Molecular Scissors: CRISPR/Cas9 Technology

CRISPR technology is a relatively simple tool that is particularly powerful with respect to editing genomes. Through this technology, DNA sequences can be altered and gene function modified which lead to a multitude of potential applications such as correcting genetic defects. Despite this, ethical concerns are raised at its promise.

Note: this article will be an introduction to CRISPR, and not too extensive - outlining the basic concepts, what it is used for, and some recent developments. Further, more in depth, articles on CRISPR are to come, so stay tuned for the series!

The CRISPR technology was adapted from the natural defence mechanisms found within bacteria and single-celled microorganisms found within the archaea domain. It was found that CRISPR-derived RNA and Cas proteins (including the noted Cas9) were key to defending in attacks by viruses or other foreign bodies by chopping up and destroying the DNA of the foreign body. CRISPRs (Clusters of Regularly Interspaced Short Palindromic Repeats) are specialised stretches of DNA, and Cas9 (a protein) is an enzyme - the "molecular scissors" which can cut strands of DNA. The ideas of CRISPR started to formulate around 2012, but no one had truly observed the process until 2017.

CRISPRs have two key distinct characteristics - the presence of nucleotide repeats and spacers. CRISPR RNA (crRNA) is the result once a spacer has been incorporated, and the virus has attacked again. Part of the CRISPR gets transcribed into crRNA - each crRNA will consist of a nucleotide repeat and a spacer portion. Cas9 is an enzyme that cuts foreign DNA, typically binding to two RNA molecules. These are crRNA, and tracrRNA (trans-activating crRNA). These two guide Cas9 to the target site where it then cuts. The DNA expanse is complementary to a section of crRNA 20 nucleotides in length. Cas9 creates a double stranded break by cutting both strands of the DNA double helix. PAMs (protospacer adjacent motifs) are short DNA sequences which are "tags" to ensure that Cas9 does not cut the target DNA sequence by sitting adjacent to them.

Uses and Limitations

CRISPR/Cas9 has dramatically gained popularity in recent years - it is relatively simple to use, and very efficient when compared with previous genome-editing tools (such as TALENS).

Some of the many uses of CRISPR/Cas9 technology:

Studies using in vitro and animal models of human diseases have shown that the CRISPR technology can be effective when used to correct genetic defects - some experimented on include Fanconi anaemia and cystic fibrosis.
Applied in food and agricultural industries to engineer probiotic cultures, and vaccinate industrial cultures against viruses.
Used in crops to improve yield, nutritional properties and tolerance to weather conditions/climate.
Create gene drives - genetic systems which increase the chances of a particular trait being inherited by offspring, thus leading to the trait spreading through entire populations over time. This technology could be used to control the spread of diseases, and eradicate invasive species amongst other things.

How about the limitations? No system is perfect, and there are certainly risks with CRISPR too - even with the uses mentioned above, there are risks. For example, creating gene drives could lead to eradication of traits in preference of others when the preferred trait does not have any significant advantage - almost veering towards genetic engineering and the prospect of things like designer babies. A couple of other limitations:

CRISPR is not 100% efficient - genome-editing efficiencies can vary especially. Some target efficiencies can be up to 80%+, whereas some may only receive efficiencies of around 50%.
Off-target effects can occur where DNA is cut at sites other than the intended target which could lead to the introduction of unintended mutations in the genome.
A Chinese group reported the first application of CRISPR/Cas9 in April 2015 to non-viable human embryos - two key issues: philosophical dilemma and safety after the National Institutes of Health had declared that they would not fund any use of genome editing technologies in human embryos - conflict potentially between researchers.

Where should the boundaries be? Ethical concerns - what should we be considering and/or asking?

Below are a few thoughts on the matter:

Ecological impacts of using gene drives - an introduced trait could spread beyond the target group to other organisms in crossbreeding
Gene drives could reduce the genetic diversity of the target population
Germline editing - editing reproductive cells / human embryos could lead to changes being passed on to subsequent generations too
Variable efficacy - safety risk
Off-target effects - significant safety risk to potentially have edits in other parts of the genome
Imprecise edits - as above
Is it ethical to make changes that affects future generations without their consent?
How would people react if germline editing was used to enhance characteristics instead of being a therapeutic tool? Designer babies?

Recent Research Interests

Numerous projects have been based around CRISPR - research discovery pace increased dramatically as a result of the technology. Some key / interesting findings are listed below for you to maybe look into (some will feature more heavily in future CRISPR-related articles!)

2014 ->: genome-wide screens to identify genes involved in resistance to cancer drugs, and to dissect immune regulatory networks
2014 (throughout): CRISP used to rapidly create mouse models of cancer arising from multiple gene alterations
2015: success with Cas9 derived from a different bacterium - Staphylococcus aureus (SaCas9) which is smaller than the original Cas9 - advantages for gene therapy
October 2015: CRISPR/Cas9 modified 60 genes in pig embryos - first step to create human transplate organs
November 2015: genetically modified mosquitoes using CRISPR/Cas9 to prevent them carrying malaria
February 2016: UK scientists authorised to genetically modify human embryos with CRISPR/Cas9
April 2017: a CRISPR molecule was programmed to find strains of viruses in blood serum, urine and saliva
May 2017: CRISPR/Cas9 shown to eliminate HIV in infected mice
August 2017: Heart disease defect successfully removed in an embryo using CRISPR
April 2018: CRISPR upgraded to edit thousands of genes at once
November 2018: first gene edited babies announced by Chinese scientist (later convicted in December 2019)
December 2018: CRISPR/Cas9 helped restore first-line chemotherapy effectiveness in lung cancer patients
March 2020: first patient received gene editing CRISPR therapy (administered directly into the body)
June 2020: speculation over safety of CRISPR/Cas9 to alter human embryos after research publication
June 2021: CRISPR/Cas9 preliminary trial results show that the CRISPR gene editing can be released directly into the body to treat a rare, fatal condition - namely transthyretin amyloidosis

I hope that this short introduction with some interesting event summaries has helped to introduce the topic of CRISPR/Cas9 to you all, and I look forward to getting down into the interesting science behind it all in upcoming parts of the CRISPR series! Tomorrow: Mass Spectrometry: Post-Translational Modifications!

References

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Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014 Jun 5;157(6):1262-78. doi:10.1016/j.cell.2014.05.010. Review. PubMed: 24906146.

Komor AC, Badran AH, Liu DR. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell. 2017 Apr 20;169(3):559. doi:10.1016/j.cell.2017.04.005. PubMed: 28431253.

Lander ES. The Heroes of CRISPR. Cell. 2016 Jan 14;164(1-2):18-28. doi:10.1016/j.cell.2015.12.041. Review. PubMed: 26771483.

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Tian, X., Gu, T., Patel, S. et al. CRISPR/Cas9 – An evolving biological tool kit for cancer biology and oncology. npj Precis. Onc.3, 8 (2019). https://doi.org/10.1038/s41698-019-0080-7

WhatisBiotechnology.org. 2021. CRISPR enables gene editing on an unprecedented scale. [online] Available at: <https://www.whatisbiotechnology.org/index.php/science/summary/crispr> [Accessed 19 September 2021].

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