Azim, T. | Search bioRxiv

bioRxiv2024

Synonymous mutations in AAV Rep enhance genome packaging in a library selection

When producing Adeno-Associated Virus (AAV) gene therapies, a significant fraction of capsids can lack the desired DNA cargo. In AAV, Rep proteins mediate DNA packaging and virus assembly, suggesting that changes in Rep activity, expression, or DNA binding might affect genome packaging. To understand how mutations in the Rep gene affect activity, we selected a library of Rep mutants for their ability to produce active virions. By sequencing the Rep gene following the purification of viruses that package AAV genomes, we identified Rep mutants having non-synonymous mutations with a range of cellular activities. Surprisingly, synonymous mutations within the p19 promoter were enriched to the greatest extent, increasing in abundance by 102 to 104-fold. When the most highly enriched mutant was used to package a synthetic DNA cargo into the AAV capsid, the packaging efficiency could not be differentiated from native Rep. These findings suggest that these synonymous mutations enhance AAV genome packaging into capsids by affecting Rep-DNA interactions. They also suggest that silent sequence changes in the DNA cargo packaged by Rep can be used to tune packaging DNA packaging efficiency. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/617207v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1ba20d0org.highwire.dtl.DTLVardef@c05a6corg.highwire.dtl.DTLVardef@70a248org.highwire.dtl.DTLVardef@ae587c_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Comprehensive analysis and genome-wide association studies of biomass, chlorophyll, seed and salinity tolerance related traits in rice highlight genetic hotspots for crop improvement

Farmland is on the decline and worldwide food security is at risk. Rice is the staple of choice for over half the Earths people. To sustain current demands and ascertain a food secure future, substandard farmland affected by abiotic stresses must be utilized. For rapid crop improvement, a broader understanding of polygenic traits like stress tolerance and crop yield is indispensable. To this end, the hidden diversity of resilient and neglected wild varieties must be traced back to their genetic roots. In this study, we separately assayed 15 phenotypes in a panel of 176 diverse accessions predominantly comprised of local landraces from Bangladesh. We compiled high resolution sequence data for these accessions. We collectively studied the ties between the observed phenotypic differences and the examined additive genetic effects underlying these variations. We applied a sophisticated fixed effect model to associate phenotypes with genotypes on a genomic scale. Discovered QTLs were mapped to known genes. Candidate genes were sorted by tissue specific gene expression profiles and protein level consequence of existing polymorphisms. Our explorations yielded 17 QTLs related to various traits in multiple trait classes. 12 identified QTLs were equivalent to findings from previous studies. Integrative analysis assumes novel functionality for 21 candidate genes on multiple evidence levels. These findings will usher novel avenues for the bioengineering of high yielding crops of the future fortified with genetic defenses against abiotic stressors.

plant biology↗