A guide to genome engineering with programmable nucleases

  title={A guide to genome engineering with programmable nucleases},
  author={Hyongbum Henry Kim and Jin-Soo Kim},
  journal={Nature Reviews Genetics},
Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) system — enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site… 

Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9

Methods for detecting nuclease off-target mutations are compared and methods for profiling genome-wide off- target effects are reviewed to discuss how to reduce or avoid off- Target mutations.

Targeted Genome Editing Using Site-Specific Nucleases

This chapter will primarily focus on ZFNs and TALENs, which are artifi cial proteins composed of a specifi c DNA-binding domain and a restriction enzyme FokI DNAcleavage domain, and review the properties and construction methods of these nucleases.

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Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating nuclease protein activity.

Genome editing comes of age

This work highlights the key advances that set the foundation for the rapid and widespread implementation of CRISPR–Cas9 genome editing approaches that has revolutionized the field.

Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN.

Probing the impact of chromatin conformation on genome editing tools

Qualitative cellular systems based on genetic reporters in which the euchromatic and heterochromatic statuses of designer nuclease target sites are stringently controlled by small-molecule drug availability demonstrate that TALENs and CRISPR/Cas9 nucleases are both significantly affected by the high-order epigenetic context of their target sequences.

Genome engineering technologies for targeted genetic modification in plants

A detailed overview of these systems is provided, the strengths and weaknesses of each are highlighted, research advances made with these technologies in model and crop plants are summarized, and their applications in plant functional genomics are discussed.

Genome-editing technologies and patent landscape overview.

This review compares key technologies of genome-editing zinc finger nucleases, transcriptional activator-like effector nucleases and CRISPR, with a focus on the race to acquire lucrative intellectual property rights, the currentCRISPR patent dispute and potential repercussions on innovation and the adoption of this promising technology by the medical community.



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The development of TALEN technology is reviewed in terms of scaffold optimization, DNA recognition, and repeat array assembly, and some perspectives on the future development of this technology are provided.

Precision genome engineering with programmable DNA-nicking enzymes

It is proposed that programmable nickases will be of broad utility in research, medicine, and biotechnology, enabling precision genome engineering in any cell or organism.

RNA-Guided Human Genome Engineering via Cas9

The type II bacterial CRISPR system is engineer to function with custom guide RNA (gRNA) in human cells to establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity

The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes.

A TALE nuclease architecture for efficient genome editing

This study identifies TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and uses them to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%.

An improved zinc-finger nuclease architecture for highly specific genome editing

Using structure-based design, two variant ZFNs are engineer that modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage.

Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly.

This work synthesized and tested hundreds of ZFNs to target dozens of different sites in the human CCR5 gene-a co-receptor required for HIV infection-and found that many of these nucleases induced site-specific mutations in the C CR5 sequence.

Targeted chromosomal deletions and inversions in zebrafish

It is demonstrated that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline and excised an entire lincRNA gene and enhancer element.

High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

It is found that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface, and off-target cleavage of CRISPR-associated (Cas)9-based RGNs is characterized.