Genetically Modified Organisms: Editing Genes with CRISPR-cas

By Rene Fester Kratz

One of the big challenges of genetic engineering is to insert a gene exactly where you want it in the genome of the cell. For example, if you wanted to give someone with a genetic disease a normal copy of her defective gene, the ideal way to do it would be to exactly replace her defective gene with the normal copy.

If you could be this precise, you would avoid the risk of having the normal gene insert itself into another gene, disrupting its code and potentially causing worse problems than the disease you’re trying to treat. (For example, disrupting genes that control cell division could lead to cancer.)

Until very recently, this kind of targeted delivery of genes was something scientists were struggling to achieve. But the recent discovery of how a system of bacterial genes works to target specific DNA sequences changed all that, and it’s already causing a revolution in the field of genetic engineering and gene therapy. The name of the bacterial gene system is the CRISPR-cas system (pronounced crisper-cass by scientists).

CRISPR stands for clustered regularly interspersed short palindromic repeats, which is a crazy-long name that you probably don’t need to try to remember. Cas stands for CRISPR-associated genes. CRISPR got its name because scientists noticed that it contained lots of small palindromic sequences repeated throughout the DNA.

A palindrome is a word or phrase that reads the same forward and backward such as “taco cat.” A palindromic DNA sequence could be “AATTAA.”

Scientists figured out that these repeated palindromes of DNA separated short stretches of viral DNA, leading to the realization that CRISPR-cas plays an immunological role that helps bacteria fight off invading viruses. Here’s the way that it works:

  1. Every time a bacterium with CRISPR survives a viral attack, it saves a little bit of viral DNA, tucking it into its CRISPR DNA (in between those palindromes).

    You can think of this like a set of Most Wanted posters that the bacterium keeps on its enemies.

  2. Bacteria use the viral codes in their CRISPR DNA to make small pieces of RNA according to the viral codes.

    Scientists call these RNA molecules CRISPR-RNA (crRNA). Their job is to recognize their matching viral DNA codes, essentially helping the bacteria keep an eye out for viruses they’ve seen before.

  3. The crRNA molecules attach to cas proteins that the bacteria make from the cas genes.

    Cas proteins have the ability to cut DNA into pieces. Once the cas proteins are armed with crRNA molecules, they can recognize viral DNA (thanks to their crRNA partner) and destroy it.

  4. When a virus attacks the bacterial cell, the armed cas enzymes are waiting. If the virus is one the cell has seen before, it gets destroyed very quickly. If it’s the first time this virus has attacked and the bacterial cell survives, the cell makes a new Most Wanted poster to add to its collection.

In addition to being a very cool bacterial defense system, CRISPR-cas has been adapted by scientists to create a targeted gene delivery system. Two brilliant scientists, Jennifer Doudna and Emmanuelle Charpentier, saw how cas enzymes use bits of crRNA to recognize viral DNA and wondered if they could teach those enzymes to recognize other genes as well.

To make a long story short, they figured out how to make a guide RNA that a cas enzyme called cas-9 could use to search out particular genes. The guide RNA contains a short piece of RNA that has the ability to recognize the target gene (just like the crRNA can recognize viral genes). When the guide RNA finds its matching gene, the cas enzyme cuts that gene.

CRISPR-cas in nature
CRISPR-cas in nature.
CRISPR_cas in lab
CRISPR-cas in the lab.

For bacteria, the CRISPR-cas system represents a kind of immunity that defends their cells against invading viral DNA. For some very clever scientists, this system represented just the gene targeting system they were looking for.

The ability of CRISPR-cas to recognize and cut specific genes has the potential to help gene therapy in at least two ways:

  • If a gene makes something that causes disease, scientists could potentially use CRISPR-cas to go in and cut those genes in order to stop them from working. One example could be genes that produce growth factors promoting the growth of cancer cells. Perhaps in the future scientists will send CRISPR-cas into tumors to destroy those genes.
  • If a person has a genetic disease because one of his genes has the wrong code, scientists may be able to use CRISPR-cas to replace the defective gene with a working copy. First, scientists would load the guide RNA with the sequence needed to locate the defective gene. They would send CRISPR-cas into the person’s cells along with copies of the normal form of the gene. CRISPR-cas would find the abnormal gene and cut it. When this happened, the cell’s DNA repair system would attempt to repair the cut. One way that cells would do this is to use a matching piece of DNA to rebuild the damaged DNA. Because scientists included a normal copy of the gene, the cells might use that normal copy for the repair, giving the cell a working copy of the gene.

Although the CRISPR-cas breakthrough is very exciting for scientists and it’s already leading to advances in research, many details need to be examined, refined, and tested before it can actually be used in gene therapy.