Sasha (Alexandra) Khristich
@khristich.bsky.social
postdoc at the Petrov lab at Stanford studying evolution & evolvability
Taken together, we show that nicks near the GAA repeat of pathogenic and pre-mutational length lead to dramatic expansions of the repeat and explore the genetic mechanism of such expansions. Check out our paper for more details and let us know what you think!
November 25, 2024 at 9:44 PM
Taken together, we show that nicks near the GAA repeat of pathogenic and pre-mutational length lead to dramatic expansions of the repeat and explore the genetic mechanism of such expansions. Check out our paper for more details and let us know what you think!
We propose that a nick upstream of the replication fork transforms into a double-strand break during replication and expansion happens during the misalignment of the GAA repeats during the repair of this break via homologous recombination. 20/n
November 25, 2024 at 9:44 PM
We propose that a nick upstream of the replication fork transforms into a double-strand break during replication and expansion happens during the misalignment of the GAA repeats during the repair of this break via homologous recombination. 20/n
Turns out, homologous recombination is mostly required for all the expansions where the replication fork bumps into the nick first. This means that the position relative to replication is what determines the mechanism of expansion and not the composition of the strand! 19/n
November 25, 2024 at 9:44 PM
Turns out, homologous recombination is mostly required for all the expansions where the replication fork bumps into the nick first. This means that the position relative to replication is what determines the mechanism of expansion and not the composition of the strand! 19/n
In our original cassette, the replication machine first bumps in the 5’-GAA nick and then into the repeat, and in the inverted cassette, the replication fork first bumps into the repeat and then into the 5’-GAA nick. The situation is mirrored for the 5’-TTC nick. 18/n
November 25, 2024 at 9:44 PM
In our original cassette, the replication machine first bumps in the 5’-GAA nick and then into the repeat, and in the inverted cassette, the replication fork first bumps into the repeat and then into the 5’-GAA nick. The situation is mirrored for the 5’-TTC nick. 18/n
We then asked: does the mechanism of nick-induced expansion depend on the composition of the strand (GAA VS TTC) or the position of the nick relative to replication? To answer this question, we inverted the repeat cassette relative to the origin of replication. 17/n
November 25, 2024 at 9:44 PM
We then asked: does the mechanism of nick-induced expansion depend on the composition of the strand (GAA VS TTC) or the position of the nick relative to replication? To answer this question, we inverted the repeat cassette relative to the origin of replication. 17/n
Even among the two 5’-nicks, the mechanisms by which they cause expansions are different: the 5’-nick near the GAA repeat strand (and much less for the TTC strand) promotes expansions that depend on the Rad52 protein and thus happen via homologous recombination. 16/n
November 25, 2024 at 9:44 PM
Even among the two 5’-nicks, the mechanisms by which they cause expansions are different: the 5’-nick near the GAA repeat strand (and much less for the TTC strand) promotes expansions that depend on the Rad52 protein and thus happen via homologous recombination. 16/n
We found that not all nicks are created equal! Turns out, the nicks at the 5’- end of either strand lead to much higher GAA repeat expansion than those on the 3’-ends. 15/n
November 25, 2024 at 9:44 PM
We found that not all nicks are created equal! Turns out, the nicks at the 5’- end of either strand lead to much higher GAA repeat expansion than those on the 3’-ends. 15/n
What is the mechanism of nick-induced GAA repeat expansion? To figure that out, we decided to induce nicks at different locations around the repeat tract: 5’- and 3’- ends on the GAA and TTC strands. 14/n
November 25, 2024 at 9:44 PM
What is the mechanism of nick-induced GAA repeat expansion? To figure that out, we decided to induce nicks at different locations around the repeat tract: 5’- and 3’- ends on the GAA and TTC strands. 14/n
Even more exciting, when we induce a nick near shorter tracts of GAA repeats, such as 40xGAA or 33xGAA repeats, we still detect a bunch of expansion events. This is the first time someone ever detected an expansion of a pre-mutational GAA repeat allele in a model system! 13/n
November 25, 2024 at 9:44 PM
Even more exciting, when we induce a nick near shorter tracts of GAA repeats, such as 40xGAA or 33xGAA repeats, we still detect a bunch of expansion events. This is the first time someone ever detected an expansion of a pre-mutational GAA repeat allele in a model system! 13/n
We found that expressing the nickase HUGELY increases the rate of long 100xGAA repeat tract expansion, by up to ∼340 fold (when I said hugely I meant it!). This makes the rate of expansion comparable with those observed in human pedigrees. 12/n
