Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements

Abstract

Although homologous recombination between transposable elements can drive genomic evolution in yeast by facilitating chromosomal rearrangements, the details of the underlying mechanisms are not fully clarified. In the genome of the yeast Saccharomyces cerevisiae, the most common class of transposon is the retrotransposon Ty1. Here, we explored how Cas9-induced double-strand breaks (DSBs) directed to Ty1 elements produce genomic alterations in this yeast species. Following Cas9 induction, we observed a significant elevation of chromosome rearrangements such as deletions, duplications and translocations. In addition, we found elevated rates of mitotic recombination, resulting in loss of heterozygosity. Using Southern analysis coupled with short- and long-read DNA sequencing, we revealed important features of recombination induced in retrotransposons. Almost all of the chromosomal rearrangements reflect the repair of DSBs at Ty1 elements by non-allelic homologous recombination; clustered Ty elements were hotspots for chromosome rearrangements. In contrast, a large proportion (about three-fourths) of the allelic mitotic recombination events have breakpoints in unique sequences. Our analysis suggests that some of the latter events reflect extensive processing of the broken ends produced in the Ty element that extend into unique sequences resulting in break-induced replication. Finally, we found that haploid and diploid strain have different preferences for the pathways used to repair double-stranded DNA breaks. Our findings demonstrate the importance of DNA lesions in retrotransposons in driving genome evolution.

Fig 1. Ectopic recombination between Ty elements resulting in chromosome rearrangements.

Ty elements are depicted with black arrows, and centromeres are shown as white ovals or circles. (A) An unequal crossover between Ty elements located on sister chromatids can produce both duplications and deletions. Deletions between direct repeats can also occur by the single-strand annealing pathway (Fig 2B). (B) A crossover between repeats on opposite chromosome arms of sister chromatids can generate dicentric and acentric chromosomes. (C) An intrachromatid crossover between repeats on opposite arms can produce an acentric linear chromosome and a centromere-containing circle. (D) A crossover between Ty elements located on opposite arms in inverted orientation can generate two isochromosomes. (E) A crossover between inverted Ty elements located on the same arm results in an inversion in the segment located between the Ty elements. (F) Recombination between repeats located on non-homologous chromosomes can generate reciprocal translocations. Depending on the segregation pattern of the chromosomes, this event can produce cells that have a coupled terminal duplication and a terminal deletion. The two homologs are shown in red and blue.

Fig 2. Pathways of homologous recombination.

(A) Repair of a double-strand break (DSB) by synthesis-dependent strand annealing (SDSA), break-induced replication (BIR), and double-stranded break repair (DSBR). All pathways are initiated by a DSB shown by the vertical arrow, followed by 5’ to 3’ resection of the broken ends; the 3’ ends of the strands are marked with an arrow. The 3’ single-strand of one broken end invades the unbroken chromosome. DNA synthesis (dotted line) displaces a strand from the unbroken template. In the SDSA pathway, the invading strand is displaced and re-pairs with one of the broken ends. Mismatch repair of the heteroduplex (duplex formed from red and blue strands) results in a region of gene conversion (boxed) in which the flanking sequences are in the original parental configuration. In the BIR pathway, one of the broken products of the “red” chromosome is lost, and replaced by DNA synthesis from the unbroken template. This conservative synthesis involves the use of the blue strand as a template for one strand (continuous dotted line), and the new strand as a template for synthesis of the second strand (multiple short fragments with dotted lines). The net result of this process is a non-reciprocal LOH event that may extend to the end of the chromosome. The DSBR pathway is characterized by pairing between the displaced blue single-strand and one of the broken red strands. The resulting double-Holliday junction is resolved by cleaving the strands that connect the two duplexes. If the strands are cut as shown by the short arrows, the resulting duplexes have a region of conversion flanked by sequences in the recombinant configuration. It should be pointed out that extensive strand resection from the DSB may allow the recombination breakpoint to be displaced from the location of the DSB by >10 kb, as observed by Hoang et al. [15]. (B) Single-strand annealing (SSA) pathway. This intrachromosomal pathway is used to repair a DSB located between two repeated sequences (shown in black) that are separated by a region of non-repeated DNA (shown in gray). Resection of both ends exposes homology within the two repeats, allowing pairing. The unpaired single-strands are removed by endonucleases. The net result of this event is loss of one repeat and the intervening non-repeated sequence. Since Ty elements are flanked by 330-bp repeats, loss of the Ty and retention of one delta sequence may reflect the SSA pathway.

Fig 3. Sequence of CRISPR/Cas9 target in the Ty element, and diagnosis of events causing loss of URA3 insertion from Ty1::URA3.

(A) Sequence in Ty1 targeted by the guide RNA (shown in red). (B) Position of marked Ty element on chromosome III, and location of PCR primers used to diagnose 5-FOA-resistant isolates. The distance between the DSB site (CRISPR/Cas9 target) and the 3’ end of URA3 is about 2.5 kb. Large black arrows show the LTRs (delta elements) associated with Ty1. (C) Different classes of 5-FOA-resistant isolates based on PCR analysis. The sizes of the fragments resulting from