{"id":5721,"date":"2024-08-20T10:35:17","date_gmt":"2024-08-20T02:35:17","guid":{"rendered":"https:\/\/fanyiming.life\/?p=5721"},"modified":"2025-12-21T01:04:34","modified_gmt":"2025-12-20T17:04:34","slug":"quick-guide-px458-primer-design","status":"publish","type":"post","link":"https:\/\/fanyiming.life\/?p=5721","title":{"rendered":"Quick Guide: PX458 primer design"},"content":{"rendered":"\n
\u4e2d\u6587\u7248\uff08\u673a\u7ffb\uff09<\/summary>\n

\u5f15\u8a00<\/strong><\/p>\n\n\n\n

pX458\u8d28\u7c92\u662fCRISPR-Cas9\u5b9e\u9a8c\u4e2d\u5e38\u7528\u7684\u5de5\u5177\u4e4b\u4e00\uff0c\u5b83\u96c6\u6210\u4e86Cas9\u6838\u9178\u9176\u548c\u5411\u5bfcRNA\uff08sgRNA\uff09\u652f\u67b6\uff0c\u8ba9sgRNA\u7684\u63d2\u5165\u53d8\u5f97\u7b80\u5355\u6613\u884c\u3002pX458\u8d28\u7c92\u901a\u8fc7\u5229\u7528BbsI\u9650\u5236\u6027\u5185\u5207\u9176\u4ea7\u751f\u7684\u7279\u5b9a\u4f4d\u70b9\uff0c\u521b\u9020\u51fa\u7cbe\u786e\u7684\u7c98\u6027\u672b\u7aef\uff0c\u65b9\u4fbf\u6211\u4eec\u8fdb\u884c\u514b\u9686\u64cd\u4f5c\u3002\u63a5\u4e0b\u6765\uff0c\u6211\u4eec\u5c31\u6765\u770b\u770b\u5982\u4f55\u6210\u529f\u5730\u5c06\u4f60\u7684sgRNA\u514b\u9686\u5230pX458\u8f7d\u4f53\u4e2d\u3002<\/p>\n\n\n\n

BbsI\u5207\u5272pX458\u4ea7\u751f\u7684\u7c98\u6027\u672b\u7aef<\/strong><\/h3>\n\n\n\n

BbsI\u662f\u4e00\u79cd\u7279\u6b8a\u7684II\u578b\u9650\u5236\u6027\u5185\u5207\u9176\uff0c\u5b83\u5728\u8bc6\u522b\u5e8f\u5217\u4e4b\u5916\u5207\u5272DNA\uff0c\u8fd9\u79cd\u72ec\u7279\u7684\u5207\u5272\u65b9\u5f0f\u80fd\u8ba9BbsI\u4ea7\u751f\u7279\u5b9a\u7684\u7c98\u6027\u672b\u7aef\uff0c\u8fd9\u4e9b\u672b\u7aef\u4e0d\u5305\u542b\u8bc6\u522b\u5e8f\u5217\u672c\u8eab\u3002<\/p>\n\n\n\n

\u5f53pX458\u88abBbsI\u9176\u5207\u540e\uff0c\u4f1a\u4ea7\u751f\u5982\u4e0b\u7c98\u6027\u672b\u7aef\uff0c\u8ba9\u4f60\u53ef\u4ee5\u8f7b\u677e\u63d2\u5165sgRNA\u5e8f\u5217\uff1a<\/p>\n\n\n\n

5' - CACCG - 3'\n3' - C - 5'\n\n5' - AAAC - 3'\n3' -      - 5'<\/code><\/pre>\n\n\n\n
    \n
  • \u6b63\u94fe\u7c98\u6027\u672b\u7aef (CACCG<\/code>)<\/strong>\uff1a\u8fd9\u4e2a\u7c98\u6027\u672b\u7aef\u7528\u4e8e\u6b63\u5411\u5f15\u7269\u3002\u5b83\u4f1a\u5728\u4f60\u7684sgRNA\u5e8f\u5217\u524d\u9762\u52a0\u4e0a\u4e00\u4e2aG\uff0c\u786e\u4fdd\u4e0eU6\u542f\u52a8\u5b50\u517c\u5bb9\u3002<\/li>\n\n\n\n
  • \u8d1f\u94fe\u7c98\u6027\u672b\u7aef (AAAC<\/code>)<\/strong>\uff1a\u8fd9\u4e2a\u7c98\u6027\u672b\u7aef\u7528\u4e8e\u53cd\u5411\u5f15\u7269\u3002\u6839\u636e\u6b63\u5411\u5f15\u7269\u4f7f\u7528\u7684\u662fCACCG<\/code>\u8fd8\u662fCACC<\/code>\uff0c\u53cd\u5411\u5f15\u7269\u53ef\u80fd\u9700\u8981\u6216\u8005\u4e0d\u9700\u8981\u57283′-\u7aef\u52a0\u4e0a\u4e00\u4e2a\u989d\u5916\u7684”C”\u3002<\/li>\n<\/ul>\n\n\n\n
