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Most people in the world of biology have heard of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated systems) genome editing by now.
This article aims to give you some ideas about what CRISPR genome editing can do for you and a brief overview of what the technology involves.
Although in some cases, CRISPR Cas9 gene editing can be used on whole organisms (e.g. mice), this article will focus on CRISPR in cultured cells. Much of the information presented here stems from work in human cells, but generally speaking, the setup is applicable to any species.
What Can CRISPR Genome Editing Be Used For?
CRISPR-Cas9 gene editing allows you to target a nuclease to cut at a specific site in the genome. Then, you let the DNA repair machinery of the cell do the rest of the work for you.
- Most often, CRISPR-Cas9 is used to generate knockouts of genes of interest.
- First, a a so-called “guide RNA” (gRNA) is designed to target the CRISPR-Cas9 complex to cut the desired site in the genome.
- In a proportion of cells, non-homologous end-joining (NHEJ) DNA repair will then make an error while repairing the DNA, resulting in the creation of a small insertion or deletion.
- If this insertion or deletion is not in a multiple of 3 bases, the mutation will lead to a frameshift in the open reading frame, resulting in a nicely scrambled protein.
Alternatively, you can use CRISPR-Cas9 to not only cut the genome, but also provide a repair template. This will allow the cells to use homology-directed repair mechanisms to repair the DNA using the modified version as a template.
As long as the modified repair template has a reasonable overlap with either side of the cut site, you can usually make significant edits. For example, you could change a single nucleotide polymorphism (SNP), or add a reporter gene or regulatory sequence. Insertions of up to several hundred bases have been achieved, but generally speaking, the smaller the change, the greater the chance of success.
So How Do You Do It?
1. Choose Your Guide
- First, decide on what you want to achieve! No, not world domination, but do you want to make a knock-out? Change a SNP?
- Next, design a gRNA to accomplish this goal. There are many free tools that can help you to do this: e.g. CRISPR design and sgRNA designer. If you’re making a SNP change or an insertion, you will also need to design a suitable repair template.
2. Get It Into Your Cells
- The next trick is to get the gRNA(s) into your cells. The simplest way to do this is to acquire or make a single vector containing a cas9 gene and a gRNA expression cassette. Addgene is a good place to start finding gRNA cassettes – just make sure you pick one with an appropriate promoter for your cell type. Once you have the cassette, clone it into the vector containing cas9 and transfect your cells.
- If you’re using a repair template, you’ll also need to clone that into a suitable expression vector and co-transfect it with the cas9/gRNA construct. For maximum success here, you’ll need to either get a reasonably good transfection efficiency (ideally >50%) OR use a CRISPR-Cas9 vector with a fluorescent tag and use flow cytometry to enrich for transfected cells.
3. Check Your Cells
- At this point, you need to find out if your CRISPR-Cas9 gene editing strategy is working. Prepare some genomic DNA from your transfected cells and use your favorite technique to look for the mutation. The most widely used tools here are:
- Next Generation Sequencing (NGS) – although this may be an expensive choice, it is very useful if you have a lot of samples and/or you also want to detect off-target edits.
- PCR – This is quick and easy and accessible by most labs. You can detect a deletion by performing PCR with primers that flank the targeted deletion region. PCR reactions from a successful CRISPR experiment should yield smaller amplicons than control samples.
- Other techniques include assays to detect homology-directed repair and mismatch cleavage assays for indel detection.
4. Go Clonal
- Assuming that your strategy is working, ~5% of the transfected cells will be mutated; the proportion may be higher if your gRNA is particularly efficient.
- Now, you need to make a clonal population to create a new cell line containing the mutation.
- For transformed cells, you can just prepare a single-cell dilution, and once the colonies are big enough, isolate the genomic DNA and continue with the colonies that contain the desired mutation(s)s.
- If it isn’t possible to make a single-cell dilution with your cells (e.g. induced pluripotent stem (iPS) cells), then you’ll need to plate out pooled cells and pick colonies for testing when they are big enough.
So Let’s Get on With It…
Once you’ve got your individual colonies screened and found the mutations, you’ve done it! You’ve got your mutant cell line.
Now the fun is only just beginning! What do you use CRISPR genome editing for? Get in touch by writing in the comments section!
Originally published in 2016. Updated and republished in 2017.Image Credit: Matt Hampel