A better alternative to immortal cells (2023)

Genetic editing is key to understanding human disease. We usually use immortal cells for in vitro experiments, but they have abnormal genomes that are not representative of the average human. Induced pluripotent stem cells can be differentiated into almost any species and genome-edited using CRISPR, creating a powerful alternative to immortal cell lines for the study of human biology.

Adjusted according toPrecise, high-efficiency stem cell processing for human biology testing and disease modeling,BitesizeBio webinar sBill Skarnes.

Studying human diseases and genetic diversity is not easy.In vitroStudies allow us to manipulate the genome of cells fairly easily, but creating model systems that are easy to work with and provide valid data is challenging.

To overcome this, we usually resort to immortal cells. But these cell lines are not normal. They have important mutations in their genome, exhibiting characteristics not found in healthy peoplelivecells, and some are of unethical origin.

Fortunately, there is an alternative to immortal cells.

Using CRISPR to edit induced pluripotent stem cells is one way to generate physiologically relevant and translational models. We will discuss this in this article.

We will cover the basics and explain the guiding principles of genome editing topics. Plus, we'll give you a bunch of practical steps to monitor and improve processing efficiency, design RNA routers, check the zygosity of your clones, and more!

A CRISPR iPSC genome editing platform for probing human biology

This hands-on approach to human biology research and disease modeling combines two Nobel Prize-winning technologies. These are:

1. Reprogramming stations.
2. CRISPR.

We'll go into this in a little more depth next. Briefly, cell reprogramming allows the creation of induced pluripotent stem cells (iPSCs) from somatic cells and, using CRISPR, we can edit the genome of iPSCs to change their phenotype.

It gives us access to a huge amount of human biology. For example:

• Early human development (embryos).
• Organogenesis (organoids).
• Hematopoiesis/innate immunity.
• Rare diseases.
• Gene therapy.
• Cell biology.
• Metabolomika.

Now let's look at these technologies in a little more detail.

Induced pluripotent stem cells

The strength of iPSCs is that they can differentiate into almost any cell type in the human body.

Furthermore, these cells have a normal genome, unlike the cells commonly used in human cell biology and biochemistry in the past, such as HeLa and HEK293 cells, which have highly abnormal genomes that do not represent those of the average human.

Despite the genomic abnormality,The non-consensus origin of HeLa cells raises a serious ethical question.

Furthermore, you can grow iPSCs indefinitely and maintain a normal diploid karyotype: a healthy set of 23 homologous pairs of chromosomes.

There are some disadvantages of iPSCs. The main ones are that the efficiency of inducing pluripotency is sometimes low and sometimes involves virus deliverycarcinogenicgenes in somatic cells.

For a more in-depth look at the pros and cons of iPSCs and how to make them, check out this paper by Medvedev, Shevchenki, and Zakian. [1]

What is CRISPR?

CRISPR is a revolutionary gene editing technology that needs little introduction to most readers. If you need an introduction, check it outBitesize Bio Simple Guide to CRISPR.

In short, it is a powerful tool for cutting and altering the DNA sequences of bothin vitroilive. A small RNA molecule called a guide RNA (gRNA) guides a nuclease (usually Cas9) to a desired target site in the genome to remove or add genes, correct mutations, and even regulate gene expression.

CRISPR offers two major advantages over conventional gene editing tools. These are:

1. You can make single base pair changes by cutting or tagging the DNA. [2]
2. You can modify both copies of a gene at the same time (biallelic mutations).

However, for CRISPR to be a valid genome editing tool, there are two key requirements:

1. The target sequence intended for modification is genomically unique.
2. The target sequence is a few base pairs upstream of the adjacent protospacer motif sequence (PAM sequence).

If any of these criteria are not met, CRISPR will not work.

See this post forlearn more about Cas9s and what wires they haveick.

What are PAM sequences?

PAM sequences are short DNA sequences that are necessary for cleavage by Cas nuclease.

They are found on a non-complementary chain: nucleic acid chain, ssame seriesas guide RNA.

And this is usually 3-4 base pairs downstream of the Cas nuclease cut site.

You will find a list of common PAM sequencesBitesize Bio guide to nucleasesand towards its bottomAddgene article on CRISPR.

A word to the wise: when designing guides for your CRISPR experiments, avoid those that introduce mutations into the PAM sequence.

Practical instructions for CRISPR iPSC genome editing projects

Now you know the benefits of creating an alternative to immortal cells using iPSCs and CRISPR, here are some practical tips on how to do it well.

Guiding principles

Five basic principles or steps that apply to any genome editing project.

1. Subclone the iPSCs

Subcloning of induced pluripotent stem cells (iPSCs) allows you to isolate a clonal population of cells with the best genetic properties for your project.

