Design any edit, across the genome, with unmatched ease and precision.

Target type: Single location

Purpose: This approach enables the discovery of novel variants that improve enzyme function. Using this unbiased strategy can uncover beneficial diversity in unexpected sites in a protein.

Example libraries:

• Full AA saturation of E. coli dapA (3^rd enzyme in the lysine biosynthesis pathway). This 292 AA enzyme can be covered with full AA saturation (20 codons, including a synonymous codon, for each AA substituted at each position) at every position in a single Onyx library with 5,840 Onyx designs.
• Full AA saturation of rpoA, the alpha subunit of the RNA polymerase, to map the surface of responses to small molecule stress from coumaric acid.
• Full AA saturation of S. cerevisiae HEM13 (6^th step in heme biosynthesis pathway). This 328 AA enzyme can be covered with full AA saturation at all structured regions, from residues 16 – 315, in a single Onyx library with 6,000 designs.

See how to design a library in InscriptaDesigner^® software: dapA full saturation library design (2:49 min)

See examples of a project using this library:

• Lysine production in E. coli – Mini webinar (16:28 min)
• Investigating antimicrobial mechanisms-of-action using automated genome-wide and gene-specific editing (Webinar)

Target type: Single location, pathway

Purpose: A hybrid between purely arational full AA saturation (20 codons tested per AA position) and targeted AA substitutions, this approach designs a specific reduced set of AA substitutions at every position and/​or targets only a reduced subset of positions.

Example of libraries:

• 11 AA​“library” (KDSQCGALMFW) was used to target all residues of 506 AA protein ZWF1 in yeast, the first gene in the pentose phosphate pathway.
• Target every 3^rd residue of ectopic enzyme XylA. Use learnings to design subsequent libraries that hit only regional​“hot-spots” of activity with full AA saturation.
• Lysine biosynthetic pathway engineering: target every third residue in a protein for saturation mutagenesis, target every residue with a defined subset of amino acids, target every residue with a randomly selected subset of amino acids

See examples of a project using this library:

• Lysine production in E. coli – Mini webinar (16:28 min)

Target type: Single location, pathway

Purpose: Keys residues or ​“hot-spots” in multiple genes can be discovered in a single experiment. Minimizing the number of Onyx edits per residue can allow you to target many genes in one experiment, and subsequent libraries can do full AA saturation on key regions.

Example of libraries:

• Alanine scanning across all genes in the valine biosynthesis pathway in E. coli

• Alanine scanning across all genes in lipid biosynthesis pathways in S. cerevisiae

Target type: Single location, pathway

Purpose: Hypothesis-guided engineering can lead to an increase in the frequency of hits. Testing multiple hypothesis-guided strategies could also lead to learnings that inform future campaigns, increasing the efficiency of forward engineering over time.
Specific AA substitutions could be chosen based on: structure-guided engineering strategies, evolutionary strategies guided by multiple-sequence alignments, prior datasets identifying key domains or key residues, etc.

Example of libraries:

• Targeted AA substitution for a green fluorescent protein (GFP) integrated into the E. coli MG1655 genome. We designed 723 specific edits targeting know mutation hotspots — residues previously implicated in improving protein stability or altering its activity.
• Structure-based libraries doing AA substitution at all​“surface” residues for the genomically-integrated AhSTS gene in a resveratrol producing E. coli strain

See examples of a project using this library:

• Heterologous protein engineering and screening of genome-wide regulatory element libraries in E. coli (application note)

Target type: Pathway, genome-wide

Purpose: Altering global regulation through post-translational modifications can cause larger impacts on cell physiology with a smaller number of more targeted edits.

Example of libraries:

• Removing all known/​predicted ubiquitylation sites in the yeast proteome

• Mutating all known/​predicted phosphorylation sites in the yeast proteome to the phosphomimetic, aspartic acid

Target type: Single location

Purpose: In forward engineering, a winning strategy is often to find beneficial variants and then consolidate those variants into a single strain to find combinations that yield further phenotypic improvements. A single Onyx edit is targeted to one site in the genome, but within that Onyx edit, multiple changes to the genome can be made at one time. For example, making 6 different, targeted AA substitutions within a 15 AA window in a gene.

