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Unicellular Organisms

CRISPR Genome Editing

Recombinant DNA was the only technique available to introduce fragment of DNA into the genome up till 2013. It borrows the restriction
Recombinant DNA CRISPR enzyme from the bacteria to cut up the plasmid or DNA at the restriction site (~ 1 / 64000 base pairs). The purpose in nature is to defence viral infection. Modern technique add a foreign segment of DNA into the opening to usher in the GMO (Genetically Modified Organisms) era for more than 40 years (Figure 11-35g1).
It turns out that the bacteria possess an "Adaptive Immune System", which keeps a segment of the viral DNA from the previous attack. It uses this piece (CRISPR) together with a cutting enzyme (Cas9) to disable re-occurrent attack. The technique to adopt this mechanism for gene editing

Figure 11-35g1 Recombinant DNA [view large image]

Figure 11-35g2 CRISPR Genome Editing [view large image]

became viable by 2013 and was AAAS's choice for breakthrough of the year in 2015 (Figure 11-35g2, also see CRISPR Timeline).

CRISPR Editing Steps
    CRISPR-Cas9 Gene Editing Steps (see Figure 11-35g3) :

  1. There are two components to be prepared. The CRISPR is a piece of RNA, which guides the complex to the site of interest and hence the name gRNA. The gRNA itself composes two sub-components - the tracrRNA which identifies the site and the crRNA which contains the DNA sequence (the spacer) to be deleted. The Cas9 is an enzyme for cutting the corresponding sequence.

  2. The CRISPR-Cas9 complex together with whatever replacement (donor) DNA's are introduced into the cell by transfection.

  3. The complex looks for a match with an additional PAM sequence (in the cell's DNA) to make sure the deletion is legitimate (a feature inherited from the bacteria).

  4. The cut (and paste) is to proceed upon positive identification. The technique should be quicker, cheaper, more precise and more efficient than the previous versions of gene editing tools such as the artificial restriction enzymes Zinc-finger nucleases and TALEN.

Figure 11-35g3 CRISPR Editing Steps [view large image]

Mini-Cas9 Actually up to the summer of 2016, CRISPR-Cas9 editing is used mostly to delete genes that cause diseases. Gene insertion is more difficult with much lower efficiency. The existing system has many problems including : the CRISPR-Cas9 package is too large to load into virus for delivery, and it depends on the detection of PAM sequence to cut. Researches are in progress to use mini-Cas9, and Cpf1 as alternatives to address the shortcomings. It is found that disabling Cas9 could alter the genetic code one base at a time, but would not cut. Then there is the controversial NgAgo protein, which slices DNA at a pre-determined site without a gRNA or a PAM sequence. Laboratories all over the world have so far failed to reproduce the result (the authors retract the claim from Nature on August, 2017).

Figure 11-35g4 Hosts of Mini-Cas9 [view large image]

Figure 11-35g4 shows some of the bacteria Staphylococcus aureus, which are the host to a smaller version of the enzyme Cas9 (mini-Cas9).

The CRISPR-Cas9 technology should have many applications as shown in Figures 11-35g2, and g3. Some of them are illustrated in Figure 11-35g5 with a brief description.

CRISPR-Cas9 Applications
  • (a) Malaria Elimination - Out of about 3400 different species of mosquitoes worldwide, the Anopheles gambiae (a major carrier of dangerous malaria parasites in sub-Saharan Africa) is selected for the wholesale elimination via the CRISPR-Cas9 technology, which removes three genes that impacted female fertility. Coupling with the application of Gene Drive, this particular species can be eliminated in a few generations (~ 2-3 weeks) when actually implemented.

    The risk of Gene Drive has drawn the attention of US Defense Research Agency. It has awarded US$65 million for the studying of bio-terrorists attack, bio-error accidents, and the development of "anti-gene drive".

Figure 11-35g5 CRISPR-Cas9 Applications
[view large image]

  • (b) Pig's Organs Transplant - The organs in pig have similar size as human's. The problem is with the PERV virus DNA (causing AIDS) riddled in its genes. It has now been demonstrated that all the 62 occurrences of PERV genes can be removed from a pig's kidney cell by CRISPR-Cas9 editing. See a 2017 Update.
  • Other problematic genes can be removed similarly. Primate trials using parts grown from pig embryos with the modified genes will start within a year or two.

  • (c) Genetically Engineered Crops - Researchers are working on CRISPR-Cas9 edited crops such as corn, soybeans, canola, rice, and wheat, with new traits like drought resistance and higher yields. It has already created a white-button mushroom that doesn't turn brown as quickly by knocking out six genes, and actually can be sold in the market. It is hopeful that such products are more acceptable by consumers since unlike the traditional GMO, the new technology does not involve the merger with foreign genes (see NBT).

  • (d) Human Germline Editing - Despite urging caution by CRISPR-Cas9 scientists, at least four labs in the US, labs in China and the UK, and a US biotechnology company announced plans or ongoing research to apply CRISPR-Cas9 gene editing technique to human embryos. In 2015, Chinese scientists reported results of unsuccessful attempt to alter the DNA of non-viable human embryos using CRISPR to correct a mutation that causes heritable problem. Another unsuccessful run in 2016 tried to make the non-viable embryos HIV resistant.

  • CRISPR-Cas9, Clinical Trial
  • (e) First Clinical Trail - In October, 2016, a clinical trial of the CRISPR product has been applied to a Chinese patient with metastatic non-small-cell lung cancer. The technique is to remove some immune cells from the patient, then uses CRISPR to disable the gene coding the PD-1 protein which normally puts a brake on the cell's immune response. The modified cells are cultured to increase their number and injected back into the patient. It is anticipated that the edited immune cells will attack and destroy the cancer (hopefully without affecting the normal cells). Figure 11-35g6 shows a Human T cells (blue) attacks a cancer cell (red).
    See original article : "CRISPR Gene-editing Tested in a Person for the First Time".
  • Figure 11-35g6 CRISPR-Cas9, Clinical Trial
    [view large image]

  • (f) Fixing Disease Gene in Viable Human Embryos - Researchers in 2017 targeted a mutation in a gene called MYBPC3, which causes the heart muscle to thicken - a condition known as hypertrophic cardiomyopathy that is the leading cause of sudden death in young athletes. The editing was successful but not implanted.

  • Embryonic OCT4 Gene Removal
  • (g) Embryonic OCT4 Gene Removal - It is reported in September 2017 that removal of the OCT4 gene, which governs the development of the embryo, suppresses further progress of the blastocyst (a ball of about 200 cells, Figure 11-35g7). The same gene editing on mice shows that the deleterious effects occur later and more focused. It suggests the need for human embryo research since the result of gene editing could be different in other species.

    See the original article "CRISPR used to peer into human embryos' first days" from Nature, 20 September 2017.
  • Figure 11-35g7 Embryonic OCT4 Gene Removal

  • (h) Evo-devo - More radical applications of the CRISPR-Cas9 editing is already in the research on evolutionary developmental biology. The technique is now applied to turn fin into feet in zebrafish by inactivating some genes (see CRISPR Gene Editing Storms Evo-Devo Labs), Others have used the technique to determine how butterflies evolved exquisite colour vision, how crustaceans acquired claws, and tracking the evoluation of snakes. Even more
  • Figure 11-35g8 Dino-chicken [view large image]

    interesting works would involve swapping genes between distantly related organisms to trace the history of life backward (Fgiure 11-35g8). Such novel application may one day make the fiction of "Jurassic Park" to come true.


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