CRISPR-Cas9

by Hoi Kiu Wong

Image from Shutterstock 

INTRODUCTION 

Recently, The 2020 Nobel Prize in Chemistry was awarded to Jennifer Duodna, an American Biochemist [1], and Emmanuelle Charpentier, a French professor and researcher in microbiology, genetics and biochemistry, for their collaborative discovery of CRISPR-Cas9 [2]. This new technology stands for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 [3]; it allows scientists worldwide to easily edit genes. In this article, we will look into what CRISPR-Cas9 is, how it works and the pivotal role it plays in the field of genetic modification - including its application in stem cell engineering, gene therapy, tissue and animal disease models, and engineering disease-resistant transgenic plants [4]. 

WHAT IS CRISPR-Cas9 

CRISPR-Cas Systems are prokaryotic adaptive immune response systems. They help the bacteria cleave nucleic acids of the invading virus; protecting itself from viral infections [3]. These systems were first discovered in the 1980s in E.coli; however, their function was unknown until 2007 by Barrangou and colleagues, who has shown that S. thermophilus acquired immunity against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus. There are many different types of CRISPR mechanisms; the most studied being type II [5]. 

So how do these systems work? In the bacterial cell, during the acquisition phase, the protospacers of the foreign DNA are incorporated into the bacterial genome at the CRISPR loci amidst a series of spacers (segments of exogenous DNA approx. 30bp in length) and repeats (approx. 20bps) [4] [5]; forming CRISPR arrays. During the transcription of the CRISPR loci, CRISPR RNA biogenesis occurs: the pre-crRNA transcribed binds with tracrRNA and together they ‘recruit’ enzymes that chop the pre-crRNA into smaller pieces to form mature crRNA (consisting of a 3’ repeat and approx. 20 nucleotide spacer). These mature crRNA (cr-RNA and tracker RNA complex, also known as gRNA) then binds to its complementary sequence in the viral DNA; in doing so, guides the Cas (CRISPR-associated) proteins (effector endonucleases), which in this case is Cas9, to where it needs to be. This occurs because the Cas protein is able to recognise a protospacer adjacent motif (PAM); a 2-5 base pair sequence close to the crRNA complementary strand. Therefore, the Cas protein can cut the DNA of the invading virus (or any foreign genetic elements such as those present within plasmids and bacteriophages) based on sequence complementarity; in turn, destroying the virus [6] (See Fig.1 and Fig. 2). 

Fig. 1 CRISPR Cas System 

Source: Interdependencies Between the Adaptation and Interference Modules Guide Efficient CRISPR-Cas Immunity - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/CRISPR-Cas-adaptive-immunity-The-three-stages-of-CRISPR-Cas-system-function-are_fig1_319010479 [accessed 14 Nov, 2020]

Fig. 2 CRISPR-Cas9 System - closer look into CRISPR Interference Step. 

Source: [4] https://www.thermofisher.com/hk/en/home/life-science/genome-editing/genome-editing-learning-center/crispr-cas9-technology-information/jcr:content/MainParsys/accordion_3192/itemspar/accordionitem_3490/itemParsys/image_44cd/foregroundimg.img.320.low.jpg/1572990663599.jpg

HOW IT WORKS 

In 2012, George Church, Jennifer Doudna, Emmanuelle Charpentier and Feng Zhang used the CRISPR-Cas system as a tool to help modify targeted regions in the genomes. They could do this because the Cas-9 gene editing tool works in various eukaryotic cells and permits targeted gene cleavage as the endonuclease cleavage specificity in CRISPR-Cas9 systems is guided by RNA sequences; editing can be directed towards virtually any genomic locus by engineering the guide RNA sequence and delivering it along with the Cas endonuclease to your target cell [4]. Once the DNA is cut (Cas 9 makes a blunt double-stranded DNA break), the researchers use the cell’s own DNA repair machinery to add or remove pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customised DNA sequence [3]. 

Non-homologous end joining is one of the DNA repair mechanisms. It ligates the break ends of the DNA without the need for a homologous template; hence, there is a loss of the complementary sequence with no replacement (the complementary sequence was removed by the Cas9 protein) [7]. The other type of DNA repair mechanism is homology directed repair; it is very useful as it allows a new gene to be inserted into the DNA - a ‘donor’ DNA would need to be delivered to the host cell (donor DNA is the gene we want to add in) and it will act like a sister chromatid as a reference to recreate the lost genetic information (forming a homologous recombination with a donor template DNA to create site-specific edits) [6] (See Fig. 2). 

Fig. 2 Genome Editing with CRISPR-Cas9 - using DNA Repair Mechanisms (Non-homologous end joining and Homology-directed repair). 

