Module 3- CRISPR and Gene Editing Flashcards
CRISPR video
https://www.youtube.com/watch?v=4YKFw2KZA5o
Bacteria’s First Line of Defense
- Think back to the Immunity and Vaccines lecture — bacteriophages are a type of virus that infect bacteria
- To prevent viral disease, CRISPR evolved as sequences of DNA that are derived from bacteriophage genes that have previously infected the
bacteria - When the bacteriophage attacks again, their newly inserted genes are recognized and removed by CRISPR-Cas9
How can we harness the CRISPR-Cas system?
- The CRISPR-Cas system gives bacteria acquired immunity… but it can also be used for other purposes
- Think about it: evolution has given us a tool that can recognize specific sequences of nucleotides and remove them from the genome
- We have genetic scissors at our disposal
CRISPR-Cas9 can be used to select specific genes and entirely remove them from a genome
- The Cas9 enzyme takes in a guide RNA of our gene of interest and the target DNA
- After unwinding the DNA, Cas9 checks for sites complementary to the 20 base pair spacer region of the guide RNA
- If the DNA substrate is complementary to the guide RNA, Cas9 cuts the DNA
crRNA and tracrRNA
- While bacterial Cas9 requires of two different RNA strands — crRNA to serve as reference and tracrRNA to activate the CRISPR mechanism…
- Engineering has allowed for a linker loop to bind the two RNAs into a singular strand. This is the form of CRISPR we use today
Cut — Copy — Paste
- In the cases of genetic diseases caused by a single gene, it may be useful to simply cut out a portion of the genome — yet we must often replace DNA as opposed to just removing it
What are the two ways in which the DNA strand can be repaired after the
cut?
- Non-Homologous End Joining mends the
broken strand right back together - Homology-Directed Repair adds a new series of nucleotides to the cut area
Timeline for Cas9 (Part 1)
Timeline for Cas9 (Part 2)
The He Jiankui Affair
- In a secret experiment, Dr. He Jiankui used CRISPR to genetically edit two twin girls known as Nana and Lulu
- The two embryos were edited of their CCR5 gene in an attempt to confer genetic resistance to HIV — this is the first and only recorded case of a genome
edited human babies - Although the twins were born healthy, the experiment was met with outrage from the scientific community. Jiankui was arrested in December of 2019 for 3 years and must pay a fine of around half a million dollars
Understanding the CCR5 Gene
- Why the outrage?
- Apart from ethical and societal considerations, the CCR5 gene is also linked to improved memory function, enhanced recovery from strokes, and cancer.
-Additionally, the gene was only deleted — leading to further risk of infection and unintended mutations
2020-2023 timeline
While the NaLu experiment was done in vivo, most medical applications are being designed to be ex vivo
Examples of things CRISPR can be used for
As of today, all CRISPR clinical trials only edit somatic tissue cells without affecting germ cells, meaning that no DNA changes can be passed onto future generations
Vertex and Blood Disease - Casgevy
- Red blood cells use hemoglobin to pick up oxygen in the lungs and carry it to all the tissues of the body. Genetic mutations in Hemoglobin to different disorders: beta thalassemia and sickle cell disease
- Current CRISPR trials these aim to increase levels of fetal hemoglobin — a form of hemoglobin only made by fetuses in the womb that can replace defective adult hemoglobin in red blood cells
First use of an ex vivo CRISPR-based therapy to cure a genetic disease
In the first use of an ex vivo CRISPR-based therapy to cure a genetic disease, one patient with beta thalassemia and one with SCD have been successfully treated.
Eye Disease
This is the retina in a person with an inherited form of blindness called Leber congenital amaurosis 10, caused by mutations in the CEP290 gene
CRISPR treatment makes a change to the patient’s faulty photoreceptor gene so makes a full-size, functional protein
Since LCA-10 is same condition as caused to the RPE65 gene and it has an approved gene therapy — why can’t we administer gene therapy for CEP290 gene the same way?
