Quick Guide: PX458 primer design

中文版（机翻）

引言

pX458质粒是CRISPR-Cas9实验中常用的工具之一，它集成了Cas9核酸酶和向导RNA（sgRNA）支架，让sgRNA的插入变得简单易行。pX458质粒通过利用BbsI限制性内切酶产生的特定位点，创造出精确的粘性末端，方便我们进行克隆操作。接下来，我们就来看看如何成功地将你的sgRNA克隆到pX458载体中。

BbsI切割pX458产生的粘性末端

BbsI是一种特殊的II型限制性内切酶，它在识别序列之外切割DNA，这种独特的切割方式能让BbsI产生特定的粘性末端，这些末端不包含识别序列本身。

当pX458被BbsI酶切后，会产生如下粘性末端，让你可以轻松插入sgRNA序列：

5' - CACCG - 3'
3' - C - 5'

5' - AAAC - 3'
3' -      - 5'
  • 正链粘性末端 (CACCG)：这个粘性末端用于正向引物。它会在你的sgRNA序列前面加上一个G，确保与U6启动子兼容。
  • 负链粘性末端 (AAAC)：这个粘性末端用于反向引物。根据正向引物使用的是CACCG还是CACC，反向引物可能需要或者不需要在3′-端加上一个额外的”C”。

为pX458设计引物

以下是为pX458质粒设计引物的方法：

  1. 从目标序列开始：你可以通过一些在线工具获得多个sgRNA序列，比如CHOPCHOP。它通常会提供一个含有23个核苷酸的序列（20个核苷酸的目标序列加上NGG PAM）。记得删掉NGG，然后再继续。
  2. 正链引物：在你的目标序列前20个核苷酸加上CACCG。
  3. 负链引物：设计反向互补序列。获取你的20个核苷酸sgRNA序列的反向互补序列，并在5’-端加上AAAC，3’-端加上一个”C”。
5' - CACCG-N20 - 3'
    3' - C-N20(反向互补)-CAAA - 5'

那个“G”的重要性

sgRNA是由U6启动子驱动的，而U6启动子偏好以G核苷酸开始转录sgRNA。如果你的目标序列不以G开头，你需要手动添加一个G。这就是为什么我们在正向引物中加入CACCG的原因——确保sgRNA能够高效转录。

  • 如果目标序列以G开头：你可以使用CACC或CACCG在正向引物中。两种方法都可以，因为G的需求已经被满足了。
  • 如果目标序列不以G开头：你必须在正向引物中使用CACCG来添加必要的G，保证sgRNA能与U6启动子有效配合。

在加入了CACCG之后，最终得到的sgRNA序列长度将会变成21个核苷酸，而不是20个。不过，这只会对靶向效率产生轻微影响，相比于最佳的20个核苷酸长度来说。因此，在实践中，你可以总是使用CACCG，而不是CACC。但是，别忘了在反向引物的3’-端加上一个”C”，与CACCG中的”G”配对。

例子

假设你从CHOPCHOP获得了以下sgRNA序列：

GGTATGACATGCCTTTCCAGAGG（23个核苷酸：20个核苷酸的目标序列+NGG）

PAM序列”NGG”不应该包含在pX458质粒中。因此，我们需要删除这部分：
GGTATGACATGCCTTTCCAG（NGG已去除）

  • 如果使用CACC：
    • 正链引物: 5'-CACCGGTATGACATGCCTTTCCAG-3'
    • 负链引物: 5'-AAACCTGGAAAGGCATGTCATACC-3' （无需额外"C"）
  • 如果使用CACCG：
    • 正链引物: 5'-**CACCG**GTATGACATGCCTTTCCAG-3'
    • 负链引物: 5'-**AAAC**CTGGAAAGGCATGTCATACC**C**-3' （3’-端加一个"C"）

引物设计小结

  1. 去掉NGG：从CHOPCHOP序列中移除NGG，得到你的20个核苷酸目标序列。
  2. 根据目标序列是否以G开头选择CACC或CACCG。
  3. 设计正链引物，加上适当的粘性末端。
  4. 设计负链引物作为反向互补序列，并在3’-端加上一个”C”（如果使用CACCG的话）。

现在，你已经准备好设计引物并将sgRNA克隆到pX458质粒中，为成功的CRISPR实验打下坚实的基础啦！

Introduction

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.

Overhang of pX458 Cut by BbsI

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.

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:

5' - CACCG - 3'
3' - C - 5'

5' - AAAC - 3'
3' -      - 5'
  • Top Strand Overhang (CACCG): 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.
  • Bottom Strand Overhang (AAAC): This overhang is used for the reverse primer. Depending on whether the forward primer used CACCG or CACC, the reverse primer may or may not require an additional “C” at the 3′-end.

Designing Primers for pX458

Here’s how to design primers for cloning your sgRNA into the pX458 plasmid:

  1. Start with your sgRNA sequence: 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 before proceeding.
  2. Top Strand Primer: Add CACCG to the first 20 nucleotides of your target sequence.
  3. Bottom Strand Primer: Design the reverse complement of the 20-nt target sequence. Get the reverse complementary sequence of your 20nt sgRNA sequence, and add AAAC to the 5’-end and a single C to the 3’-end.
5' - CACCG-N20 - 3'
    3' - C-N20(ReverseComplementary)-CAAA - 5'

The “G” Matters

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 motif in the forward primer comes into play, ensuring that your sgRNA is efficiently transcribed.

If your target sequence starts with a G: You can use either CACC or CACCG in your forward primer. Both options work because the G requirement is already met.

If your target sequence doesn’t start with a G: You must use CACCG in the forward primer to add the necessary G, ensuring the sgRNA works effectively with the U6 promoter.

After adding CACCG, 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 instead of CACC in practice. However, you must keep in mind to add a C at the 3′-end of reverse primers to pair with the G within CACCG .

Example:

You got a sgRNA Sequence directly from CHOPCHOP:

GGTATGACATGCCTTTCCAGAGG (23 nt: 20-nt target + NGG)

The “NGG” PAM sequence should not included in the PX458 plasmid. So you need to delete it for next step primer design: GGTATGACATGCCTTTCCAG (NGG removed)

  • If using CACC:
    • Top Strand Primer: 5'-CACCGGTATGACATGCCTTTCCAG-3'
    • Bottom Strand Primer: 5'-AAACCTGGAAAGGCATGTCATACC-3' (No extra "C")
  • If using CACCG:
    • Top Strand Primer: 5'-**CACCG**GTATGACATGCCTTTCCAG-3'
    • Bottom Strand Primer: 5'-**AAAC**CTGGAAAGGCATGTCATACC**C**-3' (Add an extra "C" at 3'-end)

Primer Design Recap

  1. Remove NGG from the CHOPCHOP sequence to get your 20-nt target sequence.
  2. Choose CACC or CACCG based on whether your target sequence starts with a G.
  3. Design the top strand with the appropriate overhang.
  4. Design the bottom strand as the reverse complement, adding a C at the 3′-end if using CACCG.

With this guide, you’re ready to design primers and clone your sgRNA into pX458, setting the stage for successful CRISPR experiments!