Example of libraries:

• Random combinations of AA substitutions at 56 key residues near the active site of ERG8 in the S. cerevisiae ergosterol biosynthesis pathway. An example of one design in this library could be an Onyx edit that delivers substitutions A56T, G64F, and H65Q altogether.

• Random combinations of AA substitutions at key residues in dapA in the E. coli lysine biosynthesis pathway
• Obtaining a map of per-residue impacts to stress response, the tolerance- and sensitizing edits to coumaric acid were combined in nearby combinatorial to further enhance tolerance and sensitivity.

See examples of a project using this library:

• Lysine production in E. coli – Mini webinar (16:28 min)
• Investigating antimicrobial mechanisms-of-action using automated genome-wide and gene-specific editing (webinar)

Target type: Pathway, genome-wide

Purpose: Localization of enzymes plays a key role in their biological function. Disrupting or ​“re-wiring” localization within the cell can cause significant biological changes which may be beneficial to desired phenotypes.

Example of libraries: Modify the secretory machinery of the cell by changing the localization of key enzymes to different cellular compartments. For all proteins known to be involved in secretion, replace any endogenous localization signals with an NLS (sequester in the nucleus) and Golgi localization signal.

Target type: Single location, pathway

Purpose: Evolution has found many ​“solutions” to biological problems already, but it can be challenging to synthesize and screen all known orthologs of a single enzyme. With an Onyx library, you can deliver a mix of different variants that are found in nature to a single base enzyme expressed from the genome.

Example of libraries:

• In an S. cerevisiae strain engineered to express a high-value natural product from plants, create 1000’s precise Onyx edits that each deliver 1 – 12 AA substitutions or in/​del changes found in a multiple sequence alignment of the rate-limiting enzyme.

Target type: Pathway

Purpose: If you have a protein pathway that you want to purify, instead of tagging each protein separately in different strains, you could introduce His-tags to all of those proteins at one time in a pooled format, and then purify them all together in a ​“one-pot” purification.

Example of libraries:

• His tags on key genes involved in protein synthesis, enabling in-vitro translation reactions to be performed from a single cell lysate/​purification.

Target type: Pathway, genome-wide

Purpose: Many proteins have separate functional domains, and their activity/​stability/​function can be modulated with domain truncations that would not be possible with full gene knock-outs.

Example of libraries:

• C‑term truncation of all genes predicted to be involved in stress tolerance in E. coli at three different positions: half, 2/​3rds, and 3/​4ths of the way through the gene body.

See examples of a project using this library:

• Investigating antimicrobial mechanisms-of-action using automated genome-wide and gene-specific editing (webinar)

Target type: Pathway, genome-wide

Purpose: Many proteins have separate functional domains, and their activity/​stability/​function can be modulated with domain truncations that would not be possible with full gene knock-outs. The N‑terminus is also a common site for signal and degradation signals and other regions of high biological significance to the protein’s function.

Example of libraries:

• Genome-wide library creating 18 AA N‑term truncations of all genes by deleting the first 18 codons following the start codon.

Target type: Single location, pathway

Purpose: Expression tuning can be critical for metabolic engineering and many other applications — often, even just small, localized changes in the promoter region can have large impacts on gene expression.

Example of libraries:

• HMG1 is the rate-limiting step in the mevalonate/​sterol pathway. To tune its expression in a yeast cell factory, perform full nt saturation at the ‑25 to ‑325 region

• E. coli engineered to over-express a specific lipid can be engineered with an Onyx library targeting the promoter region (-10 to ‑80) of all three key endogenous enzymes, plus the constitutive promoters of two ectopic genes.

