General Biology
Jill Thanh Tien Nguyen
November 24, 2018
CRISPR Technology
Introduction
DNA technology first emerged in the 1970's in which genetic engineering, biotechnology, and nuclear medicine revolutionized the biological sciences. Biologists began exploring the manipulation of DNA molecules by adding, removing, or alternating genes through mutagenesis. In 1987, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) sequences were discovered to act as a defense mechanism in E Coli. DNA is made up of four nucleotides in its DNA sequences, where this system utilizes 'gene targeting:' and 'gene editing' to modify the DNA instructions, enabling them to explain genome variations at a functional level. Scientists have been altering DNA for generations. They do this through a process called “selective breeding” Researchers have used the CRISPR-Cas9 system by targeting genes in different organisms such as bacteria, eukaryotic cells, and even humans in the form of GMOs, genetically modified organisms. Researchers have also used this technology for further cancer research. Considering that cancer is the second leading cause of death with about 8.8 million deaths due to cancer in 2015, this is considered a huge contribution to public health studies. (Zubair Ahmed Ratan*, 2018)
CRISPR-Cas9
The CRISPR-Cas9 is a gene editing system that was first discovered in Streptococcus pyogenes. To begin, a virus will invade the bacteria and insert its genetic material in the bacterium’s DNA and replicate itself. Specifically, when the bacteria are invaded, the virus injects its DNA sequence so that the next time the virus attacks, it is converted to an RNA copy, that carries Cas9 to the invaded viral sequence. During viral attacks, the Cas9 makes a double-stranded cut into the DNA. The bacteria "shears" or acts as a genetic scissor to edit the DNA by replacing the space sequence with another sequence. (SciToons, 2018)
Succinctly, Cas9 is the associated protein to the CRISPR system that is an adaptive immunity to invading foreign cells. These space sequences generate short CRISPR RNA which form a complex with trans-activating crRNA. This system is separated into three stages: 1) adaptation 2) expression and maturation 3) interference.
Naturally found Cas9 variants are large proteins which come in a limited package and delivery into cells with two key components: mature crRNA and tracrRNA, a trans-encoded RNA. These complexes seek the DNA sequence complementary to crRNA. Smaller Cas9 variants have greater therapeutic potentials and require more complex PAM sequences, which is a three-nucleotide-sequence. This sequence is identified and is cut upstream. (Adli, 2018)
There are two possible paths DNA takes after cutting by CRISPR: opposite terminals connecting path or homologous recombination path. The former is prone to error while the latter is designed better for DNA patterns.
Here is a depiction of the whole transaction of genome editing. Cas9 targets the DNA sequence, where tracRNA is found, which then finds complementary cRNA. The PAM site is to destabilize the adjacent sequence.
(Mahmoudian-sani, 2017)
Beyond Genome Editing
CRISPR-Cas9 can go beyond genome editing. The system has an important role in gene regulation, Epigenome editing, Chromatin imaging, Base editing, RNA targeting, and chromatin topology. Cas9 has been used for base editing without double-band breaks.
(Adli, 2018)
Improving Cas9 target recognition fidelity
As a genome engineering platform, there are 9 applications. Refer to the chart compiled from Patrick D.Hsu.
A. The Cas9 nuclease cleaves DNA via its RuvC and HNH nuclease domains
B.Two Cas9 mimics targeted DSBs via cooperative nicks
C. Expression plasmids can be directly transferred into cell line at interest
D. Purified Cas9 can be injected into fertilized zygotes for fast generation of transgenic animal models
E..Somatic genetic modification
F. Genome-scale functional screening
G. Dead Cas9 conversion into a general DNA-binding domain.
H. Illuminating dynamics of genome structure
I. Reconstituting split fragments of Cas9 via Chemicals.
(Hsu, 2015)
Real World Applications
Let’s say we wanted to experiment on mice and selectively breed white mice from black mice. The first way we would expect an offspring of white mice is by chance. If the recessive gene in mice is white, the chance we would get from a dihybrid would be ¼. If one of the parents are homozygous dominant (black), the chance would be 0. The second way is by inserting a template of DNA sequence. Accidental deletion, insertions, and replacements are common. After genome editing, scientists put the DNA template into the embryos, and into the mice, creating a progeny of white mice from their parental black mice.
Perhaps people want to change the eye color of their offspring or get rid of a hormone that releases excess proteins. Genome editing could be used in gene therapy, stem cell research, and gene drive. From all of these researches, malaria, sickle cell disease and cystic fibrosis could advance in treatment. (SciToons, 2018)
Current Events
In recent news, Dr. He Jiankui has announced that he has created the world’s first genetically-modified babies. Two Chinese girls named Lulu and Nana, have been altered to become HIV resistant. Dr. He Jiankui has edited the embryos of the two girls for “implantation”. Some have found this “disturbing” and “unethical” because researchers believe that it is too early to implant edited DNA into humans. There have been debates about whether or not human alterations through implantation should be prohibited, but conversely, this could be the next advancement to altering human genes for a more resistant human race.
(CNN, 2018)
CRISPR-cas9 Backfire
So, what happens when the CRISPR system backfires? Why is this so unethical? Studies have shown that CRISPR-Cas9 genome editing “induces a p53-mediated DNA damage response”. (Seeker, 2018) When DNA is cut, it could become damaged and kill the cell and prevent cell growth. CRISPR-Cas9 are prone to defective cells called p53 and could increase cancer in patients.
CRISPR Overview
CRISPR-Cas9 alters DNA sequences with the help of crRNA, tracRNA, in the PAM site. It has gone beyond editing and have been used to alter the offspring of organisms. This has been evident in animals, food, diseases, etc. Researchers have questioned the ethics behind genome editing especially in humans and in cases where CRISPR-Cas9 backfires. There have been current events that have demonstrated genome editing that questions these ethics.
Is CRISPR the answer for future generations and advancements to biotechnology? With the cutting and pasting of genome editing, the unprecedented rate of success of replacing a DNA strand with another is 60%. (Sanders, 2016) If we could increase that success rate, perhaps implanting in humans could be a definite answer to resistance of viral cells. It would be years before researchers will be able to examine the results in humans.
Bibliography
Adli, M. (2018). The CRISPR tool kit for genome editing and beyond. Retrieved from Nature Communications: https://www.nature.com/articles/s41467-018-04252-2
CNN. (2018, November 30). Scientist claims he helped create world's first genetically-modified babies. Retrieved from Scientist claims he helped create world's first genetically-modified babies
Hsu, P. D. (2015). Development and Applications of CRISPR-Cas9 for Genome Engineering. Retrieved from PMC: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4343198/
Mahmoudian-sani, M.-R. (2017). CRISPR genome editing and its medical applications. Retrieved from Taylor Francis Online: https://www.tandfonline.com/doi/full/10.1080/13102818.2017.1406823
Sanders, R. (2016). Advance improves cutting and pasting with CRISPR-Cas9 gene editing. Retrieved from Berkeley News: https://news.berkeley.edu/2016/01/20/advance-improves-cutting-and-pasting-with-crispr-cas9-gene-editing/
SciToons. (2018, March 30). Retrieved from What is CRISPR-Cas9?: https://www.youtube.com/watch?v=9q8AGst7KiY
Seeker. (2018, September 25). What Happens When CRISPR Backfires? Retrieved from https://www.youtube.com/watch?v=8b_d3RIJJmo
Zubair Ahmed Ratan*, Y.-J. S.-H. (2018). CRISPR-Cas9: a promising genetic engineering approach in cancer research. SageJournal.