A CRISPR look into gene editing

10 Years ago, it was first proposed in the journal Science that CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR associated systems (Cas) could be used for targeted gene editing by creating specific double stranded breaks in target DNA _[1]. In 2020, Dr Emmanuelle Charpentier and Dr Jennifer Doudna won the Nobel prize in Chemistry for their work. Since the discovery CRISPR has allowed for advancements within research into genome editing: making processes faster, cheaper, and more accurate _[2].

In prokaryotes, CRISPR is an RNA-mediated adaptive immune response used by the cell to target invading viruses, bacteriophages, and plasmids. There are 3 types of CRISPR systems which have been identified in both bacteria and Archaea _[1], the most widely researched and characterised being the CRISPR associated nuclease protein of Streptococcus pyogenes which is called Cas9. The Cas9 protein from S. pyogenes contains 2 nuclease domains and is the most widely used Cas within gene editing research. After an initial viral infection, the cell will insert part of the viral DNA into the CRISPR loci of its own genetic material (where it is called a Spacer) and the fragment sits between palindromic repeats of non-coding DNA. In a recurrent infection, the CRISPR Array within the genome undergoes translation to generate crRNA and forms an effector complex containing the Cas protein and trans-activating CRISPR RNA (tracrRNA) _[3]. When the effector complex interacts with the invading viral DNA, it binds to a Protospacer adjacent motif (PAM), the DNA unwinds and the crRNA binds to the complimentary sequence within the viral genome.  The endonucleases within the Cas9 protein will then cleave the DNA, causing the neutralisation of the virus.

Interest in using the CRISPR Cas method for genome editing in research has grown exponentially in the last decade and the technique has a large scope as to what it can achieve. For example, the system can be used for the creation of knockout genes, gene silencing and for alterations in transcription and gene expression. Another use is to deactivate the nuclease enzymes and add a different enzyme so it can be transported to the target sequence. Furthermore, CRISPR can add a fluorescent tag to target sequences to see where a gene lies within a cell and aid in the visualisation of the 3D structure of the genome.

In recent publications, Douglas et al (2021) used a bicomponent CRISPR-Cas9 strategy to produce single sex mice litters. The team created single guide RNA (sgRNA) to target the Topoisomerase 1 gene as the absence of the gene would cause early embryonic lethality. They then created mouse lines expressing Cas9 on the X and Y chromosomes. When lines were mated, the resulting litters were male- or female-only with one hundred percent efficiency _[4]. Thong et al (2021) used CRISPR Cas9 editing to rapidly produce new variants of the antibiotic Enduracidin. Gene editing was used to alter subdomains in nonribosomal peptide synthetase (NRPS) and was inserted into Streptomyces fungicidicus via plasmid. The results showed new variants of Enduracidin in sufficiently high yields and the process was found to be faster and more efficient than through conventional chemical processing _[5].  Mertaoja et al (2021) studied sporulation in Clostridium botulinum, creating optimum anaerobic growth conditions using a Whitley Workstation. The research team used CRISPR Cas9, and the double stranded break repair mechanism called homology-directed repair (HDR), to replace targeted genes with a mutant “bookmark” sequence of their own design that could act as a sgRNA target for Cas9 _[6]. The mutant strain could then be manipulated to study sporulation of the organism using different media types.

The CRISPR Cas system has the potential to revolutionise the treatment of genetic diseases and make improvements in farming and agriculture. One of the most promising areas of research is to increase the yields of crops by editing the genome and making crops more resistant to adverse conditions such as droughts and diseases.

 Written by Charlotte Austin a microbiologist in our contract laboratory.

References

1. Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna, Emmanuelle Charpentier (2012) A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity Science
2. How Does Genome Editing Work? (2017) National Human Genome Research institute genome.gov/about-genomics/policy-issues/Genome-Editing/How-genome-editing-works
3. F Ann Ran, Patrick D Hsu, Jason Wright, Vineeta Agarwala, David A Scott & Feng Zhang (2013) Genome engineering using the CRISPR-Cas9 system Nature Protocols
4. CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes (2021) Charlotte Douglas, Valdone Maciulyte, Jasmin Zohren, Daniel M. Snell, Shantha K. Mahadevaiah, Obah A. Ojarikre, Peter J. I. Ellis & James M. A. Turner Nature Communications
5. Gene editing enables rapid engineering of complex antibiotic assembly lines (2021) Wei Li Thong, Yingxin Zhang, Ying Zhuo, Katherine J. Robins, Joanna K. Fyans, Abigail J. Herbert, Brian J. C. Law & Jason Micklefield Nature Communications
6. CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research (2021) Anna Mertaoja, Maria B. Nowakowska, Gerald Mascher, Viivi Heljanko, Daphne Groothuis, Nigel P. Minton and Miia Lindström Frontiers in Microbiology