Design cas9 conditional knockout mice ------you can, you up!

The key is to construct a mouse with two Loxp sites, flox (flanked by loxP) mice.

Today we will detail how to design.

There are three steps:

In the first step, choose an exon.

The second step is to design the sgRNA.

The third step is to design the SSODN.

Sounds pretty simple? Let's talk about it in detail below.

In the first step, choose an exon.

1. Try to knock out all the exons shared by all transcripts.

We all know that eukaryotic genes have exons and introns. Using these introns and exons, one gene can splicing multiple transcripts and translating to obtain multiple proteins. When designing a conditional knockout, it is first necessary to figure out that the gene has several transcripts and generally knocks out the exons shared by all transcripts.

2. The number of bases in this exon must not be a multiple of 3.

In this way, all proteins encoded by the entire gene are frameshifted and mutated, which has the effect of knocking out the gene.

3. It is best not to choose the first exon.

Why? Because the gene may use the start codon elsewhere in the vicinity to initiate transcription. This way you knocked out the first exon and the protein can be re-translated from somewhere else. The effect of gene knockout cannot be achieved.

The second step is to design the sgRNA.

Once the exons have been selected, you can begin designing the sgRNA. The structure of sgRNA is shown in the figure below. Figure A shows the plane sequence information. Panel B shows a three-dimensional conformation. The sgRNA consists of two parts, a green target sequence part, and a red and blue skeleton part. The role is to direct the cas9 protein to cleave at a specific location on the genome. So it can be seen that the core part of sgRNA is mainly the green part. So the key to designing sgRNA is to choose the location of the green target sequence. For conditional knockout mice, the generally selected region is among the two introns adjacent to the exon. Design principles for sgRNA:

1. The sequence with similarity on the genome is the least.

The goal is to minimize off-target effects.

2. Have an NGG.

This is required for the most commonly used Cas9 protein recognition target sites.

Here you can use many online design websites to find the right place.

Quoted from Cell 156, 935–949, February 27, 2014

The third step is to design SSODN (Single-Stranded Oligo Donor).

The order of SSODN is as follows: homology arm + restriction site + loxp site + homology arm.

Homogenous arm

Generally, 40-60 bp is selected as the homology arm in the upstream and downstream of the break point. For specific operations, please refer to the following figure.

2. Add the enzyme cleavage site

Adding an enzyme cleavage site is also convenient for verification, and there is no increase or failure.

3. The direction of the Loxp locus

One point is very important, that is, the 34bp loxp site is directional, and must be in the same direction. Otherwise, when you have experienced thousands of waters and get mice to prepare for knocking out genes, you will find that you want to knock out The clip just fell off the head and was there. At this time, I just want to die. Haha, don't blame me for not reminding me.

4. SSODN and sgRNA are on the same chain.

Another point is that it is best to have SSODN and sgRNA in the same chain. Otherwise, your sgRNA has not yet played a role, and it has been occupied by SSODN. In this case, people work emotionally.

Quoted from Cell 154, 1370–1379, September 12, 2013

The fourth step, action.

If you have already come here, congratulations, you have completed the design work, you really can.

But this is the "first step in the long march."

Next is primer design, synthesis of SSODN, in vitro transcription, validation of sgRNA, microinjection, F0 verification, mouse hybridization, etc...

At this time, "a big wave of experiments is approaching you!"

Haha, don't be afraid, don't worry, Jikai has launched a conditional knockout mouse service to help you tailor it, all things are done. We are blaming, you are upgrading, so stay tuned!

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