Source: https://www.stemcell.com/genome-editing-of-human-primary-t-cells-using-the-arcitect-crispr-cas9-system.html

Not only can Cas9 be used to edit genes, but it can also silence genes. To do this, a faulty or inducible Cas9 protein (unable to chop DNA) moves to where the target gene is, and stays there - preventing transcription of this gene; silencing it [6]. 

TypeII-A Cas9s have high genome editing efficiency, but off-target cleavage at unintended genome sites can be a disadvantage. A lot of work has been put into eliminating these ‘off target’ effects, and in recent years, variants have been engineered to overcome these limitations e.g. type II-C Cas9s (has naturally higher fidelity) [8]. 

FUTURE OF GENE EDITING 

Since the discovery of the CRISPR-Cas9 Gene editing tool, a lot of research has been focused on modifying the Cas9 endonuclease to carry out targeted epigenome editing. The modified Cas9 endonuclease, also known as enzymatically dead Cas9 (dCas9), can be attached to enzymes that can alter the epigenome e.g. DNA demethylases, methylases or acetyl transferases. Transcription can be activated by demethylating DNA using enzymes such as dCas9-Tet1 or by modifying histones using dCas9 linked to the histone acetyltransferase p300 enzyme. Conversely, transcription can be repressed by methylating DNA using the enzyme DNA methyltransferase. To silence genes, dCas9 can be attached to enzymes that recruit corepressor proteins [10]. 

It is recognised that one of CRISPR-Cas9’s greatest potentials is curing human diseases. Currently, other than research on CRISPR-Cas9 epigenome editing, a lot of the research on genome editing is also done in order to understand diseases using isolated human cells and animal models. It is hoped that in the near future, once we have a in-depth understanding of the CRISPR-Cas9 technology, we can edit genes to cure genetic disorders such as Cystic Fibrosis, haemophilia and sickle cell disease. It could also be used to treat other diseases such as cancer, heart disease, and human immunodeficiency virus (HIV) infection [3]. 

As amazing as gene editing with CRISPR-Cas9 is, ethical concerns come into light; this is especially when the tool is used to alter human genomes. These ethical issues are especially serious if CRISPR-Cas9 is used to modify the DNA of egg and sperm cells; the changes made will not only affect the cell itself but consequently, the future generations as well. Hence, many countries have made germline cell and embryo genome editing illegal based on these concerns on ethics and safety [3]. A breach in Medical Ethics using CRISPR-Cas9 has been exemplified by the work of Chinese Scientist He Jiankui, where he used the gene-editing tool to engineer the birth of twin girls Lulu and Nana. As a consequence, the World Health Organisation launched a global registry in 2019 to track research on human genome editing. 

                           ©Gettyimages/elenabs 

BIBLIOGRAPHY 

[1] “Jennifer Doudna.” Wikipedia, Wikimedia Foundation, 9 Nov. 2020, en.wikipedia.org/wiki/Jennifer_Doudna. 

[2] “Emmanuelle Charpentier.” Wikipedia, Wikimedia Foundation, 9 Nov. 2020, 

en.wikipedia.org/wiki/Emmanuelle_Charpentier. 

[3] “What Are Genome Editing and CRISPR-Cas9?: MedlinePlus Genetics.” MedlinePlus, U.S. National Library of Medicine, 18 Sept. 2020, medlineplus.gov/genetics/understanding/genomicresearch/genomeediting/. 

[4] “CRISPR-Cas9.” Thermo Fisher Scientific - HK, www.thermofisher.com/hk/en/home/life-science/genome-editing/genome-editing-learning-center/crispr-cas9-technology-information.html. 

[5] Biolabs, New England. “CRISPR/Cas9 & Targeted Genome Editing: New Era in Molecular Biology: NEB.” New England Biolabs: Reagents for the Life Sciences Industry, international.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology. 

[6] “CRISPR/Cas9.” YouTube, Osmosis, 7 Sept. 2020, www.youtube.com/watch?v=Be34dclOK38. 

[7] “Non-Homologous End Joining.” Wikipedia, Wikimedia Foundation, 1 Nov. 2020, en.wikipedia.org/wiki/Non-homologous_end_joining. 

[8] “What Is CRISPR-Cas9?” Facts, The Public Engagement Team at the Wellcome Genome Campus, 19 Dec. 2016, www.yourgenome.org/facts/what-is-crispr-cas9. 

[9] Pawluk, April. CRISPR Systems: What’s the Difference?. Research Arc. 

https://www.cell.com/pb-assets/products/research-arc/infographics/CrisprVizInfo_vol1a.pdf PDF download 

[10] Ng, Daphne. “A Brief History of CRISPR-Cas9 Genome-Editing Tools.” Bitesize Bio, 3 July 2020, bitesizebio.com/47927/history-crispr/.