows:
- the sequence used in eukaryotic cells adds the KRPAATKKAGQAKKKK sequence (which is the C-terminal NLS nuclear entry sequence) to the C-terminus of the EbCas12a protein amino acid sequence, and then connects the YPYDVPDYAYPYDVPDYAYPYDVPDYA sequence (which is the 3HA sequence) with the GS sequence.
- the present invention identifies for the first time a smaller type II V CRISPR protein EbCas12a (1158AA) with gene editing effect in Erysipelotrichia bacterium strains, which is smaller than all currently reported Cas12a with gene editing function; the EbCas12a can perform gene editing on in vitro DNA and eukaryotic genomes at a fixed point under the mediation of crRNA.
- the discovery of EbCas12a further expands the types of gene editing tools, and also provides an important alternative tool for subsequent gene editing in various different situations, which plays a very important role in basic scientific research and clinical treatment.
- Figure 1 is a diagram of the CRISPR array and crRNA direct repeat of Erysipelotrichia bacteria.
- Figure 2 shows an in vitro cleavage experiment after prokaryotic expression of EbCas12a, where S represents substrate and P represents product.
- Figure 3 is an in vitro experiment verifying the PAM of EbCas12a.
- Figure 4 shows in vivo verification of EbCas12a gene editing.
- the EbCas12a in vitro different time gradient and different PAM cleavage experiments include the following experimental steps:
- EbCas12a protein The EbCas12a gene sequence (as shown below) was synthesized into the pet28a expression vector (with restriction sites of NcoI and XhoI) and a 6His tag at the C-terminus. The synthesized plasmid was then transformed into the E. coli Rosseta 2 (DE3) expression strain, and a single clone was picked. After a small-scale expression test to confirm the protein expression, a large-scale expression and purification of the protein was performed.
- the recombinant protein was purified by Ni column affinity chromatography, heparin column chromatography, and superdex 200 molecular sieve, and then stored in buffer (10 mM Tris-HCl, 200 mM NaCl, 1 mM MgCl) and frozen at -80°C for later use.
- the crRNA targeting the EGFP gene was obtained by in vitro transcription; the EbCas12a protein obtained in step (1) was mixed with the crRNA in a molar ratio of 1:1 to obtain the EbCas12a-crRNA complex.
- the Cas12a-crRNA binary complex can recognize this target and cut the 1.1 kb substrate into products with lengths of 0.4 kb and 0.7 kb, respectively.) Mix well, incubate at 37°C for 0, 2.5, 5, 8, 10, and 15 min, add appropriate amount of proteinase K, digest at 58°C for 60 min, and run on 2% agarose gel.
- PAMs PAM sequences are shown in Figure 2, the substrates are the same as above, only the four bases at PAM are different) were selected to carry out the above-mentioned cleavage experiments. The results are shown in Figure 2, and EbCas12a has good in vitro cleavage ability.
- EbCas12a can recognize different PAMs: TTTV, TCTA, TTCA, CTTA, but the optimal PAM is TTTV (V represents A, C or G).
- EbCas12a eukaryotic expression plasmid The EbCas12a gene sequence used in eukaryotic cells was constructed into the eukaryotic expression plasmid pcDNA3.1.
- the EbCas12a gene sequence used in eukaryotic cells is as follows (SEQ ID NO.4): ATGCAAGTCCAGAACCTGTACACCATCAACAAGACCCTGCGCTTCGGCCTGAAGCCCTTCGGCAAGACCCTGGAGAACTTCAACAAGACCAACCTGCTGCAGCTGGACGAGTACAAGGCCAAGCACCGCAAGGAGGTGCAGCGCCTGTTCGACGAGAACTTCAAGCAGCTGATCGAGGAGCGCCTGCGCGCGGCCTGAGCCTGGACACCCAGGCCCTGGAGGAGGCCTTCGACATCAACAAGCGCGACGCCGCCCTGATCAGCCTGAAGAAGCAGGTGACCGGCATCTGCTACGACAGCGAAATGAAGAAGACCTACCTGCAGGCCGACAAGCACTTCCAGAAGCTG
- the sequence is the C-terminal nuclear import sequence NLS, The sequence is GS link, The sequence is 3HA tag.
- AATTTCTACTGTTGTAGAT CACATAGGCCATTCAGAAAC targeting TAX1BP3
- EbCas12a eukaryotic expression plasmid 700 ng
- U6-crRNA spacer eukaryotic expression plasmid 300 ng
- PCR was performed using the surveyor primer designed in step (3) and the PCR product was purified.
- PCR product 300 ng was mixed with 1 ⁇ L 10 ⁇ T7EI buffer, and renatured according to the following PCR program: 95°C for 10 min, 95°C to 85°C -2°C/S, 85°C to 25°C -0.25°C/S, and 25°C for 1 min. After renaturation, 1 ⁇ L T7EI was added to the product, and enzyme digestion was performed at 37°C for 20 min. The product was run on a 2% agarose gel.
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Abstract
Provided is use of a compact editing tool EbCas12a in gene editing. The EbCas12a has an amino acid sequence set forth in SED ID NO. 1 or 2.
