Precision genome editing at multiple sites simultaneously with no off-target effects.

Engineering Biology

Precision genome editing at multiple sites simultaneously with no off-target effects.

Current State-of-the-Art

A variety of current tools can be used for DNA sequence edits and for non-editing-based genome engineering including gene regulation and chromatin engineering. Transcription activator-like effector nucleases (TALEN)-based or clustered regularly interspaced short palindromic repeats (CRISPR)-based genome engineering techniques introduce site-specific nicks or double-stranded breaks, which are then repaired using natural repair pathway.¹ Additional state-of-the-art editing technologies include adeno-associated virus (AAV)-mediated homologous recombination and meganuclease activity. With CRISPR and TALEN technologies, up to six distinct sites, and up to 15,000 identical sites, have been targeted simultaneously, with efficiencies ranging from 2% to 90%. Gene regulation is achieved through site-specific DNA-binding proteins (zinc-finger proteins, transcription activator-like effectors, and Cas proteins), which fuse to gene regulatory domains to carry out activation or repression of desired genes. In these cases, up to six distinct genes have been targeted for regulation, with repression magnitudes up to 300-fold (knock-down) and activation magnitudes up to 20-fold (knock-up).²

Breakthrough Capabilities

Ability to reliably create any precise, defined edit or edits (single nucleotide polymorphisms or gene replacement) with no unintended editing in any organism, with edits ranging from a single base change to the insertion of entire pathways.

Ability to generate any defined single base pair change in model organisms.

Bottleneck/Challenge: Performance of current editing technology and known-unknowns (e.g., chromatin, double-stranded break repair) and unknown-unknowns regarding basic biology.

Potential Solution: Improved base editing enzymes capable of catalyzing all possible nucleotide transitions.

Potential Solution: Engineered nucleases and recombinases to control repair of double-stranded breaks using either non-homologous end-joining or homologous recombination.

High-efficiency editing (beyond 90%) across the genome with no off-target activity.

Bottleneck/Challenge: A better understanding of canonical protospacer adjacent motif (PAM) specificities, non-canonical R-loop formation, types of double-stranded breaks, the effects of DNA supercoiling, and the double-strand DNA repair pathway mechanisms.

Potential Solution: A quantitative, predictive understanding of the coupled chromatin, editing, and repair interactions.

Potential Solution: A suite of genome editors covering all possible nucleotide (PAM) specificities.

Potential Solution: Genome editors with improved on-target and reduced off-target effects.

Potential Solution: Design algorithms for predicting single guide RNA (sgRNA) guide RNA sequences, sgRNA concentrations, and genome editor concentrations to achieve desired on-target activities with minimal off-target activities.

Bottleneck/Challenge: Need for a better understanding of chromatin effects on editing in higher order systems.

Potential Solution: Fusion of epigenetic effectors to Cas9.

High-efficiency gene insertion or deletion of moderately large changes (but less than 10 kilobases) via homologous recombination.

Bottleneck/Challenge: The ability to manipulate double-stranded DNA break repair at high efficiency in non-model cells.

Potential Solution: Single-effector base editors that catalyze insertions and deletions with high efficiency.

Potential Solution: Improving the targeted delivery of DNA repair templates to nuclei in model and non-model organisms.

Potential Solution: Inducible control of DNA break repair pathways with significantly higher efficiency.

Potential Solution: Retron-mediated synthesis of DNA repair templates from co-delivered crRNAs, sgRNAs or RNPs.

Bottleneck/Challenge: Ability to efficiently deliver large DNA constructs into cells.

Potential Solution: Expand viral packaging size.

Potential Solution: Develop and/or enhance non-viral DNA delivery tools and technologies.

Precise parallel editing or regulatory modifications (10 to 1000 modifications) across model and non-model organisms, including plants and animals.

Precise, predictable, and tunable control of gene expression for many genes inside diverse cells and organisms across different timescales.

Achieve long-lasting gene repression and activation.

Bottleneck/Challenge: Insufficient quantitative understanding of transcriptional regulation, epigenetic mechanisms, and cross-regulatory interactions.

