Publications by authors named "Jason C Lin"

11 Publications

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Electroporation-Mediated Genome Editing of Livestock Zygotes.

Front Genet 2021 13;12:648482. Epub 2021 Apr 13.

Department of Animal Science, University of California, Davis, Davis, CA, United States.

The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.
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http://dx.doi.org/10.3389/fgene.2021.648482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8078910PMC
April 2021

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.

BMC Genomics 2021 Feb 12;22(1):118. Epub 2021 Feb 12.

Department of Animal Science, University of California - Davis, Davis, CA, USA.

Background: The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells.

Results: By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template.

Conclusion: The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.
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http://dx.doi.org/10.1186/s12864-021-07418-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881600PMC
February 2021

Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos.

Sci Rep 2020 12 18;10(1):22309. Epub 2020 Dec 18.

Department of Animal Science, University of California - Davis, Davis, CA, USA.

The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.
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http://dx.doi.org/10.1038/s41598-020-78264-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749171PMC
December 2020

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos.

Sci Rep 2020 09 29;10(1):16031. Epub 2020 Sep 29.

Department of Animal Science, University of CA - Davis, Davis, CA, USA.

Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.
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http://dx.doi.org/10.1038/s41598-020-72902-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7525238PMC
September 2020

Hyperfluorescence Imaging of Kidney Cancer Enabled by Renal Secretion Pathway Dependent Efflux Transport.

Angew Chem Int Ed Engl 2021 01 26;60(1):351-359. Epub 2020 Oct 26.

Department of Chemistry and Biochemistry, The University of Texas at Dallas, 800 W. Campbell Rd., Richardson, TX, 75080, USA.

Renal tubular secretion is an active efflux pathway for the kidneys to remove molecules but has yet to be used to enhance kidney cancer targeting. We report indocyanine green (ICG) conjugated with a 2100 Da PEG molecule (ICG-PEG45) as a renal-tubule-secreted near-infrared-emitting fluorophore for hyperfluorescence imaging of kidney cancers, which cannot be achieved with hepatobiliary- and glomerular-clearable ICG. This pathway-dependent targeting of kidney cancer arises from the fact that the secretion pathway enables ICG-PEG45 to be effectively effluxed out of normal proximal tubules through P-glycoprotein transporter while being retained in cancerous kidney tissues with low P-glycoprotein expression. Tuning elimination pathways and utilizing different efflux kinetics of medical agents in normal and diseased tissues could be a new strategy for tackling challenges in disease diagnosis and treatments that cannot be addressed with passive and ligand-receptor-mediated active targeting.
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http://dx.doi.org/10.1002/anie.202010187DOI Listing
January 2021

Draft Genome Sequences of 16 Halophilic Prokaryotes Isolated from Diverse Environments.

Microbiol Resour Announc 2020 Feb 20;9(8). Epub 2020 Feb 20.

University of California, Davis, Davis, California, USA.

Halophile-specific enzymes have wide-ranging industrial and commercial applications. Despite their importance, there is a paucity of available halophile whole-genome sequences. Here, we report the draft genome sequences of 16 diverse salt-tolerant strains of bacteria and archaea isolated from a variety of high-salt environments.
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http://dx.doi.org/10.1128/MRA.01540-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033272PMC
February 2020

Tailoring Kidney Transport of Organic Dyes with Low-Molecular-Weight PEGylation.

Bioconjug Chem 2020 02 23;31(2):241-247. Epub 2019 Nov 23.

Department of Chemistry and Biochemistry , The University of Texas at Dallas , Richardson , Texas 75080 , United States.

Subtle changes in size can induce distinct responses of the body to hard nanomaterials; however, it is largely unknown whether just a few ethylene oxide unit differences in soft poly(ethylene glycol) (PEG) molecules could significantly alter the renal clearance of small molecules. By systematically investigating in vivo transport of the representative renal clearable organic dyes, IRDye800CW after being conjugated with a series of PEG molecules with molecular weight (MW) below 10 kDa, we found a MW-dependent scaling law: PEG45 (MW = 2100 Da) is an optimized MW to generate the most efficient renal clearance for IRDye800CW by expediting the glomerular filtration of organic dyes and reducing their nonspecific interactions with background tissue. Moreover, the uniqueness of PEG45 can be generalized to other organic dyes such as ZW800-1 and fluorescein. This finding highlights the importance of low-MW PEGylation in tailoring in vivo transport of organic fluorophores, which would broaden their biomedical applications.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033910PMC
February 2020

Polyphenolic glycosides and aglycones utilize opposing pathways to selectively remodel and inactivate toxic oligomers of amyloid β.