Description
本发明属于生物医学领域，具体涉及一种来自于Erysipelotrichia bacterium细菌中的II类V型CRISPR蛋白Cas12a(EbCas12a)在基因编辑中的应用。The present invention belongs to the field of biomedicine, and specifically relates to an application of a class II type V CRISPR protein Cas12a (EbCas12a) from Erysipelotrichia bacterium in gene editing.
自2013年以来，基因编辑技术取得了突破性进展，此项技术已经在基础科学研究、医药、临床、生物技术等许多领域引起了新的变革。除了具有代表性的Cas9系统之外，Cas12，又名Cpf1，作为又一种被发现的具有基因编辑效应的CRISPR系统新成员，极大的扩大了基因编辑系统靶点的可编辑范围，相比于Cas9系统，Cas12a所具有的加工前体RNA的功能，为其介导多基因编辑提供了相比于Cas9系统更为便捷高效的编辑能力。除此之外，相比于Cas9的向导RNA，Cas12a的向导RNA组成更为简单，设计更为方便。Since 2013, gene editing technology has made breakthrough progress. This technology has brought new changes in many fields such as basic scientific research, medicine, clinical, and biotechnology. In addition to the representative Cas9 system, Cas12, also known as Cpf1, is another new member of the CRISPR system with gene editing effects that has been discovered. It has greatly expanded the editable range of gene editing system targets. Compared with the Cas9 system, Cas12a has the function of processing precursor RNA, which provides it with a more convenient and efficient editing ability for mediating multi-gene editing than the Cas9 system. In addition, compared with the guide RNA of Cas9, the guide RNA of Cas12a is simpler in composition and more convenient in design.
2015年，张峰团队首次发现了Cas9系统之外的另外一种具有基因编辑能力的新成员，Cas12a，又名Cpf1，将其划分到CRISPR系统2类V型中。相比于Cas9系统，Cas12a的编辑效率与Cas9的效率相当，在有些靶点低于Cas9。Cas12a的脱靶率极低，相比于Cas9脱靶率高的特性，Cas12a是一种安全的基因编辑工具。Cas12a在切割之后形成粘性末端，而Cas9形成平末端，已有研究表明，Cas12a切割之后的粘性末端相比于Cas9的平末端而言，更容易发生同源重组修复，这也为基因的定点插入和修复提供了更好的工具。在向导RNA的加工方面，Cas12a具有明显的优势，仅仅只需要Cas12a本身就能够完成对前体RNA的加工，而Cas9系统则需要RNaseIII的加工，这极大地促进Cas12a在多基因编辑上的应用。在PAM的识别上，Cas12a识别5’-TTTN-3’或5’-KYTV-3’，Cas9则识别5’-NGG-3’。In 2015, Zhang Feng's team first discovered another new member with gene editing capabilities besides the Cas9 system, Cas12a, also known as Cpf1, and classified it into the CRISPR system type 2 type V. Compared with the Cas9 system, the editing efficiency of Cas12a is comparable to that of Cas9, and is lower than that of Cas9 at some targets. The off-target rate of Cas12a is extremely low. Compared with the high off-target rate of Cas9, Cas12a is a safe gene editing tool. Cas12a forms sticky ends after cutting, while Cas9 forms flat ends. Studies have shown that the sticky ends after Cas12a cutting are more prone to homologous recombination repair than the flat ends of Cas9, which also provides a better tool for site-specific insertion and repair of genes. In terms of guide RNA processing, Cas12a has obvious advantages. Only Cas12a itself can complete the processing of precursor RNA, while the Cas9 system requires RNaseIII processing, which greatly promotes the application of Cas12a in multi-gene editing. In terms of PAM recognition, Cas12a recognizes 5’-TTTN-3’ or 5’-KYTV-3’, while Cas9 recognizes 5’-NGG-3’.
因此，Cas12a作为一种新型基因编辑工具，与Cas9系统一道，为科学研究和疾病的治疗提供了有力的工具。基于对目前已有的Cas12a的研究，为应对将来各种情况下的基因编辑事件，发现更小更紧凑的Cas12a系统是一件具有重要意义的事情。Therefore, as a new gene editing tool, Cas12a, together with the Cas9 system, provides a powerful tool for scientific research and disease treatment. Based on the existing research on Cas12a, it is of great significance to discover a smaller and more compact Cas12a system to cope with gene editing events in various situations in the future.
发明内容Summary of the invention
本发明的目的在于针对现有技术的不足，提供一种紧凑型Cas12a编辑工具EbCas12a在基因编辑中的应用。The purpose of the present invention is to provide a compact Cas12a editing tool EbCas12a for use in gene editing in view of the deficiencies in the prior art.
为实现上述发明目的，本发明所采用的技术方案为：In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:
一种来自于Erysipelotrichia bacterium细菌中的II类V型CRISPR蛋白EbCas12a，其氨
基酸序列如SED ID NO.1或2所示。其中，SED ID NO.2所示序列为EbCas12a在真核细胞中所用序列。A class II type V CRISPR protein EbCas12a from Erysipelotrichia bacterium, its amino The amino acid sequence is shown in SED ID NO.1 or 2. Among them, the sequence shown in SED ID NO.2 is the sequence used by EbCas12a in eukaryotic cells.
