Metagenomic Breakthrough Unlovers New Retron Systems for Enhanced Gene Editing

Metagenomic Mining Reveals Novel Retron Systems

Researchers have reportedly discovered a vast array of previously unknown retron reverse transcriptases through comprehensive metagenomic analysis, according to a recent Nature Biotechnology publication. The study states that scientists developed a specialized bioinformatics pipeline to identify retron-RTs from bacterial and archaeal genomes, including approximately 2 million partially assembled bacterial genomes from the human microbiome. This approach led to the identification of more than 500 high-confidence, nonredundant retrons with well-annotated msr-msd sequences, which were subsequently classified into 11 distinct phylogenetic clades.

Metagenomic Mining Reveals Novel Retron Systems
Superior Editing Performance in Mammalian Cells
Optimized System Demonstrates Exceptional Precision
Engineering Enhancements Boost Efficiency
Expanded Targeting Capabilities
Overcoming Cellular Repair Limitations
Nickase Systems Enable DSB-Free Editing

Superior Editing Performance in Mammalian Cells

When tested in HEK293T cells, multiple novel retron systems demonstrated significantly enhanced editing capabilities, sources indicate. The report states that 32% of the 98 tested retron-RTs successfully restored RFP fluorescence in the screening assay, with ten systems showing more than double the repair activity of the previously characterized Eco1-RT. Particularly noteworthy was Mva1-RT from Myxococcus vastator, which reportedly achieved sixfold higher editing efficiency than Eco1-RT in transient repair assays. The most effective systems all belonged to clade 9, suggesting special compatibility with mammalian cellular environments.

Optimized System Demonstrates Exceptional Precision

Efe1-RT from Escherichia fergusonii emerged as the standout performer in genomic integration tests, analysts suggest. The research indicates this system restored RFP approximately tenfold better than Eco1-RT in genomically integrated reporters. Deep sequencing analysis reportedly revealed that over 99% of Efe1-RT-driven insertion events contained the intended 10-nucleotide cargo, with error rates comparable to chemically synthesized single-stranded oligodeoxynucleotides. The report states that editing fidelity appears limited by cell-intrinsic repair pathways rather than reverse transcriptase inaccuracy.

Engineering Enhancements Boost Efficiency

Further optimization through systematic engineering yielded additional improvements, according to the findings. Researchers discovered that separating the sgRNA and msr-msd into two independent transcripts increased editing efficiency, suggesting that fused designs may impair Cas9 or RT activity through structural constraints. Nuclear localization signal optimization identified that combinations of N-terminal and C-terminal bipartite SV40 NLSs provided the highest repair activity, though nuclear import didn’t appear to be the primary limiting factor for templated insertion.

Expanded Targeting Capabilities

The research demonstrates that retron editors can partner effectively with multiple CRISPR nucleases, the report states. When tested with both Cas9 and Cas12a systems, Efe1-RT maintained high insertion activity and precision. AsCas12a Ultra-RT fusions showed higher insertion activity than wild-type AsCas12a across all tested genomic loci, which analysts attribute to increased cleavage efficiency. Deep sequencing confirmed that 99% of inserts at the BRD8 locus contained the correct sequence when using Cas12a systems.

Overcoming Cellular Repair Limitations

DNA repair pathway modulation significantly enhanced retron editing performance, according to the research. Small molecule inhibitors targeting non-homologous end joining, particularly AZD7648, stimulated insertion of larger cargos by up to 8.8-fold. The report indicates that AZD7648 increased template usage for genomic repair without enhancing Cas9 cleavage activity. Fusion proteins combining Cas9 with HDR-promoting factors like CtIP and dominant-negative RNF168 increased retron editing efficiency 1.8-2.5-fold across multiple loci.

Nickase Systems Enable DSB-Free Editing

The study also explored retron editing with Cas9 nickases, potentially enabling double-strand break-free genome modification. Researchers found that Cas9(D10A) but not Cas9(H840A) retron editors could be enhanced through fusion with DNA repair proteins, achieving 3.3-fold increases in templated insertion rates. This finding suggests that specific nickase systems may provide viable pathways for precise genome editing without generating double-strand breaks.

The comprehensive characterization and engineering of retron systems described in the research reportedly establishes a robust foundation for precise genome editing applications across diverse biological contexts and target sequences.

References

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