WO2024060814A1 - 一种Cas12a变体及其在基因编辑中的应用 - Google Patents

一种Cas12a变体及其在基因编辑中的应用 Download PDF

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WO2024060814A1
WO2024060814A1 PCT/CN2023/108234 CN2023108234W WO2024060814A1 WO 2024060814 A1 WO2024060814 A1 WO 2024060814A1 CN 2023108234 W CN2023108234 W CN 2023108234W WO 2024060814 A1 WO2024060814 A1 WO 2024060814A1
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cas12a
variant
recombinant
gene editing
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竺立哲
雷湧
袁罗伟
刘慧慧
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香港中文大学（深圳）
香港中文大学(深圳)福田生物医药创新研发中心
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  • the present invention relates to the field of gene editing technology, and in particular to a Cas12a variant and its application in gene editing.
  • Cas12a As a member of the CRISPR class II effector protein, has also received widespread attention from the scientific community. Compared with Cas9 protein, which requires two molecules of crRNA and tracrRNA to cleave double-stranded DNA molecules, Cas12a only needs a single crRNA induction to complete the effective cleavage of target double-stranded DNA ( Figure 1).
  • the CRISPR/Cas12a protein remains in a catalytically active state after completing cis-cleavage of the target double-stranded DNA, and then initiates non-targeted trans-cleavage of single-stranded DNA, which has potential applications in disease and virus detection.
  • the PAM sequences preferred by Cas9 are usually G-rich, while the PAM sequences preferred by Cas12a are T-rich. This feature can complement the advantages of Cas9 and broaden the scope of gene editing.
  • the present invention proposes a Cas12a variant and its application in gene editing. application in editing.
  • the present invention focuses on FnCas12a, replacing lysine (K) at position 1065 of FnCas12a with glutamic acid (E) to obtain a new K1065E mutant, which improves its target compared with wild-type FnCas12a. towards specificity and reduce off-target.
  • the present invention provides a Cas12a variant, the amino acid sequence of the Cas12a variant is as shown in SED ID NO. 1, and the Cas12a variant protein has increased DNA cleavage selectivity relative to the corresponding wild-type Cas12a protein.
  • the present invention also provides a recombinant expression vector that expresses the Cas12a variant.
  • the recombinant expression vector includes any one of plasmid vectors, viral vectors and phage vectors.
  • the invention also provides a recombinant bacterium or recombinant cell line or recombinant virus, including the recombinant expression vector.
  • the present invention also provides a recombinant virus, which is obtained by packaging with the viral vector, and the recombinant virus includes an adenovirus or lentivirus expressing the Cas12a variant.
  • the present invention also provides the application of the Cas12a variant, the recombinant expression vector, the recombinant bacteria or recombinant cell line and the recombinant virus in gene editing.
  • the present invention also provides a CRISPR/Cas12a gene editing system including the Cas12a variant.
  • system also includes: crRNA targeting the target gene.
  • the present invention also provides a method for gene editing of a target gene in a recipient by means of the CRISPR/Cas12a system.
  • the method includes introducing the recombinant expression vector into the recipient. Cas12a variants available.