    \u4e3apX458\u8bbe\u8ba1\u5f15\u7269<\/strong><\/h3>\n\n\n\n
    \u4ee5\u4e0b\u662f\u4e3apX458\u8d28\u7c92\u8bbe\u8ba1\u5f15\u7269\u7684\u65b9\u6cd5\uff1a<\/p>\n\n\n\n
      \n
    1. \u4ece\u76ee\u6807\u5e8f\u5217\u5f00\u59cb<\/strong>\uff1a\u4f60\u53ef\u4ee5\u901a\u8fc7\u4e00\u4e9b\u5728\u7ebf\u5de5\u5177\u83b7\u5f97\u591a\u4e2asgRNA\u5e8f\u5217\uff0c\u6bd4\u5982CHOPCHOP\u3002\u5b83\u901a\u5e38\u4f1a\u63d0\u4f9b\u4e00\u4e2a\u542b\u670923\u4e2a\u6838\u82f7\u9178\u7684\u5e8f\u5217\uff0820\u4e2a\u6838\u82f7\u9178\u7684\u76ee\u6807\u5e8f\u5217\u52a0\u4e0aNGG PAM\uff09\u3002\u8bb0\u5f97\u5220\u6389NGG<\/strong>\uff0c\u7136\u540e\u518d\u7ee7\u7eed\u3002<\/li>\n\n\n\n
    2. \u6b63\u94fe\u5f15\u7269<\/strong>\uff1a\u5728\u4f60\u7684\u76ee\u6807\u5e8f\u5217\u524d20\u4e2a\u6838\u82f7\u9178\u52a0\u4e0aCACCG<\/code>\u3002<\/li>\n\n\n\n
    3. \u8d1f\u94fe\u5f15\u7269<\/strong>\uff1a\u8bbe\u8ba1\u53cd\u5411\u4e92\u8865\u5e8f\u5217\u3002\u83b7\u53d6\u4f60\u768420\u4e2a\u6838\u82f7\u9178sgRNA\u5e8f\u5217\u7684\u53cd\u5411\u4e92\u8865\u5e8f\u5217\uff0c\u5e76\u57285\u2019-\u7aef\u52a0\u4e0aAAAC<\/code>\uff0c3\u2019-\u7aef\u52a0\u4e0a\u4e00\u4e2a”C”\u3002<\/li>\n<\/ol>\n\n\n\n
      5' - CACCG-N20 - 3'\n    3' - C-N20(\u53cd\u5411\u4e92\u8865)-CAAA - 5'<\/code><\/pre>\n\n\n\n
      \u90a3\u4e2a\u201cG\u201d\u7684\u91cd\u8981\u6027<\/strong><\/h3>\n\n\n\n
      sgRNA\u662f\u7531U6\u542f\u52a8\u5b50\u9a71\u52a8\u7684\uff0c\u800cU6\u542f\u52a8\u5b50\u504f\u597d\u4ee5G\u6838\u82f7\u9178\u5f00\u59cb\u8f6c\u5f55sgRNA\u3002\u5982\u679c\u4f60\u7684\u76ee\u6807\u5e8f\u5217\u4e0d\u4ee5G\u5f00\u5934\uff0c\u4f60\u9700\u8981\u624b\u52a8\u6dfb\u52a0\u4e00\u4e2aG\u3002\u8fd9\u5c31\u662f\u4e3a\u4ec0\u4e48\u6211\u4eec\u5728\u6b63\u5411\u5f15\u7269\u4e2d\u52a0\u5165CACCG<\/code>\u7684\u539f\u56e0\u2014\u2014\u786e\u4fddsgRNA\u80fd\u591f\u9ad8\u6548\u8f6c\u5f55\u3002<\/p>\n\n\n\n
        \n
      • \u5982\u679c\u76ee\u6807\u5e8f\u5217\u4ee5G\u5f00\u5934<\/strong>\uff1a\u4f60\u53ef\u4ee5\u4f7f\u7528CACC<\/code>\u6216CACCG<\/code>\u5728\u6b63\u5411\u5f15\u7269\u4e2d\u3002\u4e24\u79cd\u65b9\u6cd5\u90fd\u53ef\u4ee5\uff0c\u56e0\u4e3aG\u7684\u9700\u6c42\u5df2\u7ecf\u88ab\u6ee1\u8db3\u4e86\u3002<\/li>\n\n\n\n
      • \u5982\u679c\u76ee\u6807\u5e8f\u5217\u4e0d\u4ee5G\u5f00\u5934<\/strong>\uff1a\u4f60\u5fc5\u987b\u5728\u6b63\u5411\u5f15\u7269\u4e2d\u4f7f\u7528CACCG<\/code>\u6765\u6dfb\u52a0\u5fc5\u8981\u7684G\uff0c\u4fdd\u8bc1sgRNA\u80fd\u4e0eU6\u542f\u52a8\u5b50\u6709\u6548\u914d\u5408\u3002<\/li>\n<\/ul>\n\n\n\n
