At NovoHelix we are constantly evaluating new technologies to advance our digital genome engineering platform. The News articles below highlight science that inspire us both here at NovoHelix and within the broader synthetic biology, genetics, and biomedical communities.
Update | 13 October 2020
On the heels of World CRISPR Day, we are pleased to announce that NovoHelix is currently accepting proposals for its inaugural grant born out of seminal work of many CRISPR pioneers including the 2020 Nobel prize in chemistry laureates Drs. Jennifer Doudna and Emmanuelle Charpentier. Rapid advancements in genome engineering with programmable nucleases have opened the capability to model in comparative animal species including large animals such as pigs, that may adequately replicate the human condition where traditional rodent models have deficiencies. NovoHelix has a reputation for advancing biomedical model generation in both rodents and livestock; and these animal models continue to play critical roles in translational research and the advancement of human health.
The winning grant applicant will receive up to $50,000 for custom animal model or cellular model generation by NovoHelix. We will also select two grant awardees for $25,000 each to supply our first-in-class DNA polymerases Incendio™ and PyroPol™ or other specified NovoHelix genome editing products to accelerate research endeavors.
For more details and eligibility requirements regarding funding opportunity NHX031078 - Genome Engineering with Programmable Nucleases for Animal Model & Cell Model Generation, please visit:
Update | 1 September 2020
Because of shorter gestational intervals than cattle and the ability to produce multiple kids per pregnancy, the domestic goat is used as a chassis to produce copious amounts of recombinant proteins in their milk, a process also known as pharming. In 1999, Baguisi and colleagues published the first successful attempts to produce live transgenic goat offspring by somatic cell nuclear transfer (SCNT). Animal cloning by SCNT is still technically challenging and encumbered by inefficiencies in the reprogramming process, so parameters to improve oocyte-based reprogramming campaigns are still needed to make SCNT feasible for commercial settings. Gavin and colleagues report a retrospective analysis of SCNT done at production scale using over 37,000 ova that yielded 203 cloned goats with an efficiency of ~0.5%. Criteria to improve SCNT technical parameters for both academic and commercial endeavors are presented and highlight how incremental improvements to the multi-step method can produce substantial gains. (Full Disclosure: I have co-authored several posters, abstracts and a publication with William Gavin regarding expression of pluripotency-determining factors OCT4/POU5F1 and NANOG in pre-implantation goat embryos).
A separate report from the lab of Guangpeng Li has demonstrated that kickstarting the reconstructed nuclear transfer genome in mice by transient overexpression of Dux enhances SCNT efficiency. A knockdown screen revealed Dux, Dppa2, and Dppa4 as key factors enhancing zygotic genome activation (ZGA) in SCNT.
Learn more: Yang L, Liu X, Song L, Di A, Su G, Bai C, Wei Z, Li G. Transient Dux expression facilitates nuclear transfer and induced pluripotent stem cell reprogramming. EMBO Rep. 2020 Jul 27:e50054. doi: 10.15252/embr.202050054. Epub ahead of print. PMID: 32715614.
Learn more: Gavin W, Buzzell N, Blash S, Chen L, Hawkins N, Miner K, Pollock D, Porter C, Bonzo D, Meade H. Generation of goats by nuclear transfer: a retrospective analysis of a commercial operation (1998-2010). Transgenic Res. 2020 Aug;29(4):443-459. doi: 10.1007/s11248-020-00207-w. Epub 2020 Jul 1. PMID: 32613547.
Learn more: Akshey YS, Malakar D, De AK, et al. Hand-made cloned goat (Capra hircus) embryos—a comparison of different donor cells and culture systems. Cell Reprogram. 2010;12(5):581-588. doi:10.1089/cell.2009.0120
Learn more: Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overström EW, Echelard Y. Production of goats by somatic cell nuclear transfer. Nat Biotechnol. 1999 May;17(5):456-61. doi: 10.1038/8632. PMID: 10331804.
Update | 16 June 2020
NovoHelix in collaboration with Andrew Dudley’s lab at the University of Virginia and UNC-AMC have generated a reporter mouse for labeling extracellular vesicles. CRISPR‐Cas9‐mediated genome editing to generate mice bearing a CD63‐emeral GFP-loxP/stop/loxP knock‐in cassette that enables the specific labeling of circulating CD63+ vesicles from any cell type when crossed with lineage‐specific Cre recombinase driver mice. As proof‐of‐principle, we have crossed these mice with Cdh5‐CreERT2 mice to generate CD63emGFP+ vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females.
McCann JV, Bischoff SR, Zhang Y, Cowley DO, Sanchez-Gonzalez V, Daaboul GD, Dudley AC. Reporter mice for isolating and auditing cell type-specific extracellular vesicles in vivo. Genesis. 2020 Jul;58(7):e23369. doi: 10.1002/dvg.23369. Epub 2020 Jun 16. PMID: 32543746; PMCID: PMC7405599.