Chembiochem 2011 Jul 10;12(11):1749-58. Epub 2011 Jun 10.

Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.

Substantial evidence suggests that soluble prefibrillar oligomers of the Aβ42 peptide associated with Alzheimer's disease are the most cytotoxic aggregated Aβ isoform. Limited previous work has revealed that aromatic compounds capable of remodeling Aβ oligomers into nontoxic conformers typically do so by converting them into off-pathway aggregates instead of dissociating them into monomers. Towards identifying small-molecule antagonists capable of selectively dissociating toxic Aβ oligomers into soluble peptide at substoichiometric concentrations, we have investigated the pathways used by polyphenol aglycones and their glycosides to remodel Aβ soluble oligomers. We find that eleven polyphenol aglycones of variable size and structure utilize the same remodeling pathway whereby Aβ oligomers are rapidly converted into large, off-pathway aggregates. Strikingly, we find that glycosides of these polyphenols all utilize a distinct remodeling pathway in which Aβ oligomers are rapidly dissociated into soluble, disaggregated peptide. This disaggregation activity is a synergistic combination of the aglycone and glycone moieties because combinations of polyphenols and sugars fail to disaggregate Aβ oligomers. We also find that polyphenolic glycosides and aglycones use the same opposing pathways to remodel Aβ fibrils. Importantly, both classes of polyphenols fail to remodel nontoxic Aβ oligomers (which are indistinguishable in size and morphology to Aβ soluble oligomers) or promote aggregation of freshly disaggregated Aβ peptide; thus revealing that they are specific for remodeling toxic Aβ conformers. We expect that these and related small molecules will be powerful chemical probes for investigating the conformational and cellular underpinnings of Aβ-mediated toxicity.
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http://dx.doi.org/10.1002/cbic.201100123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4145403PMC
July 2011

Resveratrol selectively remodels soluble oligomers and fibrils of amyloid Abeta into off-pathway conformers.

J Biol Chem 2010 Jul 28;285(31):24228-37. Epub 2010 May 28.

Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.

Misfolded proteins associated with diverse aggregation disorders assemble not only into a single toxic conformer but rather into a suite of aggregated conformers with unique biochemical properties and toxicities. To what extent small molecules can target and neutralize specific aggregated conformers is poorly understood. Therefore, we have investigated the capacity of resveratrol to recognize and remodel five conformers (monomers, soluble oligomers, non-toxic oligomers, fibrillar intermediates, and amyloid fibrils) of the Abeta1-42 peptide associated with Alzheimer disease. We find that resveratrol selectively remodels three of these conformers (soluble oligomers, fibrillar intermediates, and amyloid fibrils) into an alternative aggregated species that is non-toxic, high molecular weight, and unstructured. Surprisingly, resveratrol does not remodel non-toxic oligomers or accelerate Abeta monomer aggregation despite that both conformers possess random coil secondary structures indistinguishable from soluble oligomers and significantly different from their beta-sheet rich, fibrillar counterparts. We expect that resveratrol and other small molecules with similar conformational specificity will aid in illuminating the conformational epitopes responsible for Abeta-mediated toxicity.
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http://dx.doi.org/10.1074/jbc.M110.133108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911349PMC
July 2010

Quantitative exploration of the occurrence of lateral gene transfer by using nitrogen fixation genes as a case study.

Proc Natl Acad Sci U S A 2006 Jun 12;103(25):9584-9. Epub 2006 Jun 12.

Department of Biochemistry and Biophysics, University of California-San Francisco, 600 16th Street, San Francisco, CA 94143, USA.

Lateral gene transfer (LGT) is now accepted as an important factor in the evolution of prokaryotes. Establishment of the occurrence of LGT is typically attempted by a variety of methods that includes the comparison of reconstructed phylogenetic trees, the search for unusual GC composition or codon usage within a genome, and identification of similarities between distant species as determined by best blast hits. We explore quantitative assessments of these strategies to study the prokaryotic trait of nitrogen fixation, the enzyme-catalyzed reduction of N(2) to ammonia. Phylogenies constructed on nitrogen fixation genes are not in agreement with the tree-of-life based on 16S rRNA but do not conclusively distinguish between gene loss and LGT hypotheses. Using a series of analyses on a set of complete genomes, our results distinguish two structurally distinct classes of MoFe nitrogenases whose distribution cuts across lines of vertical inheritance and makes us believe that a conclusive case for LGT has been made.
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http://dx.doi.org/10.1073/pnas.0603534103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1480450PMC
June 2006