DNA repair mechanisms: Related to CRISPR
Several mechanisms of DNA repair exist in mammalian bodies, acting as an important defense against cell cycle malfunctioning.
In CRISPR research, how can we visualize how to not only modify genetic expression but ensure that this modification will not lead to faulty DNA repair mechanisms or a downstream deleterious mutation? Much of the talk thus far has been about eliminating sequences of nucleotides, yet we have stopped short of discussing how can DNA repair itself?
It might help to look to other instances where DNA repair takes place. Our immune system itself relies on breaking the DNA, repairing itself to diversify our antibodies, aka VDJ recombination. It is in this sequence that a plethora of proteins helps open the genetic code of the progenitor lymphoid cells that will later become immature B-cells or T-cells. If immature B-cells meet the antigen that corresponds to its receptor, antibodies will then be produced to encounter the antigen. Owing to this process is how our immune system can fend our bodies off from an assortment of different pathogens. In this case, the initial step of VDJ recombination shapes the outcome of what antibodies our B-cells will produce.
Nonhomologous end-joining (NHEJ) is a major double-strand break (DSB) pathway that undergoes this process:
a) Double-stranded DNA break (DSB) is first recognized by heterodimer consisting of Ku70 & Ku80 proteins
b) DNA-PK, another enzyme, has a high affinity for DNA ends that become even tighter when in a complex with the Ku proteins
c) “Artemis,” a nuclease protein that exists in a tight complex with DNA-PK and becomes recruited. This endonuclease is important in opening the hairpin during VDJ recombination that interacts with RAG1, and RAG2 genes
d) Pol X family polymerases add random nucleotides to the DNA ends
e) DNA Ligase IV, including XRCC4, XLF, and PAXX carry out ligation. (Pannunzio et al.)
The above process occurs during all cell cycle phases besides the S and G2 phases. Ku70/80 at high abundance increases the likelihood of Ku70/80 binding at the broken DNA end. This catalyzes the rest of the NHEJ pathway. A similar process takes place during VDJ recombination that leads to different antibody receptor production. Here, we see step (d) where the Pol X family polymerases add random nucleotides to the DNA ends. As such, this process is risky, since we are unable to assure correct base pairing matches (A — T, C — G). Unlike Homology directed repair (HDR), these random additions can lead to problematic transcription and translation events.
The HDR pathway is notably safer since it relies on a template to provide DNA repair but is less common and does not occur in VDJ recombination. It has been proposed that CYREN, a sub-peptide that affects Ku binding, can redirect the cell into either undergoing NHEJ or HDR. A dysfunctional NHEJ pathway is associated with Severe combined Immunodeficiency (SCID), which can lead to dysfunctional T-cells or B-cells. Tragic examples are demonstrated by newborns who must live inside of a bubble, isolated from the environment, to limit the incidence of infection.
The promise of safety from undergoing HDR could give way to a promising approach where we can utilize CRISPR to delete an undesirable sequence, pairing the alteration with Homologous recombination instead of Nonhomologous end-joining. The normal pathway for Homologous recombination (HDR) follows this:
a) DNA on each side of the DSB is resected in a 5’ à 3’ direction, resulting in 3’ overhangs on each of the broken ends
b) Single-stranded overhangs are stabilized by replication protein A, bound to Rad51 recombinase protein, each forming a nucleoprotein filament
c) Nucleoprotein filament invades Homologous repair template
d) Invasion forms a displacement loop, overhang binds with one strand of the template and displaces the other
e) DNA polymerase extends the 3’ end (Enzmann)
Mutations to the homologous recombination process are associated with Bloom syndrome or BRCA1/BRCA2 mutations, which both confer risk to cancers.
Berkeley Fanconi Anemia findings:
Seeing that the DNA repair commencing after CRISPR cutting has been met with several issues, one risk that people have noticed is Fanconi anemia. Interestingly, the purpose of using CRISPR is to one day hopefully find a cure for Sickle-Cell anemia, a genetic condition that involves one’s blood cells to adopt a sickle-shaped condition, which can critically damage blood flow by blocking flow completely in certain blood vessels.