上述EbCas12a识别的PAM序列主要为TTTV，也能弱识别TCTA、TTCA或CTTA，所述V表示A、C、或G。The PAM sequence recognized by the above-mentioned EbCas12a is mainly TTTV, and it can also weakly recognize TCTA, TTCA or CTTA, and the V represents A, C, or G.
上述EbCas12a具有DNA切割能力，能够定点对体外DNA和体内基因组进行基因编辑。The above-mentioned EbCas12a has the ability to cut DNA and can perform gene editing on in vitro DNA and in vivo genomes at a specific site.
上述EbCas12a在基因剪辑中的应用。所述的基因编辑包括体外基因编辑、原核生物基因编辑、真核生物基因编辑。在原核生物基因剪辑中，EbCas12a的氨基酸序列如SED ID NO.1所示；在真核生物基因剪辑中，EbCas12a的氨基酸序列如SED ID NO.2所示。The above-mentioned application of EbCas12a in gene editing. The gene editing includes in vitro gene editing, prokaryotic gene editing, and eukaryotic gene editing. In prokaryotic gene editing, the amino acid sequence of EbCas12a is shown in SED ID NO.1; in eukaryotic gene editing, the amino acid sequence of EbCas12a is shown in SED ID NO.2.
本发明所述蛋白EbCas12a的氨基酸序列如下：The amino acid sequence of the protein EbCas12a of the present invention is as follows:
EbCas12a自身的蛋白序列(SEQ ID NO.1)：
The protein sequence of EbCas12a itself (SEQ ID NO.1):
The protein sequence of EbCas12a itself (SEQ ID NO.1):
EbCas12a在真核细胞中所用序列(SEQ ID NO.2)：
Sequence used by EbCas12a in eukaryotic cells (SEQ ID NO.2):
Sequence used by EbCas12a in eukaryotic cells (SEQ ID NO.2):
真核细胞中所用序列在EbCas12a蛋白氨基酸序列的C端加入KRPAATKKAGQAKKKK序列(该序列为C端NLS入核序列)，随后用GS序列连接YPYDVPDYAYPYDVPDYAYPYDVPDYA序列(该序列为3HA序列)。The sequence used in eukaryotic cells adds the KRPAATKKAGQAKKKK sequence (which is the C-terminal NLS nuclear entry sequence) to the C-terminus of the EbCas12a protein amino acid sequence, and then connects the YPYDVPDYAYPYDVPDYAYPYDVPDYA sequence (which is the 3HA sequence) with the GS sequence.
本发明的有益效果：本发明首次在Erysipelotrichia bacterium菌株中鉴定出具有基因编辑效应的II类V型更小的CRISPR蛋白EbCas12a(1158AA)，比目前报道的具有基因编辑功能的Cas12a均要小；所述EbCas12a能够在crRNA的介导下定点对体外DNA和真核生物基因组进行基因编辑。EbCas12a的发现进一步扩大了基因编辑工具的种类，同时也为后续各种不同情况的基因编辑提供了重要的备选工具，对基础科研和临床治疗具有十分重要的作用。Beneficial effects of the present invention: The present invention identifies for the first time a smaller type II V CRISPR protein EbCas12a (1158AA) with gene editing effect in Erysipelotrichia bacterium strains, which is smaller than all currently reported Cas12a with gene editing function; the EbCas12a can perform gene editing on in vitro DNA and eukaryotic genomes at a fixed point under the mediation of crRNA. The discovery of EbCas12a further expands the types of gene editing tools, and also provides an important alternative tool for subsequent gene editing in various different situations, which plays a very important role in basic scientific research and clinical treatment.
图1为Erysipelotrichia bacterium菌株CRISPR array及crRNA direct repeat图示。Figure 1 is a diagram of the CRISPR array and crRNA direct repeat of Erysipelotrichia bacteria.
图2为原核表达EbCas12a之后，体外切割实验，S表示substrate，底物；P表示product，产物。Figure 2 shows an in vitro cleavage experiment after prokaryotic expression of EbCas12a, where S represents substrate and P represents product.
图3为体外实验验证EbCas12a的PAM。Figure 3 is an in vitro experiment verifying the PAM of EbCas12a.
图4为体内验证EbCas12a基因编辑。Figure 4 shows in vivo verification of EbCas12a gene editing.
为了更好地理解本发明，下面结合实施例进一步阐明本发明的内容，但本发明的内容不仅仅局限于下面的实施例。In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.
