Journal: bioRxiv

Article Title: Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements

doi: 10.1101/782268

Figure Lengend Snippet: A: Sequence of the SUP4 ::(CTG)n locus. The CTG trinucleotide repeat tract comes from a human DM1 patient and is shown in blue. The flanking non-repeated DNA is in black. For each guide RNA, the PAM, the gRNA sequence as well as the expected DSB site are indicated. B : Southern blots of yeast strains during the time course. Lanes labeled Sp Cas9-gRNA and Sp Cas9ΔCter+gRNA are control strains in which no DSB was visible. In the strain expressing both Sp Cas9 and the gRNA, two bands are visible in addition to the parental allele (1966 bp). One band corresponds to the 3’ end of the DSB containing a small number of triplets (821 bp), the other one corresponds to the 5’ end of the DSB containing most of the repeat tract (1145 bp). C: Quantification of 5’ and 3’ DSB signals. For each time points, the total 5’ + 3’ signals were quantified and plotted as a ratio of the total signal in the lane. Three independent time courses were run in each strain background (except rad50 Δ for which two time courses were run) and plots show the average of three (or two) time courses.

Article Snippet: The plasmid containing Enhanced Sp Cas9 (version 1.1, Addgene #71814, ) was a generous gift of Carine Giovannangeli from the Museum National d’Histoire Naturelle .

Techniques: Sequencing, Labeling, Expressing

Journal: bioRxiv

Article Title: Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements

doi: 10.1101/782268

Figure Lengend Snippet: For each strain, the same number of cells were plated on galactose and glucose plates and the survival was expressed in CFU number on galactose plates over CFU number on glucose plates. The mean and the 95% confidence interval are plotted for each strain. Significant t-test p-values when compared to wild-type Sp Cas9 survival are indicated by asterisks, as shown on the figure.

Article Snippet: The plasmid containing Enhanced Sp Cas9 (version 1.1, Addgene #71814, ) was a generous gift of Carine Giovannangeli from the Museum National d’Histoire Naturelle .

Techniques:

Journal: bioRxiv

Article Title: Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements

doi: 10.1101/782268

Figure Lengend Snippet: A : Southern blot of genomic DNA at the SUP4 locus in the wild-type strain. The probe hybridizes ∼300 bp downstream the repeat tract (see ). The dotted red line shows the initial length of the CTG repeat tract. The lane labeled “Glucose” contains a clone in which Cas9 was not induced. Lanes numbered #1 through #19 contain independent clones in which Cas9 was induced. Asterisks point to lanes in which no signal was detected, meaning that the probe containing sequence was deleted. Note that signal intensities varies among lanes, showing that the probe did not fully bind to its target sequence, due to its partial deletion. B : Some examples of chromosome rearrangements following Cas9 induction in the wild-type strain. The genomic locus surrounding SUP4 is shown on top, ARS1018 is drawn in red, delta elements are in grey, protein-coding genes are colored in blue and tRNA genes in purple. The DSB (vertical purple arrow) is induced within SUP4 ::(CTG)n. Chromosome coordinates are indicated above and the probe used for hybridization is represented by an horizontal red bar. The locus sequence was retrieved from the Saccharomyces Genome Database ( http://yeastgenome.org/ , genome version R64-2-1, released 18th November 2014). Under the reference locus are cartooned the different chromosomal structures observed in some of the survivors. A yeast colony that was grown in glucose was also sequenced as a control. For each clone, vertical dotted lines represent junctions of rearrangements observed, with deletion sizes indicated in base pairs. Asterisk: clone #2 showed a complex rearrangement with a local inverted duplication involving the δ16 LTR and the 3’ end of the KCH1 gene 5 kb upstream SUP4 . Two clones (#8 and #9) exhibit exactly the same chromosomal rearrangement at precisely the same nucleotides. Note that CDC8 is an essential gene. C : Southern blot of genomic DNA at the SUP4 locus in the rad52 Δ strain. Legend as for . Note that for this Southern blot genomic DNA was digested with EcoRV (instead of Ssp I, see Methods), therefore the expected CTG repeat length was around 1.8 kb, instead of 1 kb. D : Southern blot of genomic DNA at the SUP4 locus in the sae2 Δ strain. Legend as for .

