利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The overall goal of this procedure is to generate genomic point mutations in C.Elegans using single stranded oligonucleotide homology directed repair templates and CRISPR/Cas9 ribonucleoproteins. This method can help answer key questions in the neuroscience field such as determining the pathological consequences of genetic variance associated with neurodegenerative disease. The main advantage of this technique is that genomic point mutations can be generated rapidly allowing for the functional study of genetic variance within the context of endoginous regulatory control while avoiding the caveats of protein overexpression.

To carry out sgRNA target selection, open up the webpage shown here. Input approximately 60 base pairs of sequence flanking the desired edit of interest. After selecting the C.Elegans genome and the protospacer adjacent motif according to the text protocol, click submit.

On the results page, choose the top ranked target sequence closest to the edit of interest. Upon receipt, centrifuge the lyophilized sgRNA containing tube at room temperature and at least 12, 000 g for one minute. Add 60 microliters of nuclease free TE to the tube and use a p200 pipette to gently resuspend by pipetting up and down 15 to 20 times.

The final concentration is 50 micromolar. Design an ssODN HDR template containing the mutation of interest, a unique in frame restriction endonuclease site, a silent mutation of one or both of the Gs within the NGG PAM sequence and 50 base pair five prime and three prime homology arms flanking the first and last mutation. Upon receipt, centrifuge the lyophilized ssODN containing tube as just demonstrated then use nuclease free DD H20 to gently resuspend ssODN to a final concentration of 100 micromolar.

To prepare an injection mix, centrifuge the reagents shown here at maximum speed and four degrees celsius for two minutes. In the order listed, add the reagents to a nuclease free PCR tube. Then use a p20 pipette to gently and thoroughly mix the contents.

Incubate the tube at room temperature for ten minutes. After injecting P0 animals according to the text protocol, allow them to recover at room temperature for one to two hours on an OP50 seeded 35 millimeter NGM plate. Subsequently using a platinum wire worm pick transfer single injected P0 animals to individual OP50 seeded 35 millimeter NGM plates.

Two days post injection use a fluorescent microscope to identify P0 plates containing F1 progeny, expressing mCherry in the pharynx. Choose three P0 plates that contain the most mCherry positive F1 animals. From each of the three selected P0 plates use a worm pick to single eight to 12 mCherry positive F1 animals for a total of 24 to 36 mCherry positive F1s.

Allow individual F1s to self fertilize and lay eggs at room temperature for one to two days. After one to two days of egg laying, use a worm pick to transfer the F1s into individual PCR strip tube caps containing seven microliters of worm lysis buffer. Centrifuge the PCR tubes at maximum speed and room temperature for one minute to bring the animals to the bottom of the tube.

Then freeze the tubes at negative 80 degrees celsius for one hour. Next lyse frozen worms in a thermocycler using the following program. Then set up a PCR master mix as shown in this table.

Add 21 microliters of PCR master mix into clean PCR tubes then add four microliters of the worm lysis tube and mix well by pipetting. Then run the PCR program following the manufacturer's guidelines. Purify the PCR reactions using a DNA clean and concentrate kit following the manufacturer's instructions.

Then dilute the DNA by adding 10 microliters of water to the spin column. After setting up the restriction enzyme master mix add 10 microliters to each cleaned PCR reaction and incubate the tubes at 37 degrees celsius for one to two hours. Then separate the digested PCR products on a 1.5%agarose gel using 1x TAE buffer at 120 volts.

Examine enzyme digested samples for the presence of bands that indicate potential edited animals. After identifying potential edited animals use the remaining three microliters of worm lysis and repeat the PCR. Then immediately load the completed reactions on a 1%agarose gel before separating and extracting the band using a gel extraction kit.

Using the F1 primer carry out Sanger sequencing to identify correctly edited animals. Finally to obtain homozygous animals from verified heterozygous edited animals, single eight to 12 F2 animals and carry out genotyping and sequence verification according to the text protocol. To demonstrate the simplicity, feasibility, and efficiency of the CRISPR/Cas9 RNP based approach, the C.Elegans sod-1 gene was targeted to introduce the disease associated human G93a variant in the worm genome.

This gel image shows the genotyping results for 24 mCherry positive F1 animals. 10 contained monoallylic or biallylic modification, 10 were wild type, three were potential indels, and one was a PCR failure. To demonstrate that the fluorescent mCherry marker enriches for genome modification, eight mCherry negative animals from each of the three P0 plates were picked.

Only one animal was correctly modified whereas 23 were wild type. This figure shows the chromatogram from the full length gel extracted PCR product demonstrating that the precise nucleotide changes designed in the ssODN HDR template were faithfully incorporated into the genome of this homozygous F1 animal. Once mastered, this technique can be utilized to generate and identify genomic point mutations in C.Elegans in four to five days if it is performed properly.

While attempting this procedure it's important to remember to use nucleus free reagents and techniques including RNase free water, filter pipette tips, and gloves. Following this procedure, cellular, molecular, and behavioral assays can be performed in order to investigate phenotypes that may associate with the mutation of interest. After watching this video you should have a good understanding of how to precisely generate genomic point mutations in C.Elegans using chimeric single guide RNase and CRISPR/Cas9 ribonuclearproteins.