实施例1Example 1
EbCas12a体外不同时间梯度及不同PAM切割实验，包括以下实验步骤：The EbCas12a in vitro different time gradient and different PAM cleavage experiments include the following experimental steps:
(1)EbCas12a蛋白的表达与纯化：将EbCas12a基因序列(如下)合成到pet28a表达载体上(酶切位点为NcoI和XhoI)，在C末端带有6His标签，随后将合成好的质粒转化到E.Coli Rosseta 2(DE3)表达菌株中，挑取单克隆，小量表达检测确定蛋白表达后进行蛋白的大量表达与纯化；重组蛋白依次经过Ni柱亲和层析，heparin柱层析，superdex 200分子筛纯化后，保存在buffer(10mM Tris-HCl，200mM NaCl，1mM MgCl)中，并冻存于-80℃备用。(1) Expression and purification of EbCas12a protein: The EbCas12a gene sequence (as shown below) was synthesized into the pet28a expression vector (with restriction sites of NcoI and XhoI) and a 6His tag at the C-terminus. The synthesized plasmid was then transformed into the E. coli Rosseta 2 (DE3) expression strain, and a single clone was picked. After a small-scale expression test to confirm the protein expression, a large-scale expression and purification of the protein was performed. The recombinant protein was purified by Ni column affinity chromatography, heparin column chromatography, and superdex 200 molecular sieve, and then stored in buffer (10 mM Tris-HCl, 200 mM NaCl, 1 mM MgCl) and frozen at -80°C for later use.
EbCas12a的基因序列(SEQ ID NO.3)：
Gene sequence of EbCas12a (SEQ ID NO.3):
Gene sequence of EbCas12a (SEQ ID NO.3):
(2)使用crRNA direct repeat序列为：5’-AATTTCTACTGTTGTAGAT-3’，在体外转录获得靶向EGFP基因的crRNA；将步骤(1)所得EbCas12a蛋白与crRNA按摩尔比1:1混合得到EbCas12a-crRNA复合物。(2) Using the crRNA direct repeat sequence of 5'-AATTTCTACTGTTGTAGAT-3', the crRNA targeting the EGFP gene was obtained by in vitro transcription; the EbCas12a protein obtained in step (1) was mixed with the crRNA in a molar ratio of 1:1 to obtain the EbCas12a-crRNA complex.
(3)取100nM EbCas12a-crRNA复合物与300ng线性化的底物(PAM为TTTA，片段是以ptriEx-EGFP质粒(将EGFP基因以ptriEx载体为骨架构建)作为模板，设计上下游引物进行PCR，扩增出长度为1.1kb的底物片段。该片段上有一段PAM序列为TTTA，spacer为CGTCGCCGTCCAGCTCGACCAGG的靶点序列，Cas12a-crRNA二元复合物能识别这一靶点并将长度为1.1kb的底物切割为长度分别是0.4kb和0.7kb的产物。)混匀，37℃孵育分别孵育0、2.5、5、8、10、15min后，加入适量蛋白酶K，58℃消化60min，跑2％琼脂糖胶。另选取一些不同的PAM(PAM序列见图2，底物同上述一样，只有PAM处四个碱基不同)分别进行上述切割实验，结果如图2所示，EbCas12a具有良好的体外切割能力。(3) Take 100 nM EbCas12a-crRNA complex and 300 ng linearized substrate (PAM is TTTA, the fragment is based on ptriEx-EGFP plasmid (the EGFP gene is constructed with ptriEx vector as the backbone) as a template, design upstream and downstream primers for PCR, and amplify a substrate fragment with a length of 1.1 kb. There is a PAM sequence of TTTA and a spacer target sequence of CGTCGCCGTCCAGCTCGACCAGG on the fragment. The Cas12a-crRNA binary complex can recognize this target and cut the 1.1 kb substrate into products with lengths of 0.4 kb and 0.7 kb, respectively.) Mix well, incubate at 37°C for 0, 2.5, 5, 8, 10, and 15 min, add appropriate amount of proteinase K, digest at 58°C for 60 min, and run on 2% agarose gel. Some different PAMs (PAM sequences are shown in Figure 2, the substrates are the same as above, only the four bases at PAM are different) were selected to carry out the above-mentioned cleavage experiments. The results are shown in Figure 2, and EbCas12a has good in vitro cleavage ability.
实施例2Example 2
EbCas12a识别PAM的确定Determination of PAM recognition by EbCas12a
(1)设计NNNN四个位置随机组合的上下游引物(N表示A、G、C、T)，以ptriEx-EGFP质粒作为模板，采用overlap PCR方法进行PCR，得到256种带有不同PAM序列，但spacer序列一样的300bp的线性化底物。(1) Upstream and downstream primers with random combinations of the four positions NNNN (N represents A, G, C, and T) were designed, and the ptriEx-EGFP plasmid was used as a template. PCR was performed using the overlap PCR method to obtain 256 300-bp linearized substrates with different PAM sequences but the same spacer sequence.
(2)取100nM实施例1中的EbCas12a-crRNA复合物与300ng线性化的底物混匀，37℃分别孵育10min后，PCR扩增未切割的底物进行二代测序，结果如图3所示，EbCas12a能够识别不同的PAM：TTTV、TCTA、TTCA、CTTA，但最优PAM为TTTV(V表示A、C或G)。(2) 100 nM of the EbCas12a-crRNA complex in Example 1 was mixed with 300 ng of the linearized substrate, and the uncut substrate was amplified by PCR for second-generation sequencing. The results are shown in Figure 3. EbCas12a can recognize different PAMs: TTTV, TCTA, TTCA, CTTA, but the optimal PAM is TTTV (V represents A, C or G).
