Introduction

Genetic engineering is a powerful tool we now have as humans. For many years, however, the techniques required to genetically engineer an organism's DNA were complicated and slow. Then came CRISPR. The CRISPR Cas9 system now allows us to make precise changes to an organism's DNA quickly and cheaply.

As a result, we will see a flood of new ideas that can bring amazing benefits and immense challenges. We will struggle with the ethics and how best to apply the technology. In the not too distant future you will no doubt be affected by this technology in some way, and you will likely need to make a decision regarding its use, so it is important you have some understanding of how it works.

How Does It Work?

To undertsand CRISPR Cas9 it helps to look at how it was discovered and its role in bacteria. The story involves a collaboration between Dr. Emmanuelle Charpentier, a microbiologist researching bacterial disease, and Dr. Jennifer Doudna, a biochemist that studies RNA. What they put together was the mechanism by which bacteria are able to defend themsleves against a viral invader, called a bacterial phage.

It turns out bacteria actually keep a genetic record of past viruses that have attacked them. They do this by storing a section of the viral DNA in thier own DNA. This DNA library can then be used to target a future attack by the same virus.

Bacteria have a Library of past attacks

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How CRISPR got its name

It's the DNA library of past viral attacks that gives CRISPR its name. The location in the bacterial DNA where the library of viral DNA segments are found is characterized by segments of bacterial DNA that are palindromes. A palindrome is something that can be read forward or backward like "madam i'm adam." Each palindromic section is separated by what we call the "spacer" DNA which is the DNA from a past viral invader.

CRISPR stands for: Clustered Regularly Interspersed Short Palindromic Repeats.

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CRISPR and Cas Proteins as an Adaptive Immune System

Bacteria have the ability to cut a segment of the invading viral DNA and insert it into their own bacterial genome. Then, when that same virus attacks again, the bacterium can read off that segment from its library to use as a guide to find the invading viral DNA.

Attached to the guide DNA is a protein that can cut the viral DNA when the guide binds to it. In this way, the complex of the guide DNA and the cutting protein are like a search and destroy mechanism.

The cutting proteins are associated with the CRISPR library of viral DNA, so we call them CRISPR associated proteins, or Cas for short. There are many different Cas's and they are numbered. The one that has gained the most attention and may be of the greatest use for us is Cas9.

(click on image for a pdf that you can print out)

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Video tutorial on CRISPR Cas9 as the adaptive immune system in a bacterium

Making CRISPR Cas9 work for us

What researchers found was that they could design a guide RNA that could be attached to the Cas9 protein. This way they could send the Cas9 protein to whatever segment of DNA they wanted and it would cut the DNA at that specific location. This became a method to cut DNA exactly where we want.

(click on image for a pdf that you can print out)

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How can we use this CRISPR Cas9 complex?

Now, there are two main things we can do once we cut the target DNA.

1. We can let the cell try and repair its damaged DNA. This almost always results in an error, introducing a mutation in that segment of DNA. This can be very useful if you want to turn off a gene. We can cut the DNA somewhere inside the gene and the error prone repair will make that gene nonfunctional. This allows us to study what that gene does, or we can turn off a harmful gene.

2. We can provide an alternate segment of DNA that the cell will incorporate into its DNA where the cut is made. In this way we can insert a new trait.

    

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Where do we go from here?

As you might imagine, this opens up a massive number of potential ways we can alter the genetic code of organisms, including humans.

From genetically engineering crops, to repairing genetic disorders in people, to controlling pests, to bringing back extinct species, the possibilities are as varied as evolution itself.

One of the most promising areas for humans is the correction of genetic diseases. Sickle Cell disease is a prime example because it is caused by one single point mutation in the gene that codes for hemoglobin. By correcting that one nucleotide mistake we can change the misformed protein into a properly formed and functioning one alleviating a debilitating illness.

A critical question that arises is whether we change the DNA of just the individual by altering the gentic material of their somatic cells (body cells), or if we change all the DNA in an individual and, therefore, in their offspring as well, with a germ cell alteration (altering the sex cells or embryo).

In 2018, a Chinese scientist genetically altered embryos using CRISPR and created the first genetically altered humans. These twins, named Lulu and Nana, have been genetically changed in a gene that codes for a protein called CCR5. In addition to the normal functions of this membrane bound protein, it happens to be the doorway that HIV uses to infect an immune cell. By changing this gene, it disables this protein and HIV is no longer able to enter the cells. Lulu and Nana's father was HIV positive and their mother was HIV negative. With this geentic alteration Lulu and Nana are protected from contracting HIV.

What made this result so important and controversial is that by genetically engineering an embryo it means that not only do these twins have this trait, but they will also pass this trait on to their offspring because it is now part of every cell including their reproductive cells.

Other scientists pointed out that there was a risk of harmful mutations due to errors and the risk of the children contracting HIV was not high to begin with. Additionally, the genetic changes that damaged the CCR5 gene in Lulu and Nana were different from any natural occuring mutation in that gene in humans. This means we have no idea what might happen to these children and their offspring in the future.

The Chinese scientist who headed the project, He Jiankui, was sent to prison for 3 years and fined $430,000 for "illegal medical practice." Little is known about Lulu and Nana or a third child that resulted from the project though reports claim they are well.

This result and the scientific backlash illuminates the need to develop a clear set of guidelines and boundaries for this type of research.

Dr. Jennifer Doudna started a non-profit organization called the Innovative Genomics Institute that provides up to date information on CRISPR related projects. Here is a 2022 update on current clinical trials involving CRISPR technology.

In the following videos you will hear about some ideas that may have huge benefits to humans, and some controversies associated with this technology.

Into the Future with CRISPR Technology with Jennifer Doudna

A Promising And Still Uncertain Future For Human Gene Editing

Gene Editing & CRISPR: How Far Should We Go?

Can Genetically Engineered Mosquitoes Help Fight Disease?

Now Oxitec, Ltd. is proposing to release these mosquitoes in Tulare County, California

Continuing to hone the process

Researching CRISPR and the Cas system has been one of the most rapidly increasing areas of biology over the past 10 years. Different groups are designing guide RNA and Cas proteins that can change the rate of gene expression and replace different genes.

One of the concerns related to the use of CRISPR Cas9 has been the potential for errors. There are two categories of errors, on target and off target. On target errors are errors at the target location, and off target errors are when the complex targets the wrong section of DNA. It turns out there is some wiggle room in how the guide RNA binds to the target DNA which can result in the complex binding to a similar, but incorrect, section of DNA. A recent study found a way to decrease that likelihood without slowing down the process.

Gene editing gets safer thanks to redesigned Cas9 protein

As we decrease the potential for errors with this technique and find new ways to alter traits, there will be fewer and fewer technical obstacles to altering the genetic code of any organism and we will have to decide how far we will go.

Will we allow couples to design their children? Will this increase the disparities between the wealthy and the poor?

Will we bring back extinct species or decide to relax protections of endangered ones because we have the power to redesign them?

Will we cure cancer, diabetes, and other genetic diseases?

Will we engineer crops to tolerate a warming climate, saltier soils, and more unpredictable weather? Will that decrease our commitment to address climate change?

Science has again provided great power and shows us what we can do. We have to be mature enough as a species to determine what we should do.