Noncanonical outcomes of break-induced replication produce complex, extremely long-tract gene conversion events in yeast

Stewart, Joseph; Hillegass, Michael B; Oberlitner, Joseph H; Younkin, Ellen M.; Wasserman, Beth F; Casper, Anne M.

doi:10.1093/g3journal/jkab245

Cited by 2 publications

(4 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We postulate that the homology directed repair process initiated by the Cas9-induced DSB is likely be terminated by crossover events. In our data we find parallels to previous observations of long tract gene conversion events (Stewart et al, 2021; Neuwirth et at. 2007; Scully et al, 2019).…”

Section: Discussionsupporting

confidence: 92%

“…Long track gene conversion, a break-induced repair terminated by a crossover or a crossover-like event, is a likely mechanism behind gene conversion covering such long stretches (Kockler et al, 2021). Long track gene conversion has been observed in diploid Saccharomyces cerevisiae encompassing chromosomal regions from 6-464 kb (Stewart et al 2021). In mammalian cells, long-tract gene conversion has been observed during mitosis, encompassing a minimum of 11 kb (Neuwirth et at.…”

Section: Cas9 Expression During Meiosis Prophase I Improves Ag Effici...mentioning

confidence: 99%

“…This high degree of local sequence identity may be significant. The long-track gene conversion process is highly favored when the homology at the DSB site between the donor chromosome and the recipient is very high, for about 200 bp (Stewart et al, 2021). We speculate that having an excellent match at both ends of the DSB allows for two sites of initiation of D-loop invasion and increases the probability of starting and completing the repair leading to more successful BID and our observed high efficiency in the Cyp3A1/3A2 configuration The high homology may also provide a stable D-loop, leading to higher repair completion rates (Hunter and Kleckner, 2001;Roy and Greene, 2020).…”

Section: Cas9 Expression During Meiosis Prophase I Improves Ag Effici...mentioning

confidence: 99%

See 2 more Smart Citations

Robust and Efficient Active Genetics Gene Conversion in the Rat and Mouse

Lai¹,

Álvarez-García²,

Read³

et al. 2022

Preprint

View full text Add to dashboard Cite

The utility of Active Genetic (AG) gene conversion systems in rats and mice holds great promise for facilitating the production of complex strains harboring multiple humanizing genes. The practical application of such systems requires the identification of a robust, reusable, and highly efficient system. By characterizing twenty-eight different promoter and target site pairs we aimed to define the parameters needed to establish an efficient conversion system in male and female rats and mice. Using three specific meiosis prophase I active promoters to drive Cas9 expression. We studied several variables, including the number of Cas9 target sites, the distance between target sites, the cis versus trans configuration in linked pairs, and the effect of Cas9 copy number. In the rat, three of twelve tested configurations provided efficient AG gene conversion in the 22% - 67% range, and four others catalyzed AG in the 0.7-1% range. The rat Ddx4 (Vasa) promoter provides higher AG efficiency than the Sycp1 promoter. In mice, ten of sixteen tested configurations, using the Sycp1 and pSycp1 promoters, provided efficiency in the 0.3% - 3.2% range. In rats, Cas9 expression levels are remarkably well correlated with AG gene conversion efficiency. The rat cis rCyp3A1/rCyp3A2 locus was the most successful configuration, with gene conversion efficiencies of 0.7%-67%. This target site has a special property; the two Cas9 target sites are nearly perfectly homologous in the 100 bases around the gRNA target site. Our findings identify key parameters that improve AG efficiency, including the use of two Cas9 target sites, and efficient promoters that drive high levels of Cas9 expression, that are correctly timed during gamete development. These findings also uncover the unexpected benefit of high homology at paired gRNA target sites to promote efficiency. We provide new data to guide future efforts to develop yet further improved AG systems.

show abstract

Section: Discussionsupporting

confidence: 92%

Section: Cas9 Expression During Meiosis Prophase I Improves Ag Effici...mentioning

confidence: 99%

Section: Cas9 Expression During Meiosis Prophase I Improves Ag Effici...mentioning

confidence: 99%

See 1 more Smart Citation

Robust and Efficient Active Genetics Gene Conversion in the Rat and Mouse

Lai¹,

Álvarez-García²,

Read³

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Most of the LOH that we observed in CRISPR-modified C. parapsilosis isolates started near or at the target locus ( Fig. 2 ): this strongly suggests that LOH may be the result of homology-based repair mechanisms triggered by the Cas9-induced double-strand break (DSB), such as gene conversion (short-tract or long-tract gene conversion events) or break-induced replication (BIR; long-range LOH) ( 40 – 42 ). In two strains (e802210 and 805700Δ/Δ) LOH appears to have occurred on both sides of the Cas9 cut site.…”

Section: Discussionmentioning

confidence: 85%