实施例3Example 3
EbCas12a在哺乳动物细胞内不同基因的编辑：Editing of different genes by EbCas12a in mammalian cells:
(1)构建EbCas12a真核表达质粒：将真核细胞中用的EbCas12a基因序列构建到真核表达质粒pcDNA3.1上，真核细胞中用的EbCas12a基因序列如下(SEQ ID NO.4)：ATGAGCAAGTTCCAGAACCTGTACACCATCAACAAGACCCTGCGCTTCGGCCTGAAGCCCTTCGGCAAGACCCTGGAGAACTTCAACAAGACCAACCTGCTGCAGCTGGACGAGTACAAGGCCAAGCACCGCAAGGAGGTGCAGCGCCTGTTCGACGAGAACTTCAAGCAGCTGATCGAGGAGCGCCTGCGCGGCCTGAGCCTGGACACCCAGGCCCTGGAGGAGGCCTTCGACATCAACAAGCGCGACGCCGCCCTGATCAGCCTGAAGAAGCAGGTGACCGGCATCTGCTACGACAGCGAGATGAAGAAGACCTACCTGCAGGCCGACAAGCACTTCCAGAAGCTGCTGGCC
GCCGGCCCCAACCAGGCCATGGTGTGCACCTACGACAAGTTCAGCACCTACTTCGTGAACTTCTTCGACATCCGCACCCACATCTTCAAGGGCGACACCAGCGGCAGCATCGCCTACCGCCTGATCGACGAGAACCTGACCATCTTCAAGAAGAACGTGGACAAGATCGCCAAGCTGCCCGTGGGCCTGAAGGACGAGGTGGAGGAGCTGGCCGACATCGAGAGCCTGCAGAGCTACAACAGCTACCTGACCCAGAGCGGCATCACCGAGTACAACGAGCTGCTGGGCGGCATCGCCTACGAGGACGGCACCAAGCTGCAGGGCATCAACGAGAAGATCAACCTGTACGGCCAGAAGAACAAGCTGAAGCTGCCCCGCCTGGAGAGCCTGTACAAGATGATCCTGAGCGACCGCGAGACCCAGAGCTTCGTGCTGAGCATCATCGAGAACGACGCCGAGCTGATCGGCCAGATCAGCACCCTGCTGGAGGACGTGCTGCTGAGCAAGACCCTGGCCCTGAGCGACGTGGACGGCGTGTTCATCAAGTACACCCAGCTGGGCAACCTGCCCGGCGTGCCCTACACCGTGATCAACAGCAAGATCAACGAGGCCTTCGACGCCACCTACACCGGCAAGAAGGAGGGCGAGAAGTACAAGGTGACCAAGAAGAAGACCATCGAGAAGGACGTGTACAGCCTGAGCAAGATCGAGAAGCTGTTCCAGGACAGCGACATCAACGTGAGCGAGGCCCTGAAGAACAAGTACGGCGTGCTGATCGCCAGCTACGAGGAGGCCAAGGGCCTGTTCAACAGCATCGACTGGACCGAGATCAAGAACATCAAGCAGAGCGGCCACACCATCATCATCAAGGACGTGCTGGACGCCCTGAAGAACATCCAGTTCTTCTACAACCTGTTCGACGTGGTGGAGGAGAACCTGAACCCCAGCATCGAGTTCTACAACGAGCTGAGCCTGAACAAGAACCAGCTGGGCAACGAGTTCAACAGCACCTACAACAAGGCCCGCAACTACCTGACCAAGAAGGAGTACAGCGAGGAGAAGTTCAAGCTGAACTTCGACAGCCCCACCCTGGCCGACGGCTGGGACGTGAACAAGGAGACCGCCAACCTGACCATCCTGCTGCGCCGCTTCAACAGCGAGCGCAACAACTACGACTACTTCCTGGGCGTGTGGAAGAAGGCCGTGCCCAGCCGCGAGAAGAACCTGATCATCAACGCCGACGGCGAGTTCGAGAAGATGGACTACAAGCTGTACCCCGACCCCAGCAAGATGCTGCCCAAGCAGTTCGTGAGCGCCCAGAGCTGGTTCGACAAGTACCCCGCCAGCCCCGAGTTCATGGGCAAGTACGAGGCCGGCCTGCACAAGAAGGGCAACAACTTCGACATCGAGTTCCTGCACGAGCTGATCAACCGCTACAAGCACGGCCTGAAGCACCACGAGAACAAGTACGAGGAGACCTTCGACTTCGAGCTGAAGGAGACCGAGGAGTACAGCGAGTACAGCGAGTTCATCCAGGACGTGAGCAAGAGCAACTACAAGGTGAAGTTCAACCACGTGGCCGGCGTGGAGGAGCTGGTGGAGGAGGGCAAGCTGTACCTGTTCCAGATCTGGAGCAAGGACTTCAGCACCTTCAGCAAGGGCACCAAGAACCTGAACACCATCTACTTCGAGAGCCTGTTCAGCGAGGAGAACCTGGAGAAGCGCATCTTCAAGCTGAGCGGCGGCGCCGAGCTGTTCTACCGCCCCAAGAGCCTGACCTACACCAAGGAGCTGATGGAGAAGGGCCACCACTACAACGAGCTGAAGGACAGCTTCAACTACCCCATCATCAAGGACAAGCGCTACACCGAGGACAAGTTCATGTTCCACGTGCCCATCCAGATCAACTACGGCGCCGAGAACCTGGGCCCCGTGAAGCTGAACAACCGCATCAACGAGAACATCGACGGCTTCACCCACATCATCGGCATCGACCGCGGCGAGCGCCACCTGGTGTACATCAGCGTGGTGGAC