Yet, the treatment for this can open another complication of developing Fanconi Anemia (Sanders). If any of the genes which confer twenty-one separate proteins are damaged from this therapy, the bone marrow will be unable to make enough new blood cells, leading to anemia. One of these twenty-one proteins, FANCD2, seems to play a role in regulating the insertion of new DNA into the genome at the specific CRISPR cut site (Sanders). This begs the question, could we somehow manipulate the FANCD2 to turn on/off gene repair, or could we maximize the likelihood of the cell undergoing HDR instead of NHEJ?
Current application to Antimicrobial Resistance Genes:
Bacteria’s immune system is where CRISPR originally retrieved its name, harnessing a foreign DNA molecule (for example, a bacteriophage) that is absorbed by the nucleus and incorporated into its genome. Each subsequent encounter with the same bacteriophage or invading organism will be met with resistance since the originally injected DNA is incorporated into the bacterial genome that subsequently undergoes transcription to make complementary RNA. This complementary RNA will interact with the bacteriophage DNA, once the bacteriophage DNA is injected into the cytosol of the bacterium.
One example of utilizing CRISPR, amid the DNA repair mechanisms that exist in bacteria, is to delete antimicrobial resistance genes that are beginning to accumulate in potent pathogen-causing bacteria. As Hyunjin Shim describes Pseudomonas aeruginosa, a bacteria known to cause cystic fibrosis, can withstand many different antibiotic drugs. One of the potential treatments is utilizing CRISPR technology to delete these Antimicrobial-resistant genes, utilizing a native DNA repair mechanism to mutate the gene that will then no longer be able to withstand certain antibiotics. Balancing the interplay of CRISPR-Cas systems with DNA repair pathways during the spacer acquisition event will need to be considered. Eventually, scientists may be able to design a CRISPR-Cas system that can enter a bacterial nucleus, knock out an Antimicrobial-resistant gene, and leave the bacterium susceptible to the antibiotic.
Taking this into account, it seems there still needs to be further research into the practical usage of such technology (forgive me if you have heard this before about any cutting-edge research). The issue does not solely involve how to use CRISPR-Cas technology, but how can we control DNA repair? How can we ensure that after such deletion, the planned repair we hope to do comes to fruition?
Croton, H. (2021, Jan. 26). Federal committee recommends more research, care for patients with sickle cell. Cronkite News — Arizona PBS. https://cronkitenews.azpbs.org/2021/01/26/federal-committee-aim-to-help-patients-with-sickle-cell/.
Enzmann, B. (2019, May 30). CRISPR Editing is All About DNA Repair Mechanisms [Review of CRISPR Editing is All About DNA Repair Mechanisms]. https://www.synthego.com/blog/crispr-dna-repair-pathways#:~:text=While%20CRISPR%20components%20cut%20the%20DNA%20at%20the,a%20double-strand%20break%20%28DSB%29%20at%20that%20particular%20spot.
Pannunzio, N. R., Watanabe, G., & Lieber, M. R. (2018). Nonhomologous DNA end-joining for repair of DNA double-strand breaks. The Journal of biological chemistry, 293(27), 10512–10523. https://doi.org/10.1074/jbc.TM117.000374
Sanders, R. (2018, July 30). DNA repair after CRISPR cutting not at all what people thought. Berkeley Research; Berkeley Research. https://vcresearch.berkeley.edu/news/dna-repair-after-crispr-cutting-not-all-what-people-thought
Shim, H. (2022). Investigating the Genomic Background of CRISPR-Cas Genomes for CRISPR-Based Antimicrobials. Evolutionary Bioinformatics. https://doi.org/10.1177/11769343221103887
YouTube. (2020). Dna Repair Mechanisms: Beautiful Usmle Lectures. YouTube. Retrieved June 12, 2022, from https://www.youtube.com/watch?v=ODsBTJ1KZY0.