Target type: Single location, pathway

Purpose: Higher fold-changes in expression can often be achieved by combining nt substitutions into combinatorial Onyx edits in the promoter region. 1000s of combinations of known beneficial edits, or combinations of variants found in divergent regions of the promoter can be tested in a single Onyx library.

Example of libraries:

• Random combinations of 4 – 20 nt substitutions in the promoter of dapA, an enzyme in the lysine biosynthesis pathway.

• Targeted combinations of 5+ nt substitutions in the promoter of CIT1, the first step of the TCA cycle.

Target type: Single location, pathway

Purpose: Translation rate is often determined by the sequence adjacent to the start codon — by making synonymous substitutions at the beginning of a gene, expression can be modulated.

Example of libraries:

• Full codon saturation of FAT1, involved in the synthesis of long-chain fatty acids, at positions 2 – 30.

Target type: Pathway

Purpose: Synthetic promoters and/​or RBS elements can be used to tune the expression of genes. When targeting a pathway or set of functionally-related genes, a ​“ladder” of synthetic promoters of known strength can be used to find the ideal expression level in a single experiment.

Example of libraries:

• Five synthetic promoters, with expression levels spanning two orders of magnitude, inserted in front of the endogenous RBS for all cell cycle-related genes in E. coli

• Combinations of 10 synthetic promoters and 4 RBS sequences inserted in front of all genes in an engineered pathway inserted in the E. coli genome.

See examples of a project using this library:

• Lysine production in E. coli – Mini webinar (16:28 min)
• Identifying genome-wide targets of osmotic stress tolerance in E. coli using CRISPR-mediated forward engineering — Webinar
• Massively Parallel Genome Engineering followed by pooled growth selections for rapid target discovery in microbes-Webinar
• Massively Parallel CRISPR Genome Editing in S. cerevisiae — Webinar

Target type: Pathway, genome-wide

Purpose: Translation initiation efficiency impacts protein levels and can be regulated by editing the nucleotide sequence adjacent to the start codon.

Example of libraries:

• Genome-wide substitution of upstream Kozak sequences: genes are ​“binned” by their WT RNA and protein levels (from published genome-wide datasets). Genes with high expression have their Kozak sequences substituted for Kozak sequences from genes with low expression and vice versa to try to modulate expression directionally.

See examples of a project using this library:

• Heterologous protein engineering and screening of genome-wide regulatory element libraries in E. coli (application note and webinar)

Target type: Pathway, genome-wide

Purpose: Alternative start codons like GTG can change expression with a very small edit that can be readily performed across pathways or genome-wide.

Example of libraries:

• Substitute GTG for all ATG start codons across all E. coli metabolic pathways

Target type: Pathway

Purpose: Codon usage can impact the expression of a gene (or of adjacent genes!). Targeted codon optimization or de-optimization can be a way to modulate expression without changing enzyme activity.

Example of libraries:

• Targeted codon changes based on a prediction of optimal yeast codon usage in all key enzymes in an engineered heterologous pathway.
• Site-saturation synonymous mutation library for the CBH1 project to improve expression.

See examples of a project using this library:

• Strain improvement for increasing protein production in S. cerevisiae (application note and webinar)

Target type: Pathway

Purpose: A variety of different Onyx edits (synthetic promoter ​“ladder”, KO, RBS, UTR deletions, etc.) can be combined in a single library to modulate the expression of each gene in multiple ways within the same experiment. This can be very useful in identifying genes where phenotype increases or decreases as the expression is increased.

Example of libraries:

• In E. coli, create a library that combines KO and synthetic promoter edits targeting all transcription factor genes

Target type: Pathway, genome-wide

Purpose: Targeted codon changes based on a prediction of optimal yeast codon usage in all key enzymes in an engineered heterologous pathway.

