Reprogramming homing endonuclease specificity through computational design and directed evolution

Thyme, Summer B.; Boissel, Sandrine; Quadri, Shafat A.; Nolan, Tony; Baker, Dean A.; Park, Rachel U.; Kusak, Lara; Ashworth, Justin; Baker, David C.

doi:10.1093/nar/gkt1212

“…Several efforts have focused on the possibility of using gene drives targeting mosquitoes to block malaria transmission (Scott et al., 2002; Windbichler et al, 2007, 2008, 2011; Li et al, 2013a; Galizi et al, 2014). However, development has been hindered by the difficulty of engineering homing endonucleases to cut new target sequences (Chan et al, 2013a; Thyme et al, 2013; Takeuchi et al, 2014). Attempts to build gene drives with more easily retargeted zinc-finger nucleases and TALENs suffered from instability due to the repetitive nature of the genes encoding them (Simoni et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…Despite early hopes, it has proven difficult to engineer homing endonucleases to cleave new target sequences. Numerous laboratories have sought to accomplish this goal for well over a decade with only a few recent successes (Chan et al, 2013a; Thyme et al, 2013; Takeuchi et al, 2014). More recently, a team constructed new versions of the fruit fly gene drive using modular zinc-finger nucleases or TALENs in place of the homing endonuclease (Simoni et al, 2014), both of which can be engineered to cut new target sequences.…”

Section: Introductionmentioning

confidence: 99%

Concerning RNA-guided gene drives for the alteration of wild populations

Esvelt¹,

Smidler²,

Catteruccia

³

et al. 2014

eLife

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Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology.DOI: http://dx.doi.org/10.7554/eLife.03401.001

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“…Despite early hopes, it has proven difficult to engineer homing endonucleases to cleave new target sequences. Numerous laboratories have sought to accomplish this goal for well over a decade with only a few recent successes [13][14][15] . More recently, a team constructed new versions of the fruit fly gene drive using modular zinc-finger nucleases or TALENs in place of the homing endonuclease 16 , both of which can be engineered to cut new target sequences.…”

Section: Engineered Gene Drivesmentioning

confidence: 99%

“…Several efforts have focused on the possibility of using gene drives targeting mosquitoes to block malaria transmission [7][8][9][10][11][12] . However, development has been hindered by the difficulty of engineering homing endonucleases to cut new target sequences [13][14][15] . Attempts to build gene drives with more easily retargeted zinc-finger nucleases and TALENs suffered from instability due to the repetitive nature of the genes encoding them 16 .…”

Section: Introductionmentioning

confidence: 99%

Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations

Esvelt

¹

,

Smidler

²

,

Catteruccia

³

et al. 2014

Preprint

View full text Add to dashboard Cite

Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology.

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“…Although this behavior, as well as its impact on engineering and retargeting meganucleases for genome modification, has been well described [21,22], its structural basis is unclear. Here we describe the identification and characterization of a single-chain meganuclease, I-SmaMI, that exemplifies this behavior.…”

Section: Introductionmentioning

confidence: 99%

The Structural Basis of Asymmetry in DNA Binding and Cleavage as Exhibited by the I-SmaMI LAGLIDADG Meganuclease

Shen

¹

,

Lambert

²

,

Walker

³

et al. 2016

Journal of Molecular Biology

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LAGLIDADG homing endonucleases (“meganucleases”) are highly specific DNA cleaving enzymes that are used for genome engineering. Like other enzymes that act on DNA targets, meganucleases often display binding affinities and cleavage activities that are dominated by one protein domain. To decipher the underlying mechanism of asymmetric DNA recognition and catalysis, we identified and characterized a new monomeric meganuclease (I-SmaMI), which belongs to a superfamily of homologous enzymes that recognize divergent DNA sequences. We solved a series of crystal structures of the enzyme–DNA complex representing a progression of sequential reaction states, and we compared the structural rearrangements and surface potential distributions within each protein domain against their relative contribution to binding affinity. We then determined the effects of equivalent point mutations in each of the two enzyme active sites to determine whether asymmetry in DNA recognition is translated into corresponding asymmetry in DNA cleavage activity. These experiments demonstrate the structural basis for “dominance” by one protein domain over the other and provide insights into this enzyme's conformational switch from a nonspecific search mode to a more specific recognition mode.

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Reprogramming homing endonuclease specificity through computational design and directed evolution

Cited by 33 publications

References 69 publications

Concerning RNA-guided gene drives for the alteration of wild populations

Concerning RNA-guided gene drives for the alteration of wild populations

Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations

The Structural Basis of Asymmetry in DNA Binding and Cleavage as Exhibited by the I-SmaMI LAGLIDADG Meganuclease

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