GTGAAGACCGGCAAGATCGTGGAGCAGAAGCACCTGGACGAGATCGTGAACATCGACAGCAAGGGCAAGAAGCACTGCACCCCCTACCTGCAGAAGCTGGACGAGCGCAGCAAGACCCGCGACCAGGAGCGCAAGAGCTGGGAGGCCATCGAGACCATCAAGGAGCTGAAGGACGGCTACATCAGCCAGGTGGTGAACGAGATCTGCACCCTGCAGCAGAAGTACAACGCCCTGATCGTGATGGAGAACCTGAACCTGGGCTTCAAGCGCAGCCGCTTCAAGGTGGAGAAGCAGATCTACCAGAAGTTCGAGACCGCCCTGATCAAGAAGTTCAACTACATCATCGACAAGAAGGACAACAGCACCTACCTGCACGGCCTGCAGCTGGCCAACCCCATCCAGACCCTGAACAGCATCGGCAAGCAGAGCGGCATCATCTTCTACATCCCCGCCTGGAACACCAGCAAGATCGACCCCACCACCGGCTTCGTGAACCTGCTGTACGGCGCCGACCTGCGCTACACCAACAAGGAGCAGGCCGAGGCCTTCATCAACAAGCTGGACAAGATCTACTTCGAGGACGGCGTGTTCAAGTTCGACATCGACTTCAAGAAGTGGAACCAGCGCTACGCCAAGAGCTGCACCAAGTGGACCCTGACCAGCTACGGCACCCGCGTGGAGACCAAGCGCGACGTGATCAAGAACAACATGTGGTGCAGCAACGAGATCGACCTGACCGCCGAGTTCGAGAAGATCCTGAACAAGCGCGACGGCAGCCTGAAGACCTGCGACGTGGAGACCTACAAGCGCTTCCTGTACCTGTTCAAGCTGCTGCTGCAGATCCGCAACAGCATCACCGGCACCGACACCGACTACATGATCAGCCCCGTGATCGCCGCCGACGGCCAGCAGTTCGACAGCCGCGTGGTGGGCATGAGCCTGCCCAACGGCCTGCCCAAGGACGCCGACGCCAACGGCGCCTACAACATCGCCCGCAAGGGCCTGATGGCCGTGCACAACATCAAGGCCGGCTTCAAGAAGCCCTTCGAGATCAGCAACGAGGAGTACCTGGAGTACCTGCAGAAGAAAAGGCCGGCGGCCACG
AAAAAGGCCGGCCAGGCAAAAAAGAAAAAG
TAA。(1) Construction of EbCas12a eukaryotic expression plasmid: The EbCas12a gene sequence used in eukaryotic cells was constructed into the eukaryotic expression plasmid pcDNA3.1. The EbCas12a gene sequence used in eukaryotic cells is as follows (SEQ ID NO.4): ATGCAAGTCCAGAACCTGTACACCATCAACAAGACCCTGCGCTTCGGCCTGAAGCCCTTCGGCAAGACCCTGGAGAACTTCAACAAGACCAACCTGCTGCAGCTGGACGAGTACAAGGCCAAGCACCGCAAGGAGGTGCAGCGCCTGTTCGACGAGAACTTCAAGCAGCTGATCGAGGAGCGCCTGCGCGGCCTGAGCCTGGACACCCAGGCCCTGGAGGAGGCCTTCGACATCAACAAGCGCGACGCCGCCCTGATCAGCCTGAAGAAGCAGGTGACCGGCATCTGCTACGACAGCGAGATGAAGAAGACCTACCTGCAGGCCGACAAGCACTTCCAGAAGCTGCTGGCC GCCGGCCCCAACCAGGCCATGGTGTGCACCTACGACAAGTTCAGCACCTACTTCGTGAACTTCTTCGACATCCGCACCCACATCTTCAAGGGCGACACCAGCGGCAGCATCGCCTACCGCCTGATCGACGAGAACCTGACCATCTTCAAGAAGAACGTGGACAAGATCGCCAAGCTGCCCGTGGGCCTGAAGGACGAGGTGGAGGAGCTGGCCGACATCGAGAGCCTGCAGAGCTACAACAGCTACCTGACCCA GAGCGGCATCACCGAGTACAACGAGCTGCTGGGCGGCATCGCCTACGAGGACGGCACCAAGCTGCAGGGCATCAACGAGAAGATCAACCTGTACGGCCAGAAGAACAAGCTGAAGCTGCCCCGCCTGGAGAGCCTGTACAAGATGATCCTGAGCGACCGCGAGACCCAGAGCTTCGTGCTGAGCATCATCGAGAACGACGCCGAGCTGATCGGCCAGATCAGCACCCTGCTGGAGGACGTGCTGCTGAGCAAGA CCCTGGCCCTGAGCGACGTGGACGGCGTGTTCATCAAGTACACCCAGCTGGGCAACCTGCCCGGCGTGCCCTACACCGTGATCAACAGCAAGATCAACGAGGCCTTCGACGCCACCTACACCGGCAAGAAGGAGGGCGAGAAGTACAAGGTGACCAAGAAGAAGACCATCGAGAAGGACGTGTACAGCCTGAGCAAGATCGAGAAGCTGTTCCAGGACAGCGACATCAACGTGAGCGAGGCCCTGAAGAACAAG TACGGCGTGCTGATCGCCAGCTACGAGGAGGCCAAGGGCCTGTTCAACAGCATCGACTGGACCGAGATCAAGAACATCAAGCAGAGCGGCCACACCATCATCATCAAGGACGTGCTGGACGCCCTGAAGAACATCCAGTTCTTCTACAACCTGTTCGACGTGGTGGAGGAGAACCTGAACCCCAGCATCGAGTTCTACAACGAGCTGAGCCTGAACAAGAACCAGCTGGGCAACGAGTTCAACAGCACCTACAAC