CRISPR — General Info & Intro
I figure I should preface that I am not involved with any CRISPR development, but this would hopefully serve as an educational piece for those interested in figuring out, “What the heck is CRISPR?”
Hopefully this will serve as some help:
The players:
CRISPR = Clustered Regularly Interspersed Spacer Palindromic Repeats. We should first discuss how to parse this acronym apart.
crRNA = the sequence of RNA we will incorporate into the cell(s) that will correspond to the target genetic sequence we want to delete
Cas9 = Nuclease protein that is able to cut the target double-stranded DNA sequence. Contains two protein domains HNH and RuvC
HDR = homology-directed repair that helps to replace the broken DNA with correct nucleotides
NHEJ = non-homologous end-joining. Does not require template, but adds random nucleotides to the broken off strand
PAM = protospacer adjacent motif
The hazard:
To introduce the subject, many sources have essentially put CRISPR under the umbrella of gene-editing software that can make it seem as if we can implant any vector, or adeno-associated virus (AAV) containing genetic information that we want to express, into any organism that would bring about a target phenotype.
As it stands, this is only a half-truth. There are still lots of nooks and crannies left to be considered in this subject. For instance, the human genome is vast, with three billion base pairs in each nucleated cell in the body (recent estimate of number of human cells which make up the body is 30 trillion!). The likelihood of off-target effects (OTEs), situations where an introduced gene may accidentally target the wrong area of the genome with a similar target sequence cannot be neglected. Additionally, the repair mechanisms of DNA damage that us mammals possess, are two-fold: one being quite error-prone, and the other being more reliable.
The non-homologous end joining (NHEJ) mechanism adds random nucleotides to the fractured ends of strands until the damage has been repaired and the pairs of chromosomes end up matching again. This takes place during the G1, S, and G2 phases. In other words, think of two homologous chromosomes lined up next to each other, and one strand has a nick in the middle of its genetic sequence. NHEJ would go about solving this problem by adding random nucleotides, hoping to still keep that important homolog relationship with the other single strand after repair (Shomu’s Biology). Here, the problem is not deleting the sequence we want to delete but cleaning up after our own mess.
Meanwhile, Homology-directed repair (HDR), uses that homologous relationship as a template to anneal correct nucleotides, while keeping the all-important homolog relationship intact. This takes place during the late cell cycle phases S and G2. So how can we maximize the likelihood of this process, while inhibiting the chance that NHEJ can take effect (Douch)?
(Note: G1/G2 signifies the growth stage of a cell. S phase indicates the replication of the DNA of the cell. M phase points to the mitotic, or finally, the division of one cell into two cells. The difference between G1 and G2 is that the former prepares for DNA replication, and the latter prepares for Mitosis. G1 checks for any DNA damage and if cell has grown adequately enough, G2 precisely checks for any errors in DNA replication)
It turns out, this has been attempted through inhibiting enzymes specific to the NHEJ pathway. With some knowledge of the biochemistry involved in the NHEJ pathway, we can chemically inhibit Ku, DNA Ligase IV, DNA-PK. These molecules each play a role in the pathway of repairing DNA structures that suffer any sort of damage. One such chemical drug used to inflict such damage, Aphidicolin, increased HDR frequency in HEK293T (human embryonic kidney cells). In addition, Nocodazole, caused M cell cycle phase arrest in those same cells (Uddin et al.). Nishiyama et al., another group working on this project, edited a CAMKIIα (a calmodulin protein kinase receptor that responds to Ca++ binding) locus through HDR in postmitotic hippocampal neurons of adult mice. They used an AAV Cas9 guide RNA, along with a donor template in CAMKIIα locus, achieving successful HDR-mediated edits in ~30% of infected cells.
As such, it becomes difficult to envisage a mechanism that allows us to effectively undergo such a process without the risk of a failed repair mechanism. But the applications of its potential are revolutionary, which I will discuss here:
Cancer immunotherapy, managing AIDS, Covid assays, and human embryo modifications to prevent genetically-linked diseases are among many reasons why CRISPR is such an interesting tool to develop.
In cancer immunotherapy, scientists can genetically modify T-cells to help recognize and kill cancer cells. To do this, they can extract T-cells from the human body, invoke a chimeric antigen receptor (CAR), giving the T-cell the special ability to respond to cancer cells themselves. Once these T-cells are implanted back into the body, the target that is provided by the CAR, leads to a T-cell response against cancer. The big determining factor of a successful response would be to distinguish a receptor between a cancer cell and a regular human cell. This has been done with acute lymphoblastic leukemia, chronic lymphocytic leukemia, and B-cell non-Hodgkin’s lymphoma (Harvard). The same webpage details the risk of cytokine release syndrome, messenger molecules that if released in excess, can lead to excessive inflammation of the body.
Another application of this technology can be used in Leber Congenital Amaurosis (LCA), expressed by a biallelic loss of function mutation in the CEP290 gene. Specifically, LCA manifests in the retina that can lead to photophobia and nystagmus (involuntary movements of eyes). The EDIT-101 trial delivers CRISPR/Cas9 directly into the retina of LCA patients with intronic IVS126 mutation that will drive aberrant splicing and then a nonfunctional protein CEP290 (EDIT refers to the company EDITAS, IVS126 is in the intron region that eventually is not expressed). I will discuss the mechanism by which this happens, as illustrated in Ruan et al. paper, but if you would like to look at the research (CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10 — PMC (nih.gov)). The mutation of this IVS126 gene encodes a “cryptic” exon sequence leading to a premature stop codon. In that case, a truncated protein is produced, which the development of that leads to LCA. This therapy helps to excise the target sequence of IVS126; the authors of the study successfully “sustained generation of increased levels of wild-type CEP290” (Ruan et al.).
I will upload another blog containing more information about exactly how CRISPR works, along with some applications of these same mechanisms. As for now, this blog has exceeded the planned word limit I had anticipated.
Andrew Douch. (2022, March 16). Understanding CRISPR-Cas9 [Video]. YouTube. https://www.youtube.com/watch?v=cLMo6DYdJRE
Professor Dave Explains. (2021, October 29). CRISPR-Cas9 Genome Editing Technology [Video]. YouTube. https://www.youtube.com/watch?v=IiPL5HgPehs
Shomu’s Biology. (2020, October 9). Crispr cas9 gene editing explained [Video]. YouTube. https://www.youtube.com/watch?v=OPkPwNVNLEs
Uddin, F., Rudin, C. M., & Sen, T. (2020). CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future. Frontiers in oncology, 10, 1387. https://doi.org/10.3389/fonc.2020.01387