        \u5728\u52a0\u5165\u4e86CACCG<\/code>\u4e4b\u540e\uff0c\u6700\u7ec8\u5f97\u5230\u7684sgRNA\u5e8f\u5217\u957f\u5ea6\u5c06\u4f1a\u53d8\u621021\u4e2a\u6838\u82f7\u9178\uff0c\u800c\u4e0d\u662f20\u4e2a\u3002\u4e0d\u8fc7\uff0c\u8fd9\u53ea\u4f1a\u5bf9\u9776\u5411\u6548\u7387\u4ea7\u751f\u8f7b\u5fae\u5f71\u54cd\uff0c\u76f8\u6bd4\u4e8e\u6700\u4f73\u768420\u4e2a\u6838\u82f7\u9178\u957f\u5ea6\u6765\u8bf4\u3002\u56e0\u6b64\uff0c\u5728\u5b9e\u8df5\u4e2d\uff0c\u4f60\u53ef\u4ee5\u603b\u662f\u4f7f\u7528CACCG<\/code>\uff0c\u800c\u4e0d\u662fCACC<\/code>\u3002\u4f46\u662f\uff0c\u522b\u5fd8\u4e86\u5728\u53cd\u5411\u5f15\u7269\u76843\u2019-\u7aef\u52a0\u4e0a\u4e00\u4e2a”C”\uff0c\u4e0eCACCG<\/code>\u4e2d\u7684”G”\u914d\u5bf9\u3002<\/p>\n\n\n\n
        \u4f8b\u5b50<\/strong><\/h3>\n\n\n\n
        \u5047\u8bbe\u4f60\u4eceCHOPCHOP\u83b7\u5f97\u4e86\u4ee5\u4e0bsgRNA\u5e8f\u5217<\/strong>\uff1a<\/p>\n\n\n\n
        GGTATGACATGCCTTTCCAGAGG<\/code>\uff0823\u4e2a\u6838\u82f7\u9178\uff1a20\u4e2a\u6838\u82f7\u9178\u7684\u76ee\u6807\u5e8f\u5217+NGG\uff09<\/p>\n\n\n\n
        PAM\u5e8f\u5217”NGG”\u4e0d\u5e94\u8be5\u5305\u542b\u5728pX458\u8d28\u7c92\u4e2d\u3002\u56e0\u6b64\uff0c\u6211\u4eec\u9700\u8981\u5220\u9664\u8fd9\u90e8\u5206\uff1a
        GGTATGACATGCCTTTCCAG<\/code>\uff08NGG\u5df2\u53bb\u9664\uff09<\/p>\n\n\n\n
          \n
        • \u5982\u679c\u4f7f\u7528CACC<\/code><\/strong>\uff1a\n
            \n
          • \u6b63\u94fe\u5f15\u7269<\/strong>: 5'-CACCGGTATGACATGCCTTTCCAG-3'<\/code><\/li>\n\n\n\n
          • \u8d1f\u94fe\u5f15\u7269<\/strong>: 5'-AAACCTGGAAAGGCATGTCATACC-3' \uff08\u65e0\u9700\u989d\u5916\"C\"\uff09<\/code><\/li>\n<\/ul>\n<\/li>\n\n\n\n
          • \u5982\u679c\u4f7f\u7528CACCG<\/code><\/strong>\uff1a\n
              \n
            • \u6b63\u94fe\u5f15\u7269<\/strong>: 5'-**CACCG**GTATGACATGCCTTTCCAG-3'<\/code><\/li>\n\n\n\n
            • \u8d1f\u94fe\u5f15\u7269<\/strong>: 5'-**AAAC**CTGGAAAGGCATGTCATACC**C**-3' \uff083\u2019-\u7aef\u52a0\u4e00\u4e2a\"C\"\uff09<\/code><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n\n\n\n
              \u5f15\u7269\u8bbe\u8ba1\u5c0f\u7ed3<\/strong><\/h3>\n\n\n\n
                \n
              1. \u53bb\u6389NGG<\/strong>\uff1a\u4eceCHOPCHOP\u5e8f\u5217\u4e2d\u79fb\u9664NGG\uff0c\u5f97\u5230\u4f60\u768420\u4e2a\u6838\u82f7\u9178\u76ee\u6807\u5e8f\u5217\u3002<\/li>\n\n\n\n
              2. \u6839\u636e\u76ee\u6807\u5e8f\u5217\u662f\u5426\u4ee5G\u5f00\u5934\u9009\u62e9CACC<\/code>\u6216CACCG<\/code>\u3002<\/li>\n\n\n\n
              3. \u8bbe\u8ba1\u6b63\u94fe\u5f15\u7269\uff0c\u52a0\u4e0a\u9002\u5f53\u7684\u7c98\u6027\u672b\u7aef\u3002<\/li>\n\n\n\n