AAGGCCCGCAACTACCTGACCAAGAAGGAGTACAGCGAGGAGAAGTTCAAGCTGAACTTCGACAGCCCCACCCTGGCCGACGGCTGGGACGTGAACAAGGAGACCGCCAACCTGACCATCCTGCTGCGCCGCTTCAACAGCGAGCGCAACAACTACGACTACTTCCTGGGCGTGTGGAAGAAGGCCGTGCCCAGCCGCGAGAAGAACCTGATCATCAACGCCGACGGCGAGTTCGAGAAGATGGACTACAAGCT GTACCCCGACCCCAGCAAGATGCTGCCCAAGCAGTTCGTGAGCGCCCAGAGCTGGTTCGACAAGTACCCCGCCAGCCCCGAGTTCATGGGCAAGTACGAGGCCGGCCTGCACAAGAAGGGCAACAACTTCGACATCGAGTTCCTGCACGAGCTGATCAACCGCTACAAGCACGGCCTGAAGCACCACGAGAACAAGTACGAGGAGACCTTCGACTTCGAGCTGAAGGAGACCGAGGAGTACAGCGAGTACAGCG AGTTCATCCAGGACGTGAGCAAGAGCAACTACAAGGTGAAGTTCAACCACGTGGCCGGCGTGGAGGAGCTGGTGGAGGAGGGCAAGCTGTACCTGTTCCAGATCTGGAGCAAGGACTTCAGCACCTTCAGCAAGGGCACCAAGAACCTGAACACCATCTACTTCGAGAGCCTGTTCAGCGAGGAGAACCTGGAGAAGCGCATCTTCAAGCTGAGCGGCGGCGCCGAGCTGTTCTACCGCCCCAAGAGCCTGACC TACACCAAGGAGCTGATGGAGAAGGGCCACCACTACAACGAGCTGAAGGACAGCTTCAACTACCCCATCATCAAGGACAAGCGCTACACCGAGGACAAGTTCATGTTCCACGTGCCCATCCAGATCAACTACGGCGCCGAGAACCTGGGCCCCGTGAAGCTGAACAACCGCATCAACGAGAACATCGACGGCTTCACCCACATCATCGGCATCGACCGCGGCGAGCGCCACCTGGTGTACATCAGCGTGGTGGAC AAAAGGCCGGCGGCCACG AAAAAGGCCGGCCAGGCAAAAAAGAAAAAG TAA.
标的序列为C端入核序列NLS，标的序列为GS link，标的序列为3HA tag。Standard The sequence is the C-terminal nuclear import sequence NLS, The sequence is GS link, The sequence is 3HA tag.
(2)在哺乳动物细胞中，以293T细胞为例选择CLIC-4、VEGFA-2、PD1、DNMT1-4、TRAC、TRBC、TAX1BP3七个基因靶点，分别以这七个基因为目标，构建7个由U6启动子启动转录的U6-crRNA spacer真核表达质粒(载体骨架为pU6-As-crRNA，Addgene：#78956)。七个基因靶点的crRNA转录序列(其中AATTTCTACTGTTGTAGAT为direct repeat)分别如下：(2) In mammalian cells, seven gene targets, CLIC-4, VEGFA-2, PD1, DNMT1-4, TRAC, TRBC, and TAX1BP3, were selected using 293T cells as an example. Seven U6-crRNA spacer eukaryotic expression plasmids (the vector backbone is pU6-As-crRNA, Addgene: #78956) transcribed by the U6 promoter were constructed with these seven genes as targets. The crRNA transcription sequences of the seven gene targets (where AATTTCTACTGTTGTAGAT is a direct repeat) are as follows:
AATTTCTACTGTTGTAGATCCCTGGCTACCTCCCCTACC(靶向CLIC-4)， AATTTCTACTGTTGTAGAT CCCTGGCTACCTCCCCTACC (targeting CLIC-4),
AATTTCTACTGTTGTAGATGGAGGTCAGAAATAGGGGGTCCA(靶向VEGFA-2)， AATTTCTACTGTTGTAGAT GGAGGTCAGAAATAGGGGGTCCA (targeting VEGFA-2),
AATTTCTACTGTTGTAGATGCACGAAGCTCTCCGATGTGTTG(靶向PD1)， AATTTCTACTGTTGTAGAT GCACGAAGCTCTCCGATGTGTTG (targeting PD1),
AATTTCTACTGTTGTAGATGCTCAGCAGGCACCTGCCTCAGC(靶向DNMT1-4)，
AATTTCTACTGTTGTAGAT GCTCAGCAGGCACCTGCCTCAGC (targeting DNMT1-4),
AATTTCTACTGTTGTAGATTTGCTCCAGGCCACAGCACTGTT(靶向TRAC)， AATTTCTACTGTTGTAGAT TTGCTCCAGGCCACAGCACTGTT (targeted TRAC),
AATTTCTACTGTTGTAGATAGCCATCAGAAGCAGAGATCTCC(靶向TRBC)， AATTTCTACTGTTGTAGAT AGCCATCAGAAGCAGAGATCTCC (targeting TRBC),
AATTTCTACTGTTGTAGATCACATAGGCCATTCAGAAAC(靶向TAX1BP3)。 AATTTCTACTGTTGTAGAT CACATAGGCCATTCAGAAAC (targeting TAX1BP3).
