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.

Keywords: CRISPR/Cas9; Hammerhead ribozyme; Meganuclease; Regulatory switch; TALEN; Zinc finger nuclease.

Fig. 1

Examples of programmable genome editing tools. (a) Single-motif LAGLIDADG homing endonucleases, (b) double-motif LAGLIDADG homing endonucleases, (c) megaTAL, (d) MegaTev, (e) zinc-finger nucleases (ZFN), (f) transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems using (g) Cas9 or (h) Cpf1, (i) targetrons, (j) triplex-forming oligonucleotide (TFO) nucleases, and (k) structure-guided nucleases (SGNs). EBS = exon-binding site; IEP = intron-encoded protein. The nuclease domain of FokI is used to engineer ZNFs, TALENs, and SGNs. Elements of this figure have been adapted from Hafez et al. NRC Research Press License number: 3981970186164.

Fig. 2

Strategies used to modulate Cas9 activity. (a) Group II intron (GII)-based switch, (b) separating Cas9 into two peptides, termed split-Cas9, (c) Tetracycline-inducible and reversible expression system, and (d) ligand-dependent dimerization of split-Cas9. Note: the strategy illustrated in (a) is based on the original study conducted by Guha and Hausner on modulating expression of a meganuclease, not Cas9. A similar case is observed in (c), where Mandegar et al. modulated the expression of dCas9, not Cas9. In both cases, a similar approach might also be possible with Cas9. (e) Light-dependent dimerization of split-Cas9, termed photoactivatable Cas9 (paCas9), (f) intein-Cas9, which are activated by splicing of a ligand-dependent intein, (g) and unstable destabilizing domain-Cas9 (DD-Cas9) fusions, which are degraded unless provided with the ligand, Shield1. Abbreviations: CAG = cytomegalovirus early enhancer/chicken β-actin promoter; Cas9 = clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9; Cas9′ = partial Cas9; dCas9 = dead Cas9; FKBP = FK506 binding protein; FRB = FKBP-rapamycin binding; IPTG = isopropyl β-D-1-thiogalactopyranoside; KRAB = Krüppel-associated box; MN = meganuclease; mRNA = messenger RNA; rtTA = reverse tetracycline-controlled transcriptional activator; sgRNA = single-guide RNA; TRE = tetracycline response element; T7 = T7 RNA polymerase promoter; 4-HT = 4-hydroxytamoxifen; DD = destabilizing domain; nMag = negative Magnet; pMag = positive Magnet; sgRNA = single-guide ribonucleic acid. See text for more details.

Fig. 2

Strategies used to modulate Cas9 activity. (a) Group II intron (GII)-based switch, (b) separating Cas9 into two peptides, termed split-Cas9, (c) Tetracycline-inducible and reversible expression system, and (d) ligand-dependent dimerization of split-Cas9. Note: the strategy illustrated in (a) is based on the original study conducted by Guha and Hausner on modulating expression of a meganuclease, not Cas9. A similar case is observed in (c), where Mandegar et al. modulated the expression of dCas9, not Cas9. In both cases, a similar approach might also be possible with Cas9. (e) Light-dependent dimerization of split-Cas9, termed photoactivatable Cas9 (paCas9), (f) intein-Cas9, which are activated by splicing of a ligand-dependent intein, (g) and unstable destabilizing domain-Cas9 (DD-Cas9) fusions, which are degraded unless provided with the ligand, Shield1. Abbreviations: CAG = cytomegalovirus early enhancer/chicken β-actin promoter; Cas9 = clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9; Cas9′ = partial Cas9; dCas9 = dead Cas9; FKBP = FK506 binding protein; FRB = FKBP-rapamycin binding; IPTG = isopropyl β-D-1-thiogalactopyranoside; KRAB = Krüppel-associated box; MN = meganuclease; mRNA = messenger RNA; rtTA = reverse tetracycline-controlled transcriptional activator; sgRNA = single-guide RNA; TRE = tetracycline response element; T7 = T7 RNA polymerase promoter; 4-HT = 4-hydroxytamoxifen; DD = destabilizing domain; nMag = negative Magnet; pMag = positive Magnet; sgRNA = single-guide ribonucleic acid. See text for more details.