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Another genome editing system that has recently gained attention in research and clinical application is CRISPR/Cas9 system. The CRISPR/Cas9 system is based on CRISPR (clustered regularly interspaced short palindromic repeats) sequence and CRISPR associated (Cas) gene mechanism that are crucial for innate defense mechanism in bacteria and archaea enabling the organisms to respond to and eliminate invading genetic materials from their phages [132]. The CRISPR/Cas9 system consists of two key molecules that introduce a mutation into the DNA. First, Cas9 is an enzyme that acts as a pair of DNA scissor. It cuts the two strands of DNA at a specific location in the genome so the genome editing could be performed either addition or removal. The other molecule is guide RNA (gRNA) which consists of a small piece of predesigned RNA sequence (20 bases long) located within a long RNA scaffold. The long RNA scaffold binds to DNA and the gRNA sequence guides Cas9 to edit the specific part of genome. gRNA sequence is designed to be complementary to the target DNA sequence in the target gene in the genome. gRNA sequence consists of short palindromic repeats and the sequences that complement with the target genes. The target sequences should be present close to protospacer adjacent motif (PAM) sequence which increases the specificity of Cas9. After Cas9 nuclease enzyme site specifically cleaves double stranded DNA activating double-strand break repair machinery. If the DNA repair template is provided, the piece of DNA repair template will be inserted into the sequence of target genes [133, 134]. With this mechanism, the plasmid containing gRNA, Cas9 sequences, TracrRNA (transactivating CRISP RNA) and DNA repair template sequence can be introduced into cells or embryo of the animals by viral or nonviral delivery system [135]. Until now, there is no CRISPR/Cas9 experiment involving orthodontic tooth movement, however, this technology has been implemented in recent mineralized tissue research [136–139]. Future directions of gene therapy include the enhancement of the lentiviral vector-based approaches, fine tuning of the conditioning regimen, and the design of safer vectors or nonviral vector delivery system. In orthodontic field, the gene therapy approach will need several fundamental cell culture and animal experiments to demonstrate the safety and efficacy of the treatment concept. Clinical trials are required as the next step to ascertain the

We would like to acknowledge Ms. Pornpasdchanok Asawasuwan for all artworks in the manuscript. This manuscript was supported by ROAAP fund, the University of Illinois at Chicago, Brodie Craniofacial Endowment fund, and the National Institute of Dental and Craniofacial

clinicians and patients for efficacy of the treatments.

Acknowledgements

62 Current Approaches in Orthodontics

Research (DE024531).

Conflict of interest

The authors declare no conflict of interest.

Phimon Atsawasuwan1 \* and Sajjad Shirazi2,3

\*Address all correspondence to: patsawas@uic.edu

1 Department of Orthodontics, University of Illinois at Chicago, Chicago, Illinois, United States

