CRISPR-Cas Engineering and Bacterial Immune Systems

Here at CiteAb we’re always interested to learn more about antibodies and those technologies that use them or have an impact on their use.

For that reason, this week we’re taking a brief look at the CRISPR-CAS immune system and how more recently it has been harnessed as a technology for use in highly specific gene editing and of course how this all relates to antibodies.

What is CRISPR-Cas

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in the DNA and CRISPR Associated (Cas) proteins are a widespread bacterial and archaeal defence mechanism against invading genetic elements[1]. First discovered in 1987[2], it was a number of years before the role of CRISPR-Cas as a defense mechanism was elucidated.

CRISPR:Cas Blog ImageAppearing as arrays of short unique nucleotide spacer sequences interspersed within the CRISPR palindromic repeat regions. These spacers act as a record of previous encounters with foreign genetic elements and provide to the bacteria/archaea (when processed by Cas) the ability to degrade those foreign genetic elements containing the same sequences. In this way CRISPR-Cas endow bacteria/archaea with an adaptive response to pathogens. Along with providing a defence mechanism, there is also evidence that CRISPR-Cas may also play a role in bacterial pathogenesis[3].

Why Pay Attention to CRISPR-Cas

CRISPR-Cas as a technology for gene editing and protein knockout, is a relatively recent development and provides an alternative to the already available transcription activator-like effector nucleases (TALEN) and zinc finger methods. Unlike it’s predecessors which rely on generating custom proteins for each DNA target, CRISPR-Cas technologies rely on guide RNA molecules making them more versatile and facilitates the generation of large libraries of CRISPR-Cas transcription factors[4].

How are CRISPR-Cas Relevant to Antibodies

Successful validation of an antibody depends on being able to demonstrate that the reagent is specific, selective, and reproducible in the context for which it is intended to be used. One method by which an antibody can be validated as being specific to a target protein is by comparing wild type and knockout samples: CRISPR-CAS protein knockout can be used to create the knockout cell line which will be used as a negative control. This makes validation particularly simple, when using Western blotting for example one would expect (hope!) that the target protein band is no longer present.

So there we have it, an introduction to CRISPR-CAS! What are your opinions on CRISPR-CAS and the potential of the technology? Do you currently utilise CRISPR-CAS in your research? We’d love to hear from you – leave a message below or send us a tweet to @CiteAb!

– Matt and the CiteAb team.


Image Credits

Front page image: Crystal structure of a hypothetical protein tt1823 from thermus thermophilus. from Wikipedia
First image: Diagram of the possible mechanism for CRISPR. from Wikipedia


References

1 – Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol. 2011 Jun;9(6):467-77. doi: 10.1038/nrmicro2577. Epub 2011 May 9.
PMID: 21552286
2 – Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol. 1987 Dec;169(12):5429-33.
PMID: 3316184
3 – Sampson TR, Weiss DS. Alternative roles for CRISPR/Cas systems in bacterial pathogenesis. PLoS Pathog. 2013;9(10):e1003621. doi: 10.1371/journal.ppat.1003621. Epub 2013 Oct 17.
PMID: 24146613
4 – Lienert F, Lohmueller JJ, Garg A, Silver PA. Synthetic biology in mammalian cells: next generation research tools and therapeutics. Nat Rev Mol Cell Biol. 2014 Feb;15(2):95-107. doi: 10.1038/nrm3738. Epub 2014 Jan 17.
PMID: 24434884
Touchon M, Rocha EP. The small, slow and specialized CRISPR and anti-CRISPR of Escherichia and Salmonella. PLoS One. 2010 Jun 15;5(6):e11126. doi: 10.1371/journal.pone.0011126.
PMID: 20559554
Medina-Aparicio L, Rebollar-Flores JE, Gallego-Hernández AL, Vázquez A, Olvera L, Gutiérrez-Ríos RM, Calva E, Hernández-Lucas I. The CRISPR/Cas immune system is an operon regulated by LeuO, H-NS, and leucine-responsive regulatory protein in Salmonella enterica serovar Typhi. J Bacteriol. 2011 May;193(10):2396-407. doi: 10.1128/JB.01480-10. Epub 2011 Mar 11. PMID: 21398529
Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014 Apr;32(4):347-55. doi: 10.1038/nbt.2842. Epub 2014 Mar 2.
PMID: 24584096
Bordeaux J, Welsh A, Agarwal S, Killiam E, Baquero M, Hanna J, Anagnostou V, Rimm D. Antibody validation. Biotechniques. 2010 Mar;48(3):197-209. doi: 10.2144/000113382.
PMID: 20359301


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