We would still have the mutant gene, we would have both
RPE65 is a loss of runction, but CEP290 is not
A refresher on RPE65 gene therapy
- RPE65 is critical to resetting the visual cycle and responsible for rhodopsin production — the most abundant protein in the rod cells that is the primary photoreceptor molecule of vision.
- When RPE65 is mutated, photoreceptor cells (rods and cones) die.
Gene editing scheme for CEP290 gene
- In March 2020, a CRISPR-modified virus was injected into a set of patients’ eyes in an attempt to treat LCA.
- While the procedure didn’t work for all of the patients and none of the patients have regained normal vision, there have been notable improvements with no significant side effects have occurred.
Somatic vs. germline editing
Editing an embryo 101
Prenatal Screens and Genetic Abnormalities
- Even without genetic editing, a certain type of selective pressure has been exerted with the rise of prenatal screens that test for genetic diseases
- The suspicion of a genetic defect often leads to the termination of a pregnancy — around 92% of pregnancies in Europe in which down syndrome is detected are terminated
- What if we could just prevent it?
Reasons to edit ourselves
What are the hurdles to be resolved before CRISPR can become widespread?
- Immune responses to the bacterial parts of CRISPR may render the technology useless. A majority of tested blood samples showed existing immune responses to Cas9
- Cas9 is large, so its gene is difficult to deliver to cells via vectors such as adeno-associated viruses commonly used in gene therapy
- Off-target effects must be mediated to minimize unintended
mutations - The need for PAM sequences limits potential target sequences
Mosaicism
- Sometimes genes differ not only between individuals in a population, but also among the cells of an individual. The advent of cheap and rapid genome sequencing has revealed that this mosaicism is incredibly common
- Mosaicism is a serious problem
- How that affects the health of the resulting child would depend on which cells were edited and which were not — something that could be difficult to predict in advance
Mosaicism- Repairing the Broken
The cell’s repair processes are unpredictable
The first studies to try using the CRISPR genome-editing technique to alter the DNA of human embryos revealed edits corrected mutations in a small proportion of embryos — only 1 in 10 cells were successfully repaired
Since these embryos were very genetically abnormal, however, these experiments may not have given an accurate indication of how well the technique would work in healthier embryos
Off-target effects of Cas9
- Cas9 has been known to
cut DNA at unintended non-target sites, particularly when there are DNA sequences in the genome similar to the target - These off target cuts can result in health problems: a change to a gene that suppresses tumour growth, for example, might lead to cancer
P53 and Cancer
- Following CRISPR edits, cells have inherent mechanisms that respond quickly to this type of DNA damage — and the transcription factor p53 is at the center of these
- CRISPR has been shown to work best in p53-deficient cells, deleting or reducing p53 increased the number of surviving cells
- By selects for p53-deficient cells, edited cells are vulnerable to mutagenesis and can result in tumors
Criteria for a Safe Edit
- In 2017, the US National Academies of Sciences, Engineering, and Medicine outlined the conditions that should be met before editing a human embryo that is destined for implantation
- “At this point, we don’t really understand how embryos deal with DNA repair. A lot of work needs to be done in other kinds of embryos, just to understand the fundamentals” — Jennifer Doudna
- All considered though… can genetic editing ever be entirely safe?
USNASEM Criteria (1-5)
- Absence of reasonable alternatives
- Restriction to preventing a serious disease
- Restriction to editing genes that strongly predispose to the disease or condition
- Availability of credible preclinical or clinical data on risks and health benefits of the procedure
- Ongoing oversight to the effects of the procedure on health and safety
USNASEM Criteria (6-9)
- Concrete plans for long-term multigenerational follow-up while still respecting personal autonomy
- Absolute transparency consistent with patient privacy
- Continued reassessment of health and societal benefits and
risk, with participation and input by the public - Oversight mechanisms to prevent extension to uses other than preventing disease
The Future: Enter RNA and Base Editing
- REPAIR relies on Cas13 to edit RNA nucleosides involved in single-base diseases
- REPAIR allows for edits to be temporary (and even reversible) and the genome to remain untouched
- Research has shown success in editing RNA in human kidney cells to fix mRNA, leading to functional proteins. Cas13, just like Cas9, can be edited to select its target gene