Example of libraries:

• Insert three stop codons (TAGTGATAA) at the 15^th residue of all coding genes in the genome
• Insert three stop codons in the first 3^rd of each coding gene, with the exact location chosen to optimize the Design Score in the portal.
• Create a library of Onyx edits that knock out every known transcription factor, kinase, and phosphatase in the genome. This library can lead to larger changes impacting whole gene regulatory networks.

See how to design a library with InscriptaDesigner™ software:

See examples of a project using this library:

• Identifying genome-wide targets of osmotic stress tolerance in E. coli using CRISPR-mediated forward engineering — Webinar
• Massively Parallel Genome Engineering followed by pooled growth selections for rapid target discovery in microbes-Webinar

Target type: Pathway, genome-wide

Purpose: Synthetic promoters or terminators can lead to strong over/under-expression of genes relative to their endogenous regulation, enabling unbiased pathway-wide or genome-wide surveys to find key genes involved in a phenotype of interest.

Example of libraries:

• Insert the strong constitutive J23100 promoter with a strong RBS in front of every gene in the E. coli genome.
• Selected a synthetic terminator to be inserted at the 3’ end of every gene in yeast

Target type: Pathway, genome-wide

Purpose: Transcripts stability can play a big role in expression level — deletions of the UTR region(s) tend to decrease expression levels, which can help balance pathway flux or identify novel biological processes of interest.

Example of libraries:

• In yeast, make a 40bp deletion of the 3′ UTR (directly after the stop codon) of all genes in the genome.

• In yeast, make a ​“ladder” of 5, 15, 25, and 25 bp deletions in the 3′ UTR, to try to titrate down expression levels of all genes in the amino acid biosynthesis and the upstream shikimate pathway. This can generate a strain producing higher levels of tryptophan, which can be used as a precursor for heterologous pathways.

Target type: Pathway, genome-wide

Purpose: Gene regulation networks are ideal targets for genome engineering — a single Onyx edit in a cell can lead to key changes across a network of genes. Onyx libraries can target transcription factor genes with edits that increase and/​or decrease expression, and they can also target the transcription factor binding sites (TFBS) that are regulating the expression of individual target genes.

Example of libraries:

• In E. coli, to try to improve salt tolerance, make KO and strong promoter edits targeting all transcription factors implicated in stress response pathways. Make additional edits in all predicted binding sites for these transcription factors.
• TFBS insertions/​deletions/​substitutions in the ENO2 promoter used to drive expression of CBH1.

Target type: Single location, pathway

Purpose: ncRNAs play various roles in the eukaryotes — Onyx edits can precisely target these non-coding features to try to modulate their role in gene regulation, RNA metabolism, etc.

Example of libraries:

• Perform nt saturation at the transcription start site of SRG1, a ncRNA that regulates the expression of SER3 in yeast.

Target type: Single location, pathway

Purpose: Transposons and other mobile elements can affect the local heterochromatin state and genome stability. Onyx edits can remove certain regions of these features to impact the expression and stability of nearby regions.

Example of libraries:

• In yeast, make scanning deletions along transposon genes on the same chromosome as an integrated, heterologous pathway to try to improve its expression and genome stability at that site.

Target type: Pathway, genome-wide

Purpose: ncRNAs play various roles in the eukaryotes — Genome-wide Onyx edits can disrupt the regulatory and other functions of these ncRNAs by creating deletions that disrupt their function.

Example of libraries:

• In yeast, scan along the body of all ncRNA genes and make 50nt edits that ​“scramble” the local sequence in that region. Many of these edits will likely disrupt or modulate the function of the ncRNAs.

Target type: Single location, pathway, genome-wide

Purpose: A heterochromatin state is a critical way to regulate gene expression, and heterochromatin ​“spreading” can have far-reaching consequences on the regulation of many nearby genes. Many sequence motifs and elements are known to impact chromatin state (tRNA genes, TAD boundaries, nucleosome positioning motifs, etc).

Example of libraries:

• Create Onyx edits disrupting all known heterochromatin-promoting motifs in a region on Chr III near a safe site used for ectopic gene/​pathway integration.