Common Tools for Genome Editing in Plants

Posted by: r00ty
Date: 2018-12-01 03:40

 

Key components used for genome editing are programmable nucleases.  In the past time, multiple tools based on different mechanisms have been used. By engineering and optimization their efficiency and availability changed over time and provides promising future prospects (Kim 2016).

By induction of strand breaks in the DNA, repair mechanisms can lead to a modification on specific targets. The specifity is dependent from many different factors and represents a major hurdle. Insufficient specifity can lead to “off-targets”, which could lead to a inadvertently modification of genes. Therefore, one of the most important goals at the present time is to increase specificity through further optimisation.


Overview of reprogrammable nucleases

Meganucleases
Meganucleases are modified Homing-endonucleases. A candidate which has been previously used for gene targeting is I-SceI, originally isolated from mitochondria of yeast cells. Double strand breaks due to its nuclease activity allowed to increase homologous recombination events after transformation in Nicotiana protoplasts (Puchta et al. 1993).

Re-engineering approaches typically include the mutation and recombination of residuals or subunits. Despite of their long recognition sequence which allows a high specifity, the reprogramming of meganucleases is very challenging, making them unattractive for genome editing purposes.

Zink-Finger Nucleases (ZFNs)
ZFNs are protein fusions of zinc fingers, which are able to detect specific base triplets, and the endonuclease domain from Fok1. Fok1 requires a dimerization to provoke a double strand break, so ZFNs are typically used pairwise. Each ZFN can consists of e.g. three Zink-Finger-Proteins whereas each detects a triplet (Kim et al. 1996), allowing modularity and easier adaption to the target sequence in comparison with meganucleases. However, the construction of ZFNs is time-consuming and expensive.

Transcription Activator-Like Effector Nucleases (TALENs)
TALENs are protein fusions, consisting of TALEs and e.g. the Fok1 endonuclease domain. TALES have originally been found in several plant pathogens like Xanthomonas. By injection of TALEs, they are able to induce specific gene transcription in the host cells, increasing their vulnerability. TALEs are capable to initiate transcription after binding to promotor sequences (Boch & Bonas 2010).

The sequence specifity is based on several repeats within the TALE. Each repeat consists of about 33-35 (typically 34) amino acids which are highly conserved – except in two positions. The amino acids at these positions are called repeat-variable diresidues (RVDs). RVDs able to detect specific nucleotides. Two factors are important for the efficiency of specific DNA binding: The integrated RVDs and the number of repeats. High efficiency is e.g. given between ~ 15 to 20 repeats.

As previously mentioned, Fok1 requires dimerization in order to perform a double strand break. Therefore, TALENs are constructed to act pairwise, this also leads to a higher specifity. The nuclease is located at the C-terminus, so TALENs must be oriented accordingly, allowing a dimerization. A spacer of about 15 basepairs between both recognition sites should be considered. Higher or lower distance can affect the dimerization ability negatively, which leads to a decreased efficiency.

Reprogramming can be performed by choosing RVDs inside the repeats which match with the desired sequence. Their high flexibility, specifity and small size makes them a valuable tool for precise genome editing. Despite it requires some expertise for reprogramming of a TALEN, it requires significantly less time (< 1 week) and financial investment in contrast to Meganucleases and ZFNs.

CRISPR/Cas-System
CRISPR/Cas based Systems and their derivatives gained high relevance for genome editing applications. One of the main reasons is the simplicity of their reprogramming, since the DNA is recognized by guide RNAs. It allows to achieve programmable DNA binding without the necessity of complex protein engineering. guide RNAs can be provided as sgRNAs (single guide RNA, sometimes also called synthetic guide/short guide RNA), which consist only of a single RNA strand. Transient expression of constructs containing sgRNAs and the DNA-cleaving Cas9 protein allow to modify the target organism’s genome even without insertion of any foreign DNA.

High flexibility is achievable by several modifications. If a single strand break is preferred over double strand breaks, re-engineering allows e.g. to inactivate one of the two Cas9 nuclease regions HNH and RuvC, leading to a “nickase”. It reduces the occurrence of NHEJ (non homologous end joining) in case only HDR (homology directed repair) is desired, which is used to introduce foreign genes. Complete inactivation of Cas9 nuclease is also possible, which allows alternative applications beside genome editing: After fusion with other components, gene repression or activation can be realized for specific targets.

sgRNAs can easily be used for creation of multiplex constructs, enabling modifications of multiple targets at the same time. Involvement of ribozymes or tRNA-sequences can be used for precise transcript-processing in this manner.


Possibilities of DNA sequence recognition

The target specifity is provided by recognition of DNA sequences, whereas the recognizing components differ in the available tools.

Protein mediated

DNA can be recognized by protein structures, e.g. by interaction of amino acid residuals with DNA nucleotids.

  • Zink Finger Nucleases
    Recognition of base triplets (by assembly e.g. up to 3-6 triplets), cleavage by Fok1-domain
  • Meganucleases
    Recognition of e.g. 12-40 bp sequences
  • TALENs
    Recognition of e.g. 15 – 20 bases, cleavage by Fok1-domain
  • MegaTALs
    Fusions of TALEs and Meganucleases combines their recognition sequences

RNA mediated

RNA can bind to the complementary DNA and thereby provide target sequence specifity.

  • CRISPR/Cas-Systems
    gRNA detects e.g. sequences of 20 bp, cleavage by domains of the Cas-Protein
  • RFNs
    gRNA detects sequence, cleavage by Fok1-domain

RNA & protein mediated

The fusion of proteins allows enhancement of sequence specifity, whereas the recognition is mediated through simultaneously RNA/DNA and protein/DNA-interaction.

  • Cas9-TALE-Fusion
    sequence detection by gRNA and TALE-RVDs, cleavage by domains of Cas9
  • Cas9-ZFN-Fusion
    sequence detection by gRNA and Zinc-Finger-Protein, cleavage by Fok1-domain

 


Abbreviations:
Cas: CRISPR-associated
CRISPR: Clustered regularly interspaced short palindromic repeats
Fok1: Flavobacterium okeanokoites nuclease
RFN: RNA-guided FokI-dCas9 Nuclease
RVD: Repeat-variable diresidue (of a TALE-repeat)
TALE: Transcription activator-like effector
TALEN: Transcription activator-like effector Nuclease
ZFN: Zinc finger Nuclease

References

Kim, J. S. (2016). Genome editing comes of age. Nature protocols, 11(9): 1573-1578.

Puchta, H., Dujon, B., & Hohn, B. (1993). Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease. Nucleic acids research, 21(22): 5034-5040.

Kim, Y. G., Cha, J., & Chandrasegaran, S. (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proceedings of the National Academy of Sciences of the United States of America, 93(3): 1156-1160.

Boch, J., & Bonas, U. (2010). Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annual review of phytopathology, 48: 419-436.