Unlimited DNA Editing With Structure-Guided Nucleases

Image result for editingStructure-guided nucleases such as flap endonuclease-1 (FEN-1) bind to particular DNA structures; in the case of FEN-1 the 3′ flap structure arising during lagging strand DNA synthesis and the removal/displacement of RNA primers associated with Okazaki fragments. Xu et al. (2016) adapt FEN-1 for use as a DNA-guided DNA binding protein that can be linked to the cleavage domain of Fok1 to create a unique gene editing system.

Image result for lagging strandThe limitations of various gene editing systems are well documented. Zinc finger nucleases prefer G-rich sequences and one needs to be able to find an appropriate array of trinucleotide zinc finger targets. TALENs tend to be more versatile but Xu et al. (2016) note that they require a thymine at the 5′ end of the target sequence. RNA-guided endonucleases are extremely fascile but Cas9, for example, has requirements for the PAM sequence. So, none of these systems is DNA sequence independent, capable of cleaving any sequence. All suffer from some DNA sequence constraints, meaning that not all of the sequences in a genome are accessible to DNA editing.

This shows the creation of a 3' flap during DNA replication on the lagging strand. FEN recognizes the 3' flap structure that arises from strand displacement.

This shows the creation of a 3′ flap during DNA replication on the lagging strand. FEN recognizes the 3′ flap structure that arises from strand displacement.

The FEN-1 system described by Xu et al. (2016) potentially could be the basis for an editing system without sequence constraints.

Inactive Cas9 can be used to create RNA guided Fok1 nucleases. Structure-guided nucleases can be used in a similar way. This image is from Addgene https://www.addgene.org/crispr/jounglab/dimercrispr/. See Addgene for more information on dimeric Cas9 systems.

Inactive Cas9 can be used to create RNA guided Fok1 nucleases. Structure-guided nucleases can be used in a similar way. This image is from Addgene https://www.addgene.org/crispr/jounglab/dimercrispr/. See Addgene for more information on dimeric Cas9 systems.

FEN-1 recognized 3′ flap structures and by linking FEN-1 to the cleavage domain of Fok1 they create what they refer to as a ‘structure-guided endonuclease’. One can control this structure-guided endonuclease by providing it with a DNA oligo capable of annealing to a target sequence and creating a 3′ flap (an unpaired 3′ nucleotide at the end of an annealed DNA oligonucleotide).

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This image is from an excellent article in the same issue of Genome Biology as Xu et al. and nicely summarizes the concept of structure guided endonucleases. See Varshney GK, Burgess SM. DNA-guided genome editing using structure-guided endonucleases. Genome Biology. 2016;17:187. doi:10.1186/s13059-016-1055-4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025577/#__ffn_sectitle

Xu et al. investigate some of the critical features of the system. For example, the DNA oligonucleotide needs to be at least 20 nucleotides in length and could be as long as 60 nucleotides. The authors find that 3′ flap structures consisting of any single unpaired nucleotide will work, e.g. C upaired to a T, G unpaired to a T, and so on. There was no apparent difference in the efficiency of these various unpaired structures.

Most of the characterization of structure guided endonucleases was performed in vitro, however the authors did conduct an in vivo editing experiment in zebrafish embryos. In this case they injected mRNA encoding the structure guided endonuclease and two ‘guide DNAs’.

In vivo indel creation was inefficient. Trying to force the creation of a DNA structure such as a 3′ flap in an otherwise canonically duplexed DNA could be challenging and may be contributing to the systems current inefficiency. The authors speculate that using PNA or LNA probes as gDNAs may increase efficiency.

This is an interesting system which could be of great utility if the current efficiency rates can be substantially improved.

Xu S, Cao S, Zou B, Yue Y, Gu C, Chen X, et al. An alternative novel tool for DNA editing without target sequence limitation: the structure-guided nuclease (SGN). Genome Biol. 2016. doi:10.1186/s13059-016-1038-5. https://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-1038-5

 
Varshney GK, Burgess SM. DNA-guided genome editing using structure-guided endonucleases. Genome Biology. 2016;17:187. doi:10.1186/s13059-016-1055-4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025577/#__ffn_sectitle

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