Potential Solution: Quantitative characterization of additional genome editors with diverse protein fusions for more potent CRISPRa and CRISPRi.

Potential Solution: Systematic characterization of gene regulatory effects when changing RNP binding site locations, affinities, and RNP-PIC interactions.

Potential Solution: Engineered pioneer transcription factors that can reliably generate desired epigenetic states.

Ability to regulate expression in non-model organisms.

Bottleneck/Challenge: A quantitative understanding of promoter-specific, epigenetic-specific, and tissue-specific gene regulatory interactions.

Potential Solution: Organism- and tissue-specific promoters to express CRISPR components in desired non-model organisms.

Potential Solution: Organism- and tissue-specific protein fusion domains for potent activation or repression of genes in non-model organisms.

Potential Solution: Improved delivery and/or expression of CRISPR components into non-model organisms.

Technologies to monitor and manipulate genetic and epigenetic mechanisms controlling tissue-wide and organism-wide expression levels over time.

Bottleneck/Challenge: Insufficient methodologies for measuring and quantifying epigenetic, mRNA level, protein level, and metabolite level changes at genome-wide, single-cell resolution with sufficient precision to be relevant to clinical phenotypes.

Potential Solution: Single-cell epigenetic modification (histone modification, lncRNA, nucleosome position), protein level, and metabolite level determination.

Potential Solution: Single-cell, primary cell line measurements quantifying epigenetic modifications, protein levels, and metabolite levels, for example, using clinical tissues.

Ability to precisely regulate gene expression in whole-body organisms, including humans, with single-cell resolution using dynamic or static control.

(This capability excludes germline genome editing.)

Bottleneck/Challenge: Improved delivery and expression of large genetic constructs in primary cell lines.

Potential Solution: Coupled transfection, genome editing, and genome repair to insert large genetic constructs into site-specific regions within chromosomes.

Bottleneck/Challenge: Poorly understood tissue-specific cell states.

Potential Solution: Predictive models quantifying relationships between genetic, epigenetic states, and cellular phenotypes.

Potential Solution: Organ-, tissue-, and site-specific chromatin effectors with predictable/controllable effects on chromatin structure, gene expression, and gene accessibility.

Ability to reproducibly deliver editing cargo efficiently and specifically to a given target cells or tissues, and control dosage and timing of the editing machinery.

Improve editors to function without sequence requirements (such as protospacer adjacent motif (PAM) sequences) with activity comparable to 2019 state-of-the-art capabilities.

Routine use of editors without detectable off-target effects (less than 0.001% off-target editing).

Enhance specificity of delivery modalities for high efficiency (90% efficient) editing of cells in a defined tissue.

Bottleneck/Challenge: Current delivery modalities have low cell-type specificity.

Potential Solution: Large scale development of virus engineering for tropism/specificity.

Potential Solution: RNPs engineered with cell-type specificity via receptor interactions, and other modalities.

Bottleneck/Challenge: Editing in vivo often leads to low efficiency of edits.

Potential Solution: Enhanced viral delivery with long half-life and low immunogenicity (likely needed for every organism of interest, including plant viruses and animal viruses).

Potential Solution: Viruses with large capacity (up-to or greater than 10 kilobase capacity) needed to deliver editor, guide RNAs, and any donor DNA molecule.

Potential Solution: Engineered effector complexes (such as RNPs) that can be delivered directly in vivo and maintain activity.

Quantitative, specific, and multiplexed editing of any site, in any cell, in any organism.

Bottleneck/Challenge: Specificity, efficiency, genetic stability, and off-target effects all pose challenges.

Potential Solution: Continued improvement of delivery vectors for plants, non-model animals, and other organisms.

Potential Solution: Development of toolboxes of non-repetitive CRISPR components to enable highly-multiplexed editing without triggering genetic instability.

Potential Solution: Tools for editing in humans and animals for therapeutics, specifically those that overcome or mask the immune response.

Footnotes

Doudna, J. A., & Charpentier, E. (2014). Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), 1258096. View publication.
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173–1183. View publication.; Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., … Qi, L. S. (2013). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 154(2), 442–451. View publication.