This will reduce the genetic heterogeneity of your edited clones, increasing the consistency and reproducibility of your results.

In addition, subcloning helps eliminate unwanted off-target effects that can arise from the genome editing process.

2. Careful characterization of iPSCs

It is always wise to characterize your iPSC line to ensure that they have:

1. An intact genome. Use karyotyping, high-density SNP arrays (discussed later), and whole-genome sequencing.
2. It is not mutatedTP53. It is frequently mutated in cultured iPSCs.
3. Good differentiation potential. Use mRNA microarray, RNA sequencing or qPCR. [3]

3. Edit through

Enter only mutations relevant to your research: those found in the human population without additional changes.

Although you can introduce silent mutations that improve processing efficiency, they can affect gene transcription and cause off-target effects.

4. Label the clones after processing

Characterize your clones after processing to verify that you have introduced the desired mutation.

Also, check the cells to make sure they maintain a normal genome after treatment. Look for off-target effects that you may have unintentionally introduced due to the desired mutation.

Re-karyotype the cells and use long-range PCR to screen high-density SNP arrays to determine the zygosity of your clones (more on this later).

5. Check/Reverse

Once you've established that the cell phenotype you want to display is caused by a mutation and not something else acquired during cultivation and processing, you revert it to the wild-type sequence using CRISPR (Picture 1).

Reversion checks for off-target effects, but note that revision efficiency does not necessarily correlate with processing efficiency.

DNA repair methods in CRISPR

RNA serves as a guide to direct Cas nuclease to specific DNA target sequences, and the RNA-nuclease complex is called a ribonucleoprotein (RNP).

These complexes recognize a specific side of the genome and form a double-stranded break.

You can use one of two ways to repair the fault:

1. Non-homologous end linkage.
2. Correction based on confession.

Non-homologous end joining (NHEJ) is an error-prone process that resultsinsertion/deletion mutations;at a breakpoint in one or both alleles. This is useful for projects where you want to mutate a gene in such a way.

Homology-directed repair (HDR) involves the addition of a donor template that will integrate into the breakpoint and introduce single nucleotide variations.

Check it outFigure 2to show it graphically.

The two methods compete with each other. HDR is accurate but can be inefficient, with a low percentage of cells showing the desired mutation. This could be a problem for large projects.

NHEJ is more efficient but is prone to the imprecise types of mutations mentioned above.

Strategies to improve HDR performance

As mentioned, the yield of nuclear contamination for HDR can be low. Maybe less than 10%. Fortunately, there are ways to improve its effectiveness. Strategies to increase its effectiveness include:

• Small molecule enhancers. [4]
• Cold shock. [5]
• Final modified oligo donors. [6]

The advantage of these methods is that they involve supplying cells with reagents and conditions. No cell selection or enrichment is required.

Please note that this list is not exhaustive. You can see an overview of the strategies here. [7]

How to track efficiency improvement strategies

So you have some ideas to improve the efficiency of your gene editing, but how do you track them to know if they worked?

If you change the C to T in the histidine codon of the blue fluorescent protein BFP, you get the green fluorescent protein GFP.

• CAT → Histidine.
• TAT → tyrosine.

These proteins have different spectral properties. the first is blue, the second is green.

You can introduce this mutation using CRISPR under different iPSC conditions to monitor the efficiency of the editing process and select conditions that maximize editing efficiency.

This is a simple but powerful assay to monitor the efficiency of NHEJ and HDR. Plus, the results are easy to interpret! [8,9]

• No mutation → blue fluorescence.
• Successful HDR mutation → green fluorescence.
• Successful NHEJ mutation → no fluorescence.

The reason why NHEJ will not result in fluorescence is because of insertion or deletion mutations. It can even lead to a frame shift when the insertion or deletion length is not a multiple of three.

RNA guide design

When doing genome editing, you need to design guide RNAs that deliver the Cas nuclease to the desired target site in the genome.

WGE is an excellent tool for CRISPR RNA guide designwhich helps identify the promoters that overlap the target site and shows you their position relative to the PAM sequence. [10]

It also provides an off-target performance score that quantifies the number of off-target locations with X-base differences.

Target promoters that do not match other regions of the genome and very few mismatches of one or two bases.

And, as mentioned, avoid drivers that mutate the PAM sequence.

Screen Driver RNAin vitroilive

Drivers are wild cards in genome editing experiments and there will be variable activity between drivers.

A driver can have a great off-target score, but return only wild-type clones, or work only for (say) NHEJ. An almost identical driver (let's say just slightly shifted from the first one) can return many cells processed based on HDR with the desired mutation.