  • the present invention improves the targeting specificity of FnCas12a for double-stranded DNA cleavage and reduces its off-target probability.
  • the K1065E mutation of FnCas12a of the present invention has higher targeting specificity than wild-type FnCas12a.
  • Figure 1 is a schematic diagram of Cas12a in the prior art.
  • Figure 2 is a single mismatch in vitro cleavage experiment of wild-type and K1065E mutant FnCas12a proposed in the embodiment of the present invention.
  • Figure 3 shows the double mismatch in vitro cleavage experiment of wild-type and K1065E mutant FnCas12a proposed in the embodiment of the present invention.
  • Figure 4 is an in vitro cleavage experiment for non-classical PAM sequences of wild-type and K1065E mutant FnCas12a proposed in the embodiment of the present invention.
  • the present invention provides a novel FnCas12a mutant K1065E, the amino acid sequence of which is shown in SEQ ID NO.1.
  • the full length of the amino acid sequence of the K1065E mutant protein of FnCas12a is 1326 amino acids.
  • the mutant FnCas12a was more sensitive to single base mismatch sequences than wild-type FnCas12a. That is, a single base mismatch near the protospacer adjacent motif (PAM) region can effectively reduce potential off-target effects.
  • PAM protospacer adjacent motif
  • K1065E The mutant is able to circumvent the non-specific cleavage of the wild-type FnCas12a protein with double-site mutations at positions 5 and 6, proving that the K1065E mutant has higher targeting specificity.
  • the first part is the construction of DNA products with mismatched bases
  • the second part is the in vitro Cas12a cleavage experiment.
  • the base-complementary oligonucleotide is introduced between the EcoRI and HindIII restriction sites of plasmid pUC19.
  • the target strand of this oligonucleotide is complementary to the crRNA of FnCas12a and contains a single-base mismatch or a double-base mismatch.
  • the sequence is shown in Table 1.
  • the pUC19 plasmid after digestion and ligation was transferred into competent E. coli, plated, and single clones of E. coli were selected for sequencing to determine the sequence of the recombinant plasmid. After constructing all plasmids, take 2 ⁇ g of plasmid and add it to 1 ⁇ rCutSmart buffer.
  • EDTA and proteinase K were added to a final concentration of 80 mM and 0.8 mg/mL, respectively, and incubated at 37°C for 30 minutes to terminate the cleavage reaction.
  • the cleaved product was added to 6 ⁇ DNA loading buffer, loaded onto an agarose gel, and voltage was applied to separate it, followed by photographing.
  • the results of double mismatch in vitro cleavage are shown in Figure 3.
  • the wild-type and K1065E mutant FnCas12a have significant differences in the cleavage characteristics of consecutive double mutations at positions 5 and 6.
  • the wild-type FnCas12a protein has high nonspecific cleavage activity for consecutive double mutations at positions 5 and 6, but the K1065E mutant FnCas12a is almost unable to cleave the DNA double strands containing consecutive double mutations at positions 5 and 6, indicating that it has higher specificity than the wild type.
  • the Cas12a variant of the present invention has higher specificity than the wild type whether targeting single mismatch, double mismatch or non-canonical PAM sequence.
  • the Cas12a variant can further expand gene editing tools. types, which play an important role in the application of CRISPR/Cas gene editing systems.

