De-Extinction – Bringing Back The Wooly Mammoth

The general public was first introduced to this concept of de-extinction in the blockbuster film Jurassic Park (1993). Present efforts at de-extinction are focused on more modern animals rather than dinosaurs. The reason being the availability of complete DNA. The biology behind de-extinction actually has several components: cloning technology, gene sequencing, and gene editing (called CRISPR; say “crisper” like how you might order fries with your burger). De-extinction efforts presently involve the Wooly Mammoth and the Tasmanian Tiger (which was actually a Marsupial predator about the size of an un-related wolf). 

Wooly mammoths became extinct possibly as recent as 5000 years ago. Melting ice has exposed many frozen specimens and provided enough DNA to sequence their entire genome. Wooly mammoth’s nearest living relative is the Indian Elephant according to its genome (genome: previous blog). Efforts to re-create the Wooly Mammoth are two-fold and the two approaches differ. First is conventional “modern cloning”. South Korean scientists have successfully cloned many dogs. Use Google and search on “Suppy” a cloned Afghan dog. Suppy’s clones have been cloned. The rich and famous in Hollywood have had their dogs cloned. This approach for the Wooly Mammoth would take a Mammoth cell with intact DNA from the Mammoth and then extract the nucleus with its full set (2n) of chromosomes. An ovum from an Indian Elephant would be de-nucleated (but the remainder of sub-cellular organelles left intact in the ovum). The Wooly Mammoth’s nucleus is inserted into the Elephant ovum and proprietary technology is used to cause the ovum to start dividing. It is implanted into the uterus of an Elephant which will act as a surrogate mother. With elephants, gestation and maturation to adult body forms is a decades long process. The idea is to first produce a small herd and then allow them to grow into more herds of mammoths. 

The second approach for the Wooly Mammoth is to use the gene editing technology of CRISPR to edit into the Indian Elephant’s genome those portions of the Wooly Mammoth’s genome that are different. The Indian Elephant as well as African Elephant’s genome have been sequenced. The goal is to produce a “Wooly Elephant.” Through selective breeding and more gene editing it is believed that something pretty close to a Wooly Mammoth would be produced. CRISPR technology allows genetic engineers to cut out and replace very specific portions of a creature’s genome and either render it “non-expressed” or edit it “improved.” As long as the cut/repair is accurate all is well. If it is off-target then bad things happen such as rendering an adjacent gene non-functional. CRISPR uses bacterial enzymes to make the cut and splices. The enzymes which make the cuts utilize a manufactured guide molecule of RNA which is very specific and will only position the cutting enzyme at the intended point of the genome. The key to success being SPECIFICITY, such that changes to the genome (previous blog) are accurate. There are several  human genetic diseases that are simply one single base change in the human genome; one out of 3.2 billion! If you could apply CRISPR to fix the genetic error in a sperm or ovum, then a parent would not pass the genetic disease to their children.

How different are the genomes of Wooly Mammoth vs Indian Elephant? Asian elephants and woolly mammoths share a 99.6 percent similar DNA.  For comparison, humans and chimps share a 98.8 percent of their DNA. Despite the close percentages there are lots of differences. Humans and a banana share 44% of our genomes! This is because so much of an organism’s genome is for basic shared cellular processes related to energy metabolism.

Efforts for de-extinction of the Tasmanian Tiger (Thylacine) also known as the Tasmanian wolf, will be a bit more complicated. (Note: Tasmanian Tiger are not related to tigers and are not related to wolves. They are also not related to Australian Dingos which are derived from domesticated dogs which early man brought to Australia from neighboring islands during human population expansion. There is speculation that they may still survive in some remote regions. The last known Tasmanian Tiger died in a zoo in 1936. Tasmanian Tigers are marsupials so a marsupial will have to be selected to serve as surrogate mother. Embryonic marsupials are very poorly developed when they crawl from the uterus into the mother’s pouch. Finding a suitable surrogate from among exiting marsupials will present a challenge. An alternative approach which scientists might use is to replace the surrogate/pouch phase with “bottle” feeding milk to the tiny embryos.

CRISPR ADDENDUM FOR GENETICS STUDENTS

A Quick Overview Of CRISPR

CRISPR – stands for “clustered regularly interspaced short palindromic repeats” These sequences are present in bacterial genomes and represent fragments of former viral infections of the bacteria. They are used by the bacteria to destroy viruses that infect the bacteria. CAS-9 (or “CRISPR-associated protein 9”) is a bacterial enzyme that uses the sequences to target invading viruses and destroy them by cutting the viral genome. There are other “CAS” enzymes. So what if you could “guide” CAS-9 to a specific location in an organism’s genome and make a cut. The cut would be guided not by fragments of former viral infections as is the case in bacteria, but it would be guided by man-made guide RNA designed to align with a target area of the organism’s gene and make a cut or edit. As you will see we will need: 1) the CAS enzyme, 2) a man-made carrier or guide piece of RNA which can carry the CAS enzyme and has a man-made sequence that will base-pair with the gene at the intended cut site, and 3) a “PAM” (e.g., GCC) in the proper place (see PAM BELOW). Suppose that you want to change the genome at the location indicated by the larger font:

The bold italic portion located to the left of the intended cut provides a VERY specific 23-base pair target. Since the human genome is known and there are websites where one can search the entire human genome for a sequence one can verify that there are no un-intended portions of the genome that have the same “target.” And it has the so-called PAM (Protospacer Adjacent Motif (which is 2-6 base pairs in size) GCC at the right side of target which is specific for the cutting enzyme (CAS-x). What is needed now is a guiding molecule that will take the cutting bacterial enzyme “CAS-x” to this VERY specific point in the genome. Without the “guide” the cutting enzyme will never make a cut. Since we know the base or genome sequence at the planned cut-location we synthesize a guide piece of RNA which will match up with the DNA at the planned cut location.