              4. \u8bbe\u8ba1\u8d1f\u94fe\u5f15\u7269\u4f5c\u4e3a\u53cd\u5411\u4e92\u8865\u5e8f\u5217\uff0c\u5e76\u57283\u2019-\u7aef\u52a0\u4e0a\u4e00\u4e2a”C”\uff08\u5982\u679c\u4f7f\u7528CACCG<\/code>\u7684\u8bdd\uff09\u3002<\/li>\n<\/ol>\n\n\n\n
                \u73b0\u5728\uff0c\u4f60\u5df2\u7ecf\u51c6\u5907\u597d\u8bbe\u8ba1\u5f15\u7269\u5e76\u5c06sgRNA\u514b\u9686\u5230pX458\u8d28\u7c92\u4e2d\uff0c\u4e3a\u6210\u529f\u7684CRISPR\u5b9e\u9a8c\u6253\u4e0b\u575a\u5b9e\u7684\u57fa\u7840\u5566\uff01<\/p>\n<\/details>\n\n\n\n
                Introduction<\/strong><\/h2>\n\n\n\n
                The pX458 plasmid is a widely used tool in CRISPR-Cas9 experiments, offering an all-in-one solution that includes both the Cas9 nuclease and a guide RNA (sgRNA) scaffold. The pX458 plasmid is designed to make sgRNA insertion straightforward by utilizing BbsI restriction sites that create the precise overhangs needed for cloning. This guide will walk you through how to successfully clone your sgRNA into the pX458 vector.<\/p>\n\n\n\n
                Overhang of pX458 Cut by BbsI<\/strong><\/h2>\n\n\n\n
                BbsI is a type IIS restriction enzyme, meaning it cuts DNA outside of its recognition sequence. This unique cutting mechanism allows BbsI to generate specific overhangs that are not part of the recognition sequence itself.<\/p>\n\n\n\n
                When pX458 is digested with BbsI, the enzyme cuts at specific sites, creating overhangs that allow you to insert your sgRNA sequence. The overhangs generated are as follows:<\/p>\n\n\n\n
                5' - CACCG - 3'\n3' - C - 5'\n\n5' - AAAC - 3'\n3' -      - 5'\n<\/code><\/pre>\n\n\n\n
                  \n
                • Top Strand Overhang (CACCG<\/code>)<\/strong>: This overhang is used for the forward primer. It adds a G to the beginning of your sgRNA sequence, ensuring compatibility with the U6 promoter.<\/li>\n\n\n\n
                • Bottom Strand Overhang (AAAC<\/code>)<\/strong>: This overhang is used for the reverse primer. Depending on whether the forward primer used CACCG<\/code> or CACC<\/code>, the reverse primer may or may not require an additional “C” at the 3′-end.<\/li>\n<\/ul>\n\n\n\n
                  Designing Primers for pX458<\/strong><\/h2>\n\n\n\n
                  Here\u2019s how to design primers for cloning your sgRNA into the pX458 plasmid:<\/p>\n\n\n\n
                    \n
                  1. Start with your sgRNA sequence<\/strong>: You may get several sgRNA sequences first on plenty of online tools. CHOPCHOP, for example, provides a 23-nt sequence (20-nt target + NGG PAM). Delete the NGG<\/strong> before proceeding.<\/li>\n\n\n\n