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Abstract

一种Cas12a变体及其在基因编辑中的应用，所述Cas12a变体的氨基酸序列如SED ID NO.1所示。通过构建FnCas12a的K1065E突变体，提高FnCas12a对双链DNA切割的靶向特异性，降低其脱靶概率。所述Cas12a变体进一步扩大了基因编辑工具的种类。

Description

一种Cas12a变体及其在基因编辑中的应用 技术领域
本发明涉及基因编辑技术领域，特别涉及一种Cas12a变体及其在基因编辑中的应用。
背景技术
基于CRISPR/Cas系统的基因编辑方法自问世以来，在基础研究、疾病诊断和临床治疗等领域得到了广泛的应用。除了最早被应用于基因编辑的CRISPR/Cas9系统，Cas12a作为CRISPRⅡ类效应蛋白的成员，亦受到了科学界的广泛关注。相比于Cas9蛋白需要crRNA和tracrRNA两个分子才实现对双链DNA分子的切割，Cas12a只需要单一的crRNA诱导即可完成对目标双链DNA的有效切割(如图1)。同时，CRISPR/Cas12a蛋白在完成对目标双链DNA的顺式切割后保持催化活化状态，随后开启对单链DNA的非靶向反式切割，具有疾病和病毒检测的潜在应用。此外，通常Cas9偏好的PAM序列通常富含G，而Cas12a所偏好的PAM序列则富含T，此特性更能与Cas9优势互补，拓宽基因编辑的范围。目前，仅有Francisella tularensis Cas12a(FnCas12a)、Acidaminococcus sp.Cas12a(AsCas12a)与Lachnospiraceae bacterium Cas12a(LbCas12a)三种Cas12a蛋白被报道具有核酸内切酶和基因组编辑的功能。尽管现有的野生型Cas12a的脱靶率低于Cas9，但其脱靶问题依然存在，即Cas12a可以耐受部分靶标区域序列的碱基错配，可识别并切割非完全互补的靶向区域。因此如何降低CRISPR/Cas基因编辑系统的脱靶概率是亟待解决的问题。
发明内容
针对现有技术中的缺陷，本发明提出了一种Cas12a变体及其在基因编 辑中的应用。本发明聚焦于FnCas12a，将FnCas12a的1065号位点的赖氨酸(K)替换成谷氨酸(E)，得到一种新型的K1065E突变体，相较于野生型的FnCas12a，提高了其靶向特异性，减少脱靶。
本发明提供一种Cas12a变体，所述Cas12a变体的氨基酸序列如SED ID NO.1所示，所述Cas12a变体蛋白具有增加的相对于相应的野生型Cas12a蛋白的DNA切割选择性。
本发明还提供一种重组表达载体，所述重组载体表达所述的Cas12a变体。
进一步的，所述重组表达载体包括质粒载体、病毒载体和噬菌体载体中的任意一种。
本发明还提供一种重组菌或重组细胞系或重组病毒，包含所述的重组表达载体。
本发明还提供一种重组病毒，所述重组病毒采用所述的病毒载体包装获得，所述重组病毒包括表达所述的Cas12a变体的腺病毒或慢病毒。
本发明还提供所述的Cas12a变体、所述的重组表达载体、所述的重组菌或重组细胞系和所述的重组病毒在基因编辑中的应用。
本发明还提供一种包括所述Cas12a变体的CRISPR/Cas12a基因编辑系统。
进一步的，所述系统还包括：靶向目标基因的crRNA。
本发明还提供一种对受体中的目的基因进行基因编辑的方法，借助CRISPR/Cas12a系统对受体中的目的基因进行基因编辑，所述方法包括通过将所述的重组表达载体导入受体提供Cas12a变体。
综上，与现有技术相比，本发明达到了以下技术效果：
本发明通过构建FnCas12a的K1065E突变体，提高FnCas12a对双链DNA切割的靶向特异性，降低其脱靶概率。本发明FnCas12a的K1065E突 变体具有比野生型FnCas12a更高的靶向特异性。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为现有技术中Cas12a的示意图。
图2为本发明实施例提出的野生型和K1065E突变型FnCas12a的单一错配体外切割实验。
图3为本发明实施例提出的野生型和K1065E突变型FnCas12a的双错配体外切割实验。
图4为本发明实施例提出的野生型和K1065E突变型FnCas12a的针对非经典PAM序列体外切割实验。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
本发明提供一种新型的FnCas12a突变体K1065E，氨基酸序列如SEQ ID NO.1所示。FnCas12a的K1065E突变体氨基酸序列蛋白质全长为1326个氨基酸。体外双链DNA酶切实验显示，该突变体FnCas12a比野生型FnCas12a对单一碱基错配序列更敏感。即在靠近protospacer adjacent motif(PAM)区域的单一碱基错配即可有效降低潜在的脱靶效应。进一步的实验显示K1065E 突变体能够规避野生型FnCas12a蛋白在第5和第6位双位点突变的非特异性切割，证明K1065E突变体具有更高的靶向特异性。
除特殊说明外，本发明采用的所有的试剂、原材料、技术服务和仪器设备均可通过市场购买获得。
实施例
使用野生型和K1065E突变型的FnCas12a分别对WT位点(无错配)，1至7号错配位点的质粒进行切割，具体操作如下，分为两个部分：
第一部分为碱基不匹配的DNA产物构建；
第二部分为体外的Cas12a切割实验。
(一)碱基不匹配的DNA产物构建
碱基互补的寡核苷酸经退火后，导入质粒pUC19的EcoRⅠ和HindIII酶切位点之间。该寡核苷酸的靶标链与FnCas12a的crRNA互补，含有单一碱基的不匹配，或含有双碱基的不匹配，序列如表1所示。将酶切连接后的pUC19质粒转入感受态大肠杆菌中，涂板，挑选大肠杆菌单克隆做测序，确定重组后质粒的序列。构建好所有的质粒后，取2μg质粒，加入到1×的rCutSmart缓冲液，使用40个单位的SspI-HF限制性内切酶在37℃下反应2小时，使其充分线性化质粒，最终的反应体积为20μL。随后，65℃加热20分钟，使SspI-HF酶失活。即可得到Cas12a切割所用的DNA产物。
表1 crRNA和寡核苷酸序列


