Programmable Genome Editing Tools and their Regulation for Efficient Genome Engineering

Abstract

Targeted genome editing has become a powerful genetic tool for studying gene function or for modifying genomes by correcting defective genes or introducing genes. A variety of reagents have been developed in recent years that can generate targeted double-stranded DNA cuts which can be repaired by the error-prone, non-homologous end joining repair system or via the homologous recombination-based double-strand break repair pathway provided a suitable template is available. These genome editing reagents require components for recognizing a specific DNA target site and for DNA-cleavage that generates the double-stranded break. In order to reduce potential toxic effects of genome editing reagents, it might be desirable to control the in vitro or in vivo activity of these reagents by incorporating regulatory switches that can reduce off-target activities and/or allow for these reagents to be turned on or off. This review will outline the various genome editing tools that are currently available and describe the strategies that have so far been employed for regulating these editing reagents. In addition, this review will examine potential regulatory switches/strategies that can be employed in the future in order to provide temporal control for these reagents.

Three new active members of the I-OnuI family of homing endonucleases

“…In conclusion, this study presents three new members of the I-OnuI family of LHEs and although recently alternative genome editing reagents have become very popular, the low off-target activities make LHEs an attractive set of enzymes that warrants further exploration (Cox et al 2015;Lambert et al 2016). Engineering modular meganucleases by combining the LHE DNA cutting domains with more programmable DNA binding domains might be a promising direction for utilizing LHEs that belong to the IOnuI family (Hafez and Hausner 2012;Wolfs et al 2014Wolfs et al , 2016Boissel et al 2014;Romano Ibarra et al 2016;Guha et al 2017). prns-sub2 to prns-sub9) containing various length versions of the target region for I-CcaI.…”

Naming CRISPR alleles: endonuclease-mediated mutation nomenclature across species

“…Single-nucleotide deletions, insertions and substitutions, specific sequence replacement or insertion, large deletions or genomic rearrangements (e.g., inversions or translocations), up-regulation of specific endogenous genes, altered histone modifications or DNA methylation, or insertion of fluorescent proteins have all been achieved by these methods (reviewed in Guha et al 2017, reviewed in; Sander and Joung 2014; Wolfs et al 2016; Zhang et al 2017). In addition to biasing repair toward NHEJ, HDR or modification of histones, CRISPR/Cas technology can be used to target more than one gene at a time (reviewed in Rocha-Martins et al 2015).…”

“…All three technologies induce targeted double-strand breaks that are repaired through either error-prone, non-homologous end joining (NHEJ) or homology-directed recombination (HDR) with a donor template depending on experimental conditions to produce a variety of mutations ranging from insertions and deletions to sequence replacement ( Fig. 1; reviewed in Carroll 2014; Chen et al 2014; Guha et al 2017; Harrison et al 2014 ) . Thus, a single targeting sequence can produce an array of alleles from those containing a single-nucleotide deletion or insertion to alleles with large gene deletion (65 kb) or cassette insertion up to 5 kb (Zhang et al 2015).…”

“…Thus, a single targeting sequence can produce an array of alleles from those containing a single-nucleotide deletion or insertion to alleles with large gene deletion (65 kb) or cassette insertion up to 5 kb (Zhang et al 2015). The ability to generate so many mutations from a single targeting sequence makes CRISPR/Cas9 a powerful tool for reverse genetics, targeted allele generation, and reversion of existing mutation (reviewed in Guha et al 2017). In addition, the number and diversity of mutations and alleles created presents a challenge to unambiguous identification, integration of data within organism-specific databases, and cross-species data mining using external resources available at InterMine and the fledgling Alliance of Genome Resources (see “Summary”).…”

Application of Genome Editing Techniques in Immunology

“…Targeted genome editing (reviewed in Guha et al 2017) constitutes a powerful tool for biological research and potential approach for genetic therapy. The most general concept behind genome editing is the introduction of double-strand breaks within DNA sequence in a region of interest, followed by an action of endogenous repair machinery to induce targeted mutations.…”