Article Snippet: The plasmid containing Enhanced Sp Cas9 (version 1.1, Addgene #71814, ) was a generous gift of Carine Giovannangeli from the Museum National d’Histoire Naturelle .

Techniques: Southern Blot, Labeling, Clone Assay, Sequencing, Hybridization

Journal: bioRxiv

Article Title: Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements

doi: 10.1101/782268

Figure Lengend Snippet: Left : The twelve different possible outcomes following Sp Cas9 induction are shown, subdivided in local and extensive rearrangements (see text for details). The SUP4 locus is pictured and shows the position of each genetic element on yeast chromosome X. The probe used on Southern blots is shown, as well as both primer couples used to amplify the locus. In order to assess a given clone to a rearrangement type, the following rules were followed: i) when a band was detected by Southern blot, primers su47 and su48 were used to amplify the locus an sequence it. These events corresponded to types I-V. The absence of a PCR product indicated that primer su48 genomic sequence was probably deleted and therefore primers su23 and su42 were used to amplify and sequence the locus. These were classified as types IV-V events; ii) when no band was detected by Southern blot, primers su 23 and su42 were directly used to amplify and sequence the locus. These events were classified as types VI-X and XII. When no PCR product was obtained, it meant that at least one of the two primers genomic sequence was probably deleted and these events were classified as type XI. Note that this last category may also contain rare-but possible-chromosomal translocations that ended up in puting each primer in a separate chromosome, making unobtainable the PCR product. The extent of type XI deletions cannot go downstream the su42 primer, since the CDC8 gene is essential. Right : The proportion of each type or event recovered is represented for wild type and mutants. Altogether, 220 surviving clones were sequenced, distributed as follows: WT: 51, rad52 Δ: 29, dnl4 Δ: 61, sae2 Δ: 32, dnl4 Δ sae2 Δ: 47.

Article Snippet: The plasmid containing Enhanced Sp Cas9 (version 1.1, Addgene #71814, ) was a generous gift of Carine Giovannangeli from the Museum National d’Histoire Naturelle .

Techniques: Southern Blot, Sequencing, Clone Assay

Journal: bioRxiv

Article Title: Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements

doi: 10.1101/782268

Figure Lengend Snippet: See for legend. e Sp Cas9 #1 and #2 refer respectively to guide RNAs #1 and #2 described in . The following number of surviving clones were analyzed: Sp Cas9: 51, e Sp Cas9 #1: 49, e Sp Cas9 #2: 48. See text for details.

Article Snippet: The plasmid containing Enhanced Sp Cas9 (version 1.1, Addgene #71814, ) was a generous gift of Carine Giovannangeli from the Museum National d’Histoire Naturelle .

Techniques: Clone Assay

Journal: bioRxiv

Article Title: Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements

doi: 10.1101/782268

Figure Lengend Snippet: A : Couples of primers used to amplify each EcoRV site are indicated above and can be found in Supplemental Table 2. EcoRV sites are shown by vertical arrows. Resection graphs are plotted for each primer pair. Average relative values of resection as compared to the total DSB amount detected on Southern blots are shown at 6 hours (in blue) and 8 hours (in red), along with standard deviations. B : Mechanistic model for chromosomal rearrangements following a Cas9-induced DSB. See text for details. Resulting rearrangement types are indicated in red near each pathway.

Article Snippet: The plasmid containing Enhanced Sp Cas9 (version 1.1, Addgene #71814, ) was a generous gift of Carine Giovannangeli from the Museum National d’Histoire Naturelle .

Techniques: