Biology subjects

Sharrar, A.

Publications and source records attributed to Sharrar, A..

7 recordsLinked to original sources

bioRxiv2024

Viral delivery of compact CRISPR-Cas12f for in vivo gene editing applications

Treating human genetic conditions in vivo requires efficient delivery of the CRISPR gene editing machinery to the affected cells and organs. The gene editing field has seen clinical advances with ex vivo therapies and with in vivo delivery to the liver using lipid nanoparticle technology. Adeno-associated virus (AAV) serotypes have been discovered and engineered to deliver genetic material to nearly every organ in the body. However, the large size of most CRISPR-Cas systems limits packaging into the viral genome and reduce drug development flexibility and manufacturing efficiency. Here, we demonstrate efficient CRISPR gene editing using a miniature CRISPR-Cas12f system with expanded genome targeting packaged into AAV particles. We identified efficient guides for four therapeutic gene targets and encoded the guides and the Cas12f nuclease into a single AAV. We then demonstrate editing in multiple cell lines, patient fibroblasts, and primary hepatocytes. We then screened the cells for off-target editing, demonstrating the safety of the therapeutics. These results represent an important step in applying in vivo CRISPR editing to diverse genetic sequences and organs in the body.

molecular biology↗

bioRxiv2023

Discovery and engineering of AiEvo2, a novel Cas12a nuclease for human gene editing applications

The precision of gene editing technology is critical to creating safe and effective therapies for treating human disease. While the programmability of CRISPR-Cas systems has allowed for rapid innovation of new gene editing techniques, the off-target activity of these enzymes has hampered clinical development for novel therapeutics. Here we report the identification and characterization of a novel CRISPR-Cas12a enzyme from Acinetobacter indicus (AiCas12a). We then engineer the nuclease (termed AiEvo2) for increased specificity, PAM recognition, and efficacy on a variety of human clinical targets. AiEvo2 is highly precise and able to efficiently discriminate between normal and disease-causing alleles in Huntingtons patient derived cells by taking advantage of a single nucleotide polymorphism on the disease-associated allele. AiEvo2 efficiently edits several liver-associated target genes including PCSK9 and TTR when delivered to primary hepatocytes as mRNA encapsulated in a lipid nanoparticle. The enzyme also engineers an effective CD19 CAR-T therapy from primary human T cells using multiplexed simultaneous editing and CAR insertion. To further ensure precise editing, we engineered an anti-CRISPR protein (ErAcr) to selectively inhibit off-target gene editing while retaining therapeutic on-target editing. The engineered AiEvo2 nuclease coupled with a novel ErAcr protein represents a new way to control the fidelity of editing and improve the safety and efficacy of gene editing therapies.

biochemistry↗

bioRxiv2023

Casμ, a novel class of miniature type-V Cas12f nucleases with diverse PAM

Small CRISPR-Cas effectors are key to developing gene editing therapies due to the packaging constraints in viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type-V Cas12f effectors present a solution given their small size. Here we describe Cas, a novel class of miniature (<490AA) type-V Cas12f nucleases that cleave double stranded DNA in human cells. We determined their optimal trans-activating RNA (tracrRNA) empirically through rational modifications which resulted in an optimal single guide RNA (sgRNA). We show that the Cas nucleases have broader PAM preferences than previously known Cas12f effectors. The unique characteristics of these novel nucleases add to the diversity of the miniature CRISPR-Cas toolbox while the expanded PAM allows for the editing of genomic locations that could not be accessed with existing Cas12f nucleases.

biochemistry↗

bioRxiv2021

Soils and sediments host novel archaea with divergent monooxygenases implicated in ammonia oxidation

Copper membrane monooxygenases (CuMMOs) play critical roles in the global carbon and nitrogen cycles. Organisms harboring these enzymes perform the first, and rate limiting, step in aerobic oxidation of ammonia, methane, or other simple hydrocarbons. Within archaea, only organisms in the order Nitrososphaerales (Thaumarchaeota) encode CuMMOs, which function exclusively as ammonia monooxygenases. From grassland and hillslope soils and aquifer sediments, we identified 20 genomes from distinct archaeal species encoding divergent CuMMO sequences. These archaea are phylogenetically clustered in a previously unnamed Thermoplasmatota order, herein named the Ca. Angelarcheales. The CuMMO proteins in Ca. Angelarcheales are more similar in structure to those in ammonia-oxidizing archaea than those of bacteria, and they contain all functional residues required for activity. Similarly to the Nitrososphaerales, Ca. Angelarcheales genomes are significantly enriched in blue copper proteins (BCPs) relative to sibling lineages, including plastocyanin-like electron carriers and divergent nitrite reductase-like (nirK) 2-domain cupredoxin proteins co-located with electron transport machinery. Angelarcheales do not have identifiable genes for methanol oxidation or carbon fixation, encode significant capacity for peptide/amino acid uptake and degradation, and share numerous electron transport mechanisms with the Nitrososphaerales. In the studied soils and sediments Ca. Angelarcheales were at least as abundant as ammonia-oxidizing Nitrososphaerales. Thus, we predict that Angelarcheales live a mixotrophic lifestyle based on oxidation of ammonia liberated from peptide and amino acid degradation. This work expands the known diversity of Thermoplasmatota and of CuMMO enzymes in archaea and suggests that these organisms are important and previously unaccounted for contributors to nitrogen cycling.