Update | 1 June 2020
Multiplexed recombineering (recombination-mediated genetic engineering) is improved 5 to 10-fold in E. coli by replacing single-stranded DNA binding protein Redβ, which promotes annealing of two complementary DNA molecules at the replication fork, with a CspRecT from Collinsella stercoris phage using the tool pORTMAGE-Ec1 This improvement is an exciting development originating from the laboratories of George M. Church and Csaba Pál. Wannier et al, 2020 elaborate that a major limitation of high-efficiency recombineering is the inability to port the phage homologous recombination machinery to non-host microbial strains as Redβ displays ‘host tropism’. By screening a library of single-stranded annealing proteins referred to as the Broad SSAP and RecT Libraries, they uncover SSAP homologs to efficiently promote recombineering.
A preprint of the article has been published on bioRxiv:
The final peer-reviewed manuscript is available at PNAS:
A full description of the pORTMAGE-Ec1 (plasmid ID 138474) sequence is available at Addgene:
The Collinsella stercoris phage recombination protein CstRecT(CspRecT) sequence is provided in GenPept via accession WP_006720782 or link:
Update | 28 May 2020
A series of papers by the genome aggregation database (gnomAD) consortium have been published in Nature, and have cataloged human DNA structural variation (SV) from > 125, 000 exomes and nearly 16,000 whole-genomes. In some individuals, the SV seemingly inactivates both genetic copies known as alleles, and this results in loss of gene function (LoF), thereby effectively creating a natural human ‘knockout’. Generation of animal knockouts akin to the natural human LoF SV would help to elucidate the functional consequences, if any, of this newly uncovered genetic variation. Indeed, the NovoHelix team continues to invest in multiplexing technologies that enhance our ability to scale DNA-writing and DNA-editing to tease out genetic variation and its consequent physiological impact. Whether it’s a single gene edit needed to create an isogenic normal human and diseased cell model or an animal model with a humanized allele mimicking the natural human DNA structural variation, we are developing the tools and pipelines to modify any genomic locus.
A primer on DNA structural variation is available at dbVAR:
See the series of papers at Nature:
Collins et al, 2020:
Vallabh Minikel et al, 2020:
Karczewski et al, 2020:
The Genome Aggregation Database (gnomAD):
Update | 26 May 2020
The deployment of site-specific recombinases (SSR) facilitates construction of genetic circuits to control spatial, temporal or conditional gene expression, to incorporate switches or logic gates and to tune promoter expression. Additional molecular tools are needed to write even more advanced genetic circuits, such as modifying entire metabolic pathways or tuning polygenic expression. Gomide et al, 2020 explore a set of new irreversible large serine type phage (LSTP) integrases and compare with established Bxb1 and PhiC31 integrases in multiple eukaryotic cell lines including human and mouse embryonic stem cells, neural stem cells, and bovine fibroblasts. Of the newly described integrases evaluated, the LSTP integrase from Bacillus cytotoxicus which the authors refer to as Int-13, demonstrated functionality in ‘disease-in-a-dish’ cell line models. The integrase was previously evaluated for activity in E. coli as demonstrated from Christopher Voigt’s lab.
For more information, see Gomide et al, 2020 at Nature – Communications Biology :
Yang et al, 2014 in Nature Methods describe phage integrase activity in E. coli:
Protein accession, e.g. WP_012095429, for LSTP integrase from Bacillus cytotoxicus:
Update | 3 May 2020
Back in October 2019, David Lui’s lab introduced prime editing as a precision alternative to gene editing. The components of the prime editing system are a fusion protein of Cas9 nickase and reverse transcriptase with a CRISPR pegRNA (prime editing guide RNA). Once the target site is nicked, this nicked DNA strand is used as a primer on the custom pegRNA to initiate reverse transcription. The pegRNA contains a template to recode or edit the target DNA site. The prime edited strand is cleaved by a flap endonuclease, and the successfully edited strand and wildtype DNA strand trigger mismatch repair which converts the wildtype DNA to the successfully prime edited strand. Prime editing has recently been extended to mouse models. Check out the recent publication by Liu et al 2020:
Liu et al, 2020 – primed editing in mouse models:
Anzalone et al, 2019: the original prime editing manuscript:
Update | 1 May 2020
A cardinal goal of reproductive medicine is to be able to generate functional human germ cells for basic research and clinical value. Deconvolution of the combinatorial input factors and differentiation steps to drive output of functional human germ cells has been extremely challenging. Building on their work studying mouse oocyte biology, Saito and colleagues now present a detailed protocol for generation of human oogonia from induced pluripotent stem cells. Check out their seminal paper in Science and their newly released Nature Protocol:
Yamashiro et al, 2018 Science:
Yamashiro et al, 2020 Nature Protocols:
Update | 25 April 2020
Celebrate National DNA Day with NovoHelix on April 25th. Check out our DNA cloning and vector construction services and receive a hefty discount!
For more information about National DNA Day
What is National DNA Day and why is it important?
National DNA Day commemorates the successful completion of the Human Genome Project in 2003 and the discovery of DNA's double helix by James Watson and Francis Crick in 1953. National DNA Day is officially celebrated on April 25th and began after the first session of the 108th Congress passed concurrent resolutions designating the day in 2003. This annual celebration offers students, teachers, scientists and the public many exciting opportunities to learn about the latest advances in genomic research and explore what they may mean for their lives.