(3)分别针对这七个基因的切割靶点附近设计surveyor primer，并验证PCR引物的特异性。(3) Surveyor primers were designed near the cutting target sites of these seven genes and the specificity of PCR primers was verified.
(4)消化293T细胞，适当浓度铺24孔板，每孔500μL。(4) Digest 293T cells and plate them in a 24-well plate at an appropriate concentration, with 500 μL per well.
(5)24孔板共转EbCas12a真核表达质粒(700ng)和U6-crRNA spacer真核表达质粒(300ng)，48h后裂解细胞，取1μL裂解液作为模板、以步骤(3)设计的surveyor primer引物进行PCR，纯化PCR产物。(5) EbCas12a eukaryotic expression plasmid (700 ng) and U6-crRNA spacer eukaryotic expression plasmid (300 ng) were co-transfected in a 24-well plate. After 48 hours, the cells were lysed and 1 μL of the lysate was used as a template. PCR was performed using the surveyor primer designed in step (3) and the PCR product was purified.
(6)取300ng PCR产物与1μL 10×T7EI buffer混匀，按以下PCR程序进行复性95℃10min，95℃至85℃-2℃/S，85℃到25℃-0.25℃/S，25℃持续1min，复性之后产物加入1μL T7EI，37℃酶切20min，跑2％琼脂糖胶，结果如图4所示在CLIC-4、VEGFA-2、PD1、DNMT1-4、TRAC、TRBC、TAX1BP3能够进行基因编辑，编辑效率分别为9％、17％、32％、16％、8％、7％、6％(本实施例中的七个基因只是作为代表进行列举)。
(6) 300 ng of PCR product was mixed with 1 μL 10×T7EI buffer, and renatured according to the following PCR program: 95°C for 10 min, 95°C to 85°C -2°C/S, 85°C to 25°C -0.25°C/S, and 25°C for 1 min. After renaturation, 1 μL T7EI was added to the product, and enzyme digestion was performed at 37°C for 20 min. The product was run on a 2% agarose gel. As shown in FIG4 , gene editing was performed in CLIC-4, VEGFA-2, PD1, DNMT1-4, TRAC, TRBC, and TAX1BP3, and the editing efficiencies were 9%, 17%, 32%, 16%, 8%, 7%, and 6%, respectively (the seven genes in this example are listed as representatives).
Claims (5)
- 一种II类V型CRISPR蛋白EbCas12a在基因编辑中的应用，其特征在于：所述的EbCas12a的氨基酸序列如SEQ ID NO.1或2所示。An application of a class II type V CRISPR protein EbCas12a in gene editing, characterized in that the amino acid sequence of the EbCas12a is as shown in SEQ ID NO.1 or 2.
- 根据权利要求1所述的应用，其特征在于：EbCas12a识别的PAM序列为TTTV、TCTA、TTCA或CTTA，其中V表示A、C或G。The use according to claim 1, characterized in that the PAM sequence recognized by EbCas12a is TTTV, TCTA, TTCA or CTTA, wherein V represents A, C or G.
- 根据权利要求1所述的应用，其特征在于：所述的基因编辑包括体外基因剪辑、原核生物基因编辑、真核生物基因编辑。The use according to claim 1 is characterized in that: the gene editing includes in vitro gene editing, prokaryotic gene editing, and eukaryotic gene editing.
- 根据权利要求3所述的应用，其特征在于：在原核生物基因剪辑中，EbCas12a的氨基酸序列如SED ID NO.1所示。The application according to claim 3 is characterized in that: in prokaryotic gene editing, the amino acid sequence of EbCas12a is as shown in SED ID NO.1.
- 根据权利要求3所述的应用，其特征在于：在真核生物基因剪辑中，EbCas12a的氨基酸序列如SED ID NO.2所示。 The use according to claim 3, characterized in that: in eukaryotic gene editing, the amino acid sequence of EbCas12a is as shown in SED ID NO.2.
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