microbiology↗

bioRxiv2020

Meanders as a scaling motif for understanding of floodplain soil microbiome and biogeochemical potential at the watershed scale

Biogeochemical exports of C, N, S and H2 from watersheds are modulated by the activity of microorganisms that function over micron scales. This disparity of scales presents a substantial challenge for development of predictive models describing watershed function. Here, we tested the hypothesis that meander-bound regions exhibit patterns of microbial metabolic potential that are broadly predictive of biogeochemical processes in floodplain soils along a river corridor. We intensively sampled floodplain soils located in the upper, middle, and lower reaches of the East River in Colorado and reconstructed 248 draft quality genomes representative at a sub-species level. Approximately one third of the representative genomes were detected across all three locations with similar levels of abundance, and despite the very high microbial diversity and complexity of the soils, ~15% of species were detected in two consecutive years. A core floodplain microbiome was enriched in bacterial capacities for aerobic respiration, aerobic CO oxidation, and thiosulfate oxidation with the formation of elemental sulfur. We did not detect systematic patterns of gene abundance based on sampling position relative to the river. However, at the watershed scale meander-bound floodplains appear to serve as scaling motifs that predict aggregate capacities for biogeochemical transformations in floodplain soils. Given this, we conducted a transcriptomic analysis of the middle site. Overall, the most highly transcribed genes were amoCAB and nxrAB (for nitrification) followed by genes involved in methanol and formate oxidation, and nitrogen and CO2 fixation. Low soil organic carbon correlated with high activity of genes involved in methanol, formate, sulfide, hydrogen, and ammonia oxidation, nitrite oxidoreduction, and nitrate and nitrite reduction. Thus, widely represented genetic capacities did not predict in situ activity at one time point, but rather they define a reservoir of biogeochemical potential available as conditions change.

microbiology↗

bioRxiv2019

Bacterial secondary metabolite biosynthetic potential in soil varies with phylum, depth, and vegetation type

Bacteria isolated from soils are major sources of specialized metabolites, including antibiotics and other compounds with clinical value that likely shape interactions among microbial community members and impact biogeochemical cycles. Yet, isolated lineages represent a small fraction of all soil bacterial diversity. It remains unclear how the production of specialized metabolites varies across the phylogenetic diversity of bacterial species in soils, and whether the genetic potential for production of these metabolites differs with soil type. We sampled soils and saprolite from three sites in a northern California Critical Zone Observatory with varying vegetation and bedrock characteristics and used metagenomic sequencing and assembly to reconstruct 1,334 microbial genomes containing diverse biosynthetic gene clusters (BGCs) for secondary metabolite production. We obtained genomes for prolific producers of secondary metabolites, including novel groups within the Actinobacteria, Chloroflexi and candidate phylum Dormibactereota. Surprisingly, one genome of a Candidate Phyla Radiation bacterium encoded for a ribosomally synthesized linear azole/azoline-containing peptide, a capacity we found in other publicly available CPR bacterial genomes. Overall, bacteria with higher biosynthetic potential were enriched in shallow soils and grassland soils, with patterns of abundance of BGC type varying by taxonomy.

microbiology↗

bioRxiv2017

Recovery of genomes from metagenomes via a dereplication, aggregation, and scoring strategy

Microbial communities are critical to ecosystem function. A key objective of metagenomic studies is to analyse organism-specific metabolic pathways and reconstruct community interaction networks. This requires accurate assignment of assembled genome fragments to genomes. Existing binning methods often fail to reconstruct a reasonable number of genomes and report many bins of low quality and completeness. Furthermore, the performance of existing algorithms varies between samples and biotopes. Here, we present a dereplication, aggregation and scoring strategy, DAS Tool, that combines the strengths of a flexible set of established binning algorithms. DAS Tool applied to a constructed community generated more accurate bins than any automated method. Further, when applied to environmental and host-associated samples of different complexity, DAS Tool recovered substantially more near-complete genomes, including novel lineages, than any single binning method alone. The ability to reconstruct many near-complete genomes from metagenomics data will greatly advance genome-centric analyses of ecosystems.

microbiology↗