                  2. Top Strand Primer<\/strong>: Add CACCG<\/code> to the first 20 nucleotides of your target sequence.<\/li>\n\n\n\n
                  3. Bottom Strand Primer<\/strong>: Design the reverse complement of the 20-nt target sequence. Get the reverse complementary sequence of your 20nt sgRNA sequence, and add AAAC<\/code> to the 5\u2019-end and a single C<\/code> to the 3\u2019-end.<\/li>\n<\/ol>\n\n\n\n
                    5' - CACCG-N20 - 3'\n    3' - C-N20(ReverseComplementary)-CAAA - 5'\n<\/code><\/pre>\n\n\n\n
                    The \u201cG\u201d Matters<\/strong><\/h2>\n\n\n\n
                    The sgRNA is driven by the U6 promoter, which strongly prefers to start sgRNA transcription with a G nucleotide. If your sgRNA target sequence doesn’t naturally start with a G, you’ll need to add one. This is where the CACCG<\/code> motif in the forward primer comes into play, ensuring that your sgRNA is efficiently transcribed.<\/p>\n\n\n\n
                    If your target sequence starts with a G<\/strong>: You can use either CACC<\/code> or CACCG<\/code> in your forward primer. Both options work because the G requirement is already met.<\/p>\n\n\n\n
                    If your target sequence doesn\u2019t start with a G<\/strong>: You must use CACCG<\/code> in the forward primer to add the necessary G, ensuring the sgRNA works effectively with the U6 promoter.<\/p>\n\n\n\n
                    After adding CACCG<\/code>, the final length of it would be 21nt instead of 20nt in the resulted PX458 plasmid. However, it only bring slight influence on the targeting efficiency, compared with the optimal 20-nt length. Thus, you can always use CACCG<\/code> instead of CACC<\/code> in practice. However, you must keep in mind to add a C<\/code> at the 3′-end of reverse primers to pair with the G<\/code> within CACCG<\/code> .<\/p>\n\n\n\n
                    Example:<\/strong><\/h2>\n\n\n\n
                    You got a sgRNA Sequence directly from CHOPCHOP<\/strong>:<\/p>\n\n\n\n
                    GGTATGACATGCCTTTCCAGAGG<\/code> (23 nt: 20-nt target + NGG)<\/p>\n\n\n\n
                    The \u201cNGG\u201d PAM sequence should not included in the PX458 plasmid. So you need to delete it for next step primer design: GGTATGACATGCCTTTCCAG<\/code> (NGG removed)<\/p>\n\n\n\n
                      \n
                    • If using CACC<\/code><\/strong>:\n
                        \n
                      • Top Strand Primer<\/strong>: 5'-CACCGGTATGACATGCCTTTCCAG-3'<\/code><\/li>\n\n\n\n
