Expert of Antibody Production and Genetic Modification

CRISPR-TALEN-ZFN

CRISPR/Cas9 system

(Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR associated protein)

Nowadays, the CRISPR/Cas9 genome editing method is a worldwide current biotechnology tool, which is used for rapid, easy and efficient modification of endogenous genes in a wide variety of biomedically important cell types and in different organisms.

Our company also uses this technology reliably and routinely for producing rabbit, rat and mouse animal models.

The fundamental principle of CRISPR/Cas9 genome editing system is originated from prokaryotes. Bacteria and the most of archeas have developed this adaptive immune response against invading DNA molecules, like phages.

Modern biotechnology uses a modified system optimized for mammals. It consists of two components:

  • engineered “single guide” RNA (sgRNA)
  • non-specific CRISPR-associated endonuclease (Cas9)

The specifically designed sgRNA can bind to the Cas9 endonuclease which can cause double-strand breaks in the target DNA [1].

 

 

The CRISPR effector complex can recognize the adequate nucleotide sequence, if it presents upstream a specific motif (Protospacer Adjacent Motif, PAM).

The PAM sequence is usually 5’ NGG 3’.

Generated double-strand breaks are repaired in eukaryotic cells by non-homologous end joining (NHEJ) or homologous recombination (HR). These pathways can scars in the form of insertion/deletion (indel) mutations, which can lead to frameshift mutations and premature stop codons [2]. Additionally, we can mediate larger deletions in the genome with creation of multiple DSBs [3]. In the presence of an exogenously template DNA, the process of HR offers possibility to generate precise, defined knock-in modifications at the target locus [4].

 

 

Originally, the CRISPR technology was employed to knock-out target genes, but for today it is also usable to induce specific sequence insertions, like fused epitope tags or other functional domains, such as fluorescent proteins.

Nuclease technology is a powerful and often used specific tool for the generation of knock-out and knock-in animals, but it could be limited by off-target mutations. Therefore, the off-target analysis is always recommended [5].

CRISPR/Cas9 system is not the only available technology for genome editing however its simplicity efficiency and cheapness resulted its sudden spread.

ImmunoGenes also offers TALEN and ZFN genome editing for special requests:

TALEN (transcription activator-like effector nucleases)

This programmable nuclease is composed of a DNA-sequence-specific transcription-activator-like effector (TALE) protein originating from a plant pathogen Xanthomonas sp. [6] and a nuclease domain derived from the FokI restriction endonuclease.

The DNA binding domain

It contains a repeated highly conserved 33–35 amino acid sequence. The DNA binding specificity is determined by the amino acids at positions 12 and 13 within each repeat. These two positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and able to recognize specific nucleotides [7].

The DNA cleavage domain

In the widely used version of TALEN, the non-specific DNA cleavage domain is FokI endonuclease. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for creating a double-stranded break at a user-specified location in the genome [8].

 

ZFN (Zinc Finger Nucleases)

It is site-specific endonuclease designed to bind and cleave DNA at specific positions. The ZFN is an artificial fused enzyme with a zinc finger DNA-binding part and a DNA-cleavage domain. The DNA-binding domains of ZFNs typically contain from three to six zinc finger repeats and can each recognize between 9 and 18 base pairs [9].

The non-specific restriction endonuclease FokI is used as the cleavage domain in ZFNs. This domain must dimerize in order to cleave DNA and thus a pair of ZFNs are required to target non-palindromic DNA sites [10].

 
The constructions of nuclease systems


[1]      M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A Programmable Dual-RNA – Guided,” vol. 337, no. August, pp. 816–822, 2012.
 
[2]      K. Valerie and L. F. Povirk, “Regulation and mechanisms of mammalian double-strand break repair.,” Oncogene, vol. 22, no. 37, pp. 5792–5812, 2003.
 
[3]      L. Cong, F. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. Hsu, X. Wu, W. Jiang, L. Marraffini, and F. Zhang, “Multiplex Genome Engineering Using CRISPR/Cas Systems,” Science (80-. )., vol. 339, no. February, pp. 819–822, 2013.
 
[4]      T. O. Auer, K. Duroure, A. De Cian, J. Concordet, and F. Del Bene, “Highly efficient CRISPR / Cas9-mediated knock-in in zebrafish by homology-independent DNA repair,” Genome Res., vol. 24, pp. 142–153, 2014.
 
[5]      S. W. Cho, S. Kim, Y. Kim, J. Kweon, H. S. Kim, S. Bae, and J. Kim, “Sup2,” pp. 132–141, 2014.
 
[6]      J. Boch and U. Bonas, “Xanthomonas AvrBs3 family-type III effectors: discovery and function.,” Annu. Rev. Phytopathol., vol. 48, no. 1, pp. 419–36, 2010.
 
[7]      J. Boch, H. Scholze, S. Schornack, A. Landgraf, S. Hahn, S. Kay, T. Lahaye, A. Nickstadt, and U. Bonas, “Breaking the code of DNA binding specificity of TAL-type III effectors.,” Science, vol. 326, no. 5959, pp. 1509–12, 2009.
 
[8]      M. Christian, T. Cermak, E. L. Doyle, C. Schmidt, F. Zhang, A. Hummel, A. J. Bogdanove, and D. F. Voytas, “Targeting DNA double-strand breaks with TAL effector nucleases,” Genetics, vol. 186, no. 2, pp. 756–761, 2010.
 
[9]      M. L. Maeder, S. Thibodeau-beganny, A. Osiak, D. A. Wright, R. M. Anthony, M. Eichtinger, T. Jiang, J. E. Foley, R. J. Winfrey, J. A. Townsend, E. Unger-wallace, J. D. Sander, F. Fu, J. Pearlberg, C. Göbel, J. P. Dassie, M. H. Porteus, D. C. Sgroi, A. J. Iafrate, P. B. M. Jr, T. Cathomen, D. F. Voytas, and J. K. Joung, “NIH Public Access,” Mol. Ther., vol. 31, no. 2, pp. 294–301, 2008.
 
[10]    J. C. Miller, M. C. Holmes, J. Wang, D. Y. Guschin, Y.-L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. a Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar, “An improved zinc-finger nuclease architecture for highly specific genome editing.,” Nat. Biotechnol., vol. 25, no. 7, pp. 778–85, 2007.