November 25, 2024 at 9:44 PM
We found that expressing the nickase HUGELY increases the rate of long 100xGAA repeat tract expansion, by up to ∼340 fold (when I said hugely I meant it!). This makes the rate of expansion comparable with those observed in human pedigrees. 12/n
Here, we tested what would happen if we induced a DNA nick (a lesion in which one of the two DNA strands is broken) near a GAA repeat tract with a CRISPR Cas9 nickase. 11/n
November 25, 2024 at 9:44 PM
Here, we tested what would happen if we induced a DNA nick (a lesion in which one of the two DNA strands is broken) near a GAA repeat tract with a CRISPR Cas9 nickase. 11/n
What about the medium-length, pre-mutational GAA repeat alleles? Up to this day, no one has ever detected them expanding in a model system (at least that we know of!) so the mechanism of how pre-mutational alleles transform into pathogenic alleles has remained a mystery. 10/n
November 25, 2024 at 9:44 PM
What about the medium-length, pre-mutational GAA repeat alleles? Up to this day, no one has ever detected them expanding in a model system (at least that we know of!) so the mechanism of how pre-mutational alleles transform into pathogenic alleles has remained a mystery. 10/n
To overcome this problem, the @semirkin.bsky.social lab previously developed an extremely sensitive system in yeast that allows to ‘catch’ GAA repeat expansion happening with a probability of up to 10^-7 per cell per generation. 9/n
November 25, 2024 at 9:44 PM
To overcome this problem, the @semirkin.bsky.social lab previously developed an extremely sensitive system in yeast that allows to ‘catch’ GAA repeat expansion happening with a probability of up to 10^-7 per cell per generation. 9/n
Even though the long, pathogenic GAA repeat alleles readily expand in human pedigrees, they are pretty stable in model organisms like yeast and mice and thus detecting such expansion events to study their mechanism is not easy. 8/n
November 25, 2024 at 9:44 PM
Even though the long, pathogenic GAA repeat alleles readily expand in human pedigrees, they are pretty stable in model organisms like yeast and mice and thus detecting such expansion events to study their mechanism is not easy. 8/n
Some people also have an intermediate number of GAA repeats, between ∼34-70 repeats, so-called ‘pre-mutational’ alleles. These alleles are benign, but they do expand with a low probability and occasionally transform into the long, pathogenic alleles in future generations. 7/n
November 25, 2024 at 9:44 PM
Some people also have an intermediate number of GAA repeats, between ∼34-70 repeats, so-called ‘pre-mutational’ alleles. These alleles are benign, but they do expand with a low probability and occasionally transform into the long, pathogenic alleles in future generations. 7/n
The short GAA repeat alleles remain stable during intergenerational transitions. In contrast, the long, pathogenic alleles change their length with almost 100% probability and often become longer in children compared to their parents. 6/n
November 25, 2024 at 9:44 PM
The short GAA repeat alleles remain stable during intergenerational transitions. In contrast, the long, pathogenic alleles change their length with almost 100% probability and often become longer in children compared to their parents. 6/n
While most people have less than 33 GAA repeats in this location, Friedreich’s ataxia patients have more than 70 repeats, and occasionally more than 1000 GAA repeats! Such a large expansion turns off the FXN gene and leads to disability and ultimately death of a patient. 5/n
November 25, 2024 at 9:44 PM
While most people have less than 33 GAA repeats in this location, Friedreich’s ataxia patients have more than 70 repeats, and occasionally more than 1000 GAA repeats! Such a large expansion turns off the FXN gene and leads to disability and ultimately death of a patient. 5/n
Why do we care about GAA repeats? This is because expansion of the GAA repeat tract in the FXN gene leads to a severe disease called Friedreich’s ataxia. 4/n
November 25, 2024 at 9:44 PM
Why do we care about GAA repeats? This is because expansion of the GAA repeat tract in the FXN gene leads to a severe disease called Friedreich’s ataxia. 4/n
Other amazing students who contributed to this project are Shem Scott, an undergraduate at Tufts, and Jill Armenia, now a PhD student at Vanderbilt. 3/n
November 25, 2024 at 9:44 PM
Other amazing students who contributed to this project are Shem Scott, an undergraduate at Tufts, and Jill Armenia, now a PhD student at Vanderbilt. 3/n
This work was led by Liangzi Li, an extremely talented PhD student whom I was lucky to supervise back at the @semirkin.bsky.social lab!
Here is a copy of my original x tread about this work:
Here is a copy of my original x tread about this work:
November 25, 2024 at 9:44 PM
This work was led by Liangzi Li, an extremely talented PhD student whom I was lucky to supervise back at the @semirkin.bsky.social lab!
Here is a copy of my original x tread about this work:
Here is a copy of my original x tread about this work: