Biology subjects

Lareau, L.

Publications and source records attributed to Lareau, L..

3 recordsLinked to original sources

bioRxiv2026

Reprogramming Cas9 PAM Recognition for Allele-Specific Editing

The therapeutic potential of CRISPR-Cas9 genome editing is fundamentally constrained by the requirement for specific short DNA sequences (PAMs) flanking the target site, limiting access to many clinically relevant genomic loci. This stringent PAM requirement is particularly problematic in applications which require precise positioning, such as base editing and allele-specific editing. Although PAM-relaxed variants have expanded the targetable genome, they incur trade-offs in on-target activity, off-target editing, and cleavage kinetics. This highlights an unmet need for variants that are re-targeted to alternative PAMs in order to maintain the specificity and enzymatic performance inherent to stringent dinucleotide PAM recognition. To overcome these limitations, we developed a yeast selection platform to engineering SpCas9 variants with re-specified PAM recognition. Using a clinically relevant Huntingtons disease gene (HTT) SNP as a proof-of-concept target, we engineered variants with reciprocal NGC and NGT PAM selectivity, as a step toward allele-specific editing in a large percentage of Huntingtons disease patients. These yeast-selected SpCas9 variants retained their modified activity across multiple endogenous HEK293T loci, demonstrating that this specificity is robust across diverse genomic contexts. The variants surpassed PAM-broadened variants on their respective on-target PAM while displaying broad loss of activity across alternative PAMs, effectively re-specifying PAM recognition toward a single dinucleotide sequence. Retargeted variants recovered on-target cleavage kinetics approaching that of wild-type SpCas9, even under competing substrate conditions, demonstrating that PAM re-specification can simultaneously restore catalytic efficiency and improve specificity. Beyond NGC and NGT, we leveraged our high-throughput platform to engineer Cas9 with re-specified activity across multiple additional non-canonical PAMs in yeast, further demonstrating its utility as a general and programmable framework for expanding the therapeutic reach of precision genome editing.

molecular biology↗

bioRxiv2026

Prefoldin function links meiotic chromosome segregation with cellular remodeling and reveals tubulin sensitivity of the meiotic spindle

Faithful chromosome segregation is essential for producing viable gametes during meiosis, a specialized type of cell division compared to mitosis. Unlike mitosis, meiosis involves two consecutive chromosome segregation events without an intervening round of DNA replication. Here we identify Gim3, a subunit of the ubiquitously expressed and conserved prefoldin complex, as a critical regulator of meiotic but not mitotic chromosome segregation in budding yeast. Loss of Gim3 causes profound defects in chromosome segregation and gamete viability through reduced tubulin protein levels, which are also associated with reduced spindle length. In mitosis, however, GIM3 deletion minimally affects spindle length and chromosome segregation, despite similarly reduced tubulin levels in both contexts, highlighting a previously unrecognized difference between the sensitivity of meiotic and mitotic spindles to tubulin abundance. In addition to chromosome segregation defects, gim3{Delta} cells exhibit aberrant meiotic cellular remodeling, including defects in exclusion of age-associated protein aggregates from newly forming gametes. Importantly, experimentally induced meiotic chromosome mis-segregation similarly disturbs cellular remodeling. Together, our findings identify Gim3 as a key factor required for maintaining chromosome segregation integrity during meiosis and reveal a previously unrecognized link between chromosome segregation and meiotic cellular remodeling.

cell biology↗

bioRxiv2017

Accurate design of translational output by a neural network model of ribosome distribution

Synonymous codon choice can have dramatic effects on ribosome speed, RNA stability, and protein expression. Ribosome profiling experiments have underscored that ribosomes do not move uniformly along mRNAs, exposing a need for models of coding sequences that capture the full range of empirically observed variation. We present a method, Ixnos, that models this variation in translation elongation using a feedforward neural network to predict the translation elongation rate at each codon as a function of its sequence neighborhood. Our approach revealed sequence features affecting translation elongation and quantified the impact of large technical biases in ribosome profiling. We applied our model to design synonymous variants of a fluorescent protein spanning the range of possible translation speeds predicted with our model. We found that levels of the fluorescent protein in yeast closely tracked the predicted translation speeds across their full range. We therefore demonstrate that our model captures information determining translation dynamics in vivo, and that control of translation elongation alone is sufficient to produce large, quantitative differences in protein output.

genomics↗