(二)Cas12a的体外切割实验
首先将FnCas12a和crRNA在镁离子存在的条件下于37℃孵育10分钟，随后再加入(一)中所得到的DNA产物，进行切割反应。具体的反应比例如表2所示：
表2
切割反应在37℃的条件下反应10分钟后，加入EDTA和蛋白酶K至终浓度分别为80mM和0.8mg/mL，在37℃条件下孵育30分钟，使其终止切割反应。将切割后的产物加入6×的DNA上样缓冲液，上样到琼脂糖胶中，施加电压，使其分离，随后拍照成像。
单一错配体外切割的结果如图2所示，野生型和K1065E突变型FnCas12a对无错配的质粒都可以完全切割，得到切割产物1和2。但野生型的FnCas12a对第1至第7号错配位点依然有切割，而K1065E突变型FnCas12a对第1至第7号错配位点的切割较少，说明其相对于野生型具有更高的特异性。
双错配体外切割的结果如图3所示，野生型和K1065E突变型FnCas12a对第5和第6位的连续双突变的切割特性具有显著区别。野生型的FnCas12a蛋白对第5和第6位的连续双突变具有较高的非特异性切割活性，但K1065E突变型FnCas12a几乎无法对含有第5和第6位的连续双突变的DNA双链实施切割，说明其相对于野生型具有更高的特异性。
非经典PAM序列体外切割的结果如图4所示，野生型和K1065E突变型FnCas12a针对非经典PAM(TTTN)的其他PAM序列(如CTTN，TCTN，TTCN，CCTN)切割特异性具有显著区别。K1065E突变型FnCas12a蛋白可有效降低针对TCTN，TTCN，CCTN三种PAM序列的非特异性切割，说明其相对于野生型具有更高的特异性。
综合以上实施例，本发明的Cas12a变体不论是针对单一错配、双错配还是非经典PAM序列时相对于野生型均具有更高的特异性，所述Cas12a变体能够进一步扩大基因编辑工具的种类，在CRISPR/Cas基因编辑系统的应用中具有重要作用。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种Cas12a变体，其特征在于，所述Cas12a变体的氨基酸序列如SED ID NO.1所示，所述Cas12a变体蛋白具有增加的相对于相应的野生型Cas12a蛋白的DNA切割选择性。
  2. 一种重组表达载体，其特征在于，所述重组载体表达权利要求1所述的Cas12a变体。
  3. 根据权利要求2所述的重组表达载体，其特征在于，所述重组表达载体包括质粒载体、病毒载体和噬菌体载体中的任意一种。
  4. 一种重组菌或重组细胞系或重组病毒，其特征在于，包含权利要求2所述的重组表达载体。
  5. 一种重组病毒，其特征在于，所述重组病毒采用权利要求3所述的病毒载体包装获得，所述重组病毒包括表达权利要求1所述的Cas12a变体的腺病毒或慢病毒。
  6. 权利要求1所述的Cas12a变体、权利要求2～3任一项所述的重组表达载体、权利要求4所述的重组菌或重组细胞系和权利要求5所述的重组病毒在基因编辑中的应用。
  7. 一种包括权利要求1所述Cas12a变体的CRISPR/Cas12a基因编辑系统。
  8. 根据权利要求7所述的CRISPR/Cas基因编辑系统，其特征在于，所述系统还包括：靶向目标基因的crRNA。
  9. 一种对受体中的目的基因进行基因编辑的方法，其特征在于，借助CRISPR/Cas12a系统对受体中的目的基因进行基因编辑，所述方法包括通过将权利要求2～3任一项所述的重组表达载体导入受体提供Cas12a变体。
PCT/CN2023/108234 2022-09-21 2023-07-19 一种Cas12a变体及其在基因编辑中的应用 WO2024060814A1 (zh)

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