In most cases, one or more potential guide RNAs will guide the Cas9 nuclease to the target sequence for cleavage.

When that happens, check them outin vitrousing a mobility shift assay to see which guides best cleave the target sequence.

Then, when using the Genome Editing Experiments Guides,Record your sequencing results to see which drivers give you homozygous biallelic mutations, heterozygous mutations, and no mutations (wild type).

You can also check if the drivers for HDE, NHEJ or both are working.

You may find that the guide will only work for one repair method. This will give you an idea of ​​how well a particular mutation is tolerated or whether it is lethal. For example, no mutations are introduced via HDR, but you get a lot of wild-type and successful NHEJ-edited clones.

How to check the zygosity of clones

Depending on what you're studying (especially if you're working on genetic diseases), you may want to check the zygosity of your edited clones.

In other words, check whether you are introducing a mutation in one or both alleles.

An effective strategy may be to add inactivated Cas9 (also called dead Cas9 or dCas9) with the guide RNA during the nuclear infection step. [9]

dCas9 will bind to the target sequence, but no cleavage will occur.

Thus, dCas9 protects the cleavage and modification site from active Cas9 on any gene copy.

Note that the amount of dead Cas9 you add is critical to the result. So add a mixture of Cas9 and dCas9 to your experiments. For example, 1:1, 1:1.5, 1:2, 1:4, ratios etc.

Then measure the dose response of your cells to dCas9 and plot the percentage of homozygous, heterozygous, and wild-type clones versus dose to choose the sweet spot for the desired effect.

You can also monitor and quantify this optimization using the BFP assay mentioned above, as dCas9 will increase the percentage of wild-type clones, leading to less conversion of BFP to GFP.

Quality control: Check homozygous clones

They must be checked for adverse effects on the target that may occur during the processing process.

For example, one allele contains the desired mutation, while the other is completely lost.

To test whether a homozygous clone is indeed homozygous, you can do a long-range PCR, examineheterozygoussingle nucleotide polymorphisms (SNPs) on either side of the target sequence and verify that they are still heterozygous after editing. [11]

Loss of heterozygosity of a SNP indicates that the allele has been deleted or transposed. Or there is spam.

So be careful!

In short, CRISPR iPSC alternative to immortal cells

It's a lot to summarize, but I'll try!

Significant problems arise when using immortal cells, but CRISPR and cell reprogramming combine to create an alternative to immortal cells.

We explored the power of this technology and how it can be used to study human disease and genetic variation.

And readers involved in existing genome editing projects now have an arsenal of ideas, tips, analysis and resources for adding additional levels of control.

Here are more impressive results and discoveries from them.

Do you have anything to add? Let me know in the comments section below.

bibliographical references

1. Medvedev SP, Shevchenko AI i Zakian SM (2010.)Induced pluripotent stem cells: problems and advantages of their application in regenerative medicine.Law Nat 2(2): 18–28
2. Rees HA i Liu DR (2018.)Basic editing: precision chemistry in the genome and transcriptome of living cells. Nat Rev Genet 19(12):770-88
3. Liu LP i Zheng YW (2019.)Predicting the differentiation potential of human pluripotent stem cells: opportunities and challenges.Stem cells of the world J 11(7):375-382
4. Yu C, Liu Y, Ma T,et al. (2015)Small molecules enhance CRISPR genome editing in pluripotent stem cells.Stem cell cells 16(2): 142–7
5. Guo Q, Mintier G, Ma-Edmonds, M,et al. (2018)'Cold shock' increases frequency of homology-directed repair gene processing in induced pluripotent stem cells.Sci Rep 8:2080
6. Ghanta KS, Chen Z, Aamir Mir,et al. (2021.)5'-Modifications improve the potency and efficiency of donor DNA for precise genome editing.eLife 10:e72216
7. Liu M, Rehman S, Tang X, Gu K, Fan Q, Chen D i Ma W (2019.)Methodologies for improving HDR performance. Front Genet 9:691
8. Skarnes WC, Pellegrino I McDonough JA (2019.)Improving efficiency of homology-directed repair in human stem cells.Method 164–165 (view, professional).:18–28
9. Skarnes WC, Ning G, Giansiracusa S,et al.Control of homology-directed repair effects in human stem cells with dCas9.bioRxiv
10. Hodgkins A, Farne A, Perera S, Grego T, Parry-Smith DJ, Skarnes WC i Iyer V (2015.)WGE: CRISPR Database for Genome Engineering.Bioinformatics 31(18):3078-80
11. Wisdom I, Kroeger JA, Malik R, Klimmt J, et al. (2020)Detection of off-target effects after CRISPR editing via HDR.Tail. station 31(8)