                      • Bottom Strand Primer<\/strong>: 5'-AAACCTGGAAAGGCATGTCATACC-3' (No extra \"C\")<\/code><\/li>\n<\/ul>\n<\/li>\n\n\n\n
                      • If using CACCG<\/code><\/strong>:\n
                          \n
                        • Top Strand Primer<\/strong>: 5'-**CACCG**GTATGACATGCCTTTCCAG-3'<\/code><\/li>\n\n\n\n
                        • Bottom Strand Primer<\/strong>: 5'-**AAAC**CTGGAAAGGCATGTCATACC**C**-3' (Add an extra \"C\" at 3'-end)<\/code><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n\n\n\n
                          Primer Design Recap<\/strong><\/h2>\n\n\n\n
                            \n
                          1. Remove NGG<\/strong> from the CHOPCHOP sequence to get your 20-nt target sequence.<\/li>\n\n\n\n
                          2. Choose CACC<\/code> or CACCG<\/code> based on whether your target sequence starts with a G.<\/li>\n\n\n\n
                          3. Design the top strand with the appropriate overhang.<\/li>\n\n\n\n
                          4. Design the bottom strand as the reverse complement, adding a C<\/code> at the 3′-end if using CACCG<\/code>.<\/li>\n<\/ol>\n\n\n\n
                            With this guide, you’re ready to design primers and clone your sgRNA into pX458, setting the stage for successful CRISPR experiments!<\/p>\n","protected":false},"excerpt":{"rendered":"
                            \u8fd9\u7bc7\u7b14\u8bb0\u8be6\u7ec6\u4ecb\u7ecd\u4e86\u4e3aCRISPR-Cas9\u5e38\u7528\u8f7d\u4f53pX458\u8bbe\u8ba1sgRNA\u5f15\u7269\u7684\u6838\u5fc3\u6d41\u7a0b\u3002\u4f5c\u8005\u91cd\u70b9\u89e3\u6790\u4e86BbsI\u9176\u5207\u4ea7\u751f\u7684\u7c98\u6027\u672b\u7aef\u3001\u6b63\u53cd\u5411\u5f15\u7269\u8bbe\u8ba1\u89c4\u5219\uff0c\u5e76\u5f3a\u8c03\u4e86U6\u542f\u52a8\u5b50\u5bf9\u8d77\u59cbG\u7684\u504f\u597d\u6027\u53ca\u5bf9\u5e94\u7684\u5f15\u7269\u8c03\u6574\u7b56\u7565\u3002<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[4],"tags":[],"class_list":["post-5721","post","type-post","status-publish","format-standard","hentry","category-note"],"_links":{"self":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/5721","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=5721"}],"version-history":[{"count":2,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/5721\/revisions"}],"predecessor-version":[{"id":13943,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/5721\/revisions\/13943"}],"wp:attachment":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=5721"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=5721"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=5721"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}