The National Human Genome Research Institute (NHGRI), one of 27 institutes and centers that form the National Institutes of Health (NIH), is encouraging organizations to host events celebrating National DNA Day from January through May of each year.
Update | 23 April 2020
Recombineering allows synthetic biologists to introduce targeted mutations in bacterial artificial chromosomes (BACs). To date, most researchers have utilized counter-selection approaches to introduce targeted point mutations, but counter-selection suffers from limited robustness due to frequent escapees and many clones often have to be screened and validated. Bird and colleagues have devised an ingenious solution to circumvent counter-selection when introducing targeted point mutations: positive selection with a synthetic intron cassette containing a selectable marker and adjacent to the intended mutation site. They call this technique ESI (Exogenous/Synthetic Intronization) mutagenesis, and demonstrate that the cells receiving the BAC transgene efficiently splice out the synthetic intron. Check out their preprint publication on bioRxiv: https://www.biorxiv.org/content/10.1101/844282v2
For a review of recombineering counter-selection techniques from the labs of Don Court, George M. Church or Francis Stewart, see the following publications:
tolC, SDS, colicin E1, vancomycin:
tetA-sacB cassette, tetracycline, sucrose, fusaric acid:
bacterial toxin-antitoxin system, namely ccdB counter-selection with ccdA antidote:
optimized P. luminescens rpsL gene, rpsL-neo cassette, streptomycin:
Update | 22 April 2020
Large-scale genome editing is possible with a variety of dead-Cas9 base editor (dBE) variants. This work, originating from George Church's lab, pushes the current limits of base editing to tens of thousands of edited loci per cell as demonstrated in 293T and human induced pluripotent stem cells (hiPSCs). Notably, nickase base editors (nBE) were too toxic for large-scale editing. For more information, check out the published work in Nucleic Acid Research or the preprint server bioRxiv:
Update | 12 April 2020
Skryabin and colleagues show that long-range PCR validation is insufficient to screen for a common CRISPR-mediated knock-in error, namely head-to-tail insertions of DNA repair templates.
At NovoHelix we have observed this head-to-tail insertion phenomena across multiple projects and have developed strategies to collapse the multi-copy arrays into a single useable targeted knock-in. While these gene-editing errors have been observed by the University of Muenster team and known to the NovoHelix scientists since 2013, many newcomers to CRISPR have overlooked this common error and will need to re-validate knock-in animal models generated by CRISPR. We are excited that these multi-copy knock-in arrays have been independently observed and reported, and as a direct consequence we hope that the animal modeling community can devise approaches to mitigate these knock-in errors.
The final peer-reviewed manuscript is available at Science Advances: https://advances.sciencemag.org/content/6/7/eaax2941
A preprint of the article has been published on bioRxiv:
Update | 4 April 2020
Programmable DNA cleavage by a suite of compact CRISPR nucleases (class 2 subtype V-F1) are demonstrated from the labs of Virginijus Siksnys and Corteva Agriscience.
Update | 12 March 2020
To achieve polycistronic expression in mammalian cell lines, often two different genetic tools are harnessed from viruses: IRES's or 2A self-cleaving peptides. Woltjen and colleagues show that the residual N-terminal proline after 2A-cleavage can affect protein stability. In the context of deploying OKSM polycistronic vectors, e.g. drug-inducible piggyBac (PB) transposon reprogramming system, for nuclear reprogramming of adult cells to pluripotency, cellular reprogramming trajectories are hindered due to destabilized KLF4. Inclusion of a charged amino acid such as glutamic acid (E-) or lysine (K+) after the residual N-terminal proline (P) enhances reprogramming phenotypes. This paper builds on previously published work from Woltjen's lab in Kim et al, 2015 demonstrating that the longer KLF4 isoform by nine N-terminal amino acids improves initiation and stabilization phases of iPSC derivation.
Update | 11 March 2020
NovoHelix and Bruce D. Weintraub's Trophogen Inc. enter into a tentative agreement to improve superovulation protocols for rodents. Robust production of large numbers of viable oocytes and zygotes is integral to downstream applications such as genome engineering with CRISPR-Cas to develop relevant biomedical animal models. The most common methods for rodent superovulation use eCG/PMSG to mimic FSH stimulation and hCG to mimic LH surge to induce ovulation. NovoHelix and Trophogen are developing methods to increase the number of high-quality oocytes for animal model generation.
Update | 14 February 2020
Multiplexed recombineering (recombination-mediated genetic engineering) is improved 5 to 10-fold in E. coli by replacing single-stranded DNA binding protein Redβ, which promotes annealing of two complentary DNA molecules, with an CspRecT from Collinsella stercoris phage using the tool pORTMAGE-Ec1. This improvement is an exciting development originating from the laboratories of George M. Church and Csaba Pál.
Update | 21 January 2020
UK POST (the Parliamentary Office of Science and Technology) releases human germline editing brief
Update | 16 January 2020
Guidelines for research on human embryo models formed from stem cells
Update | 21 October 2019
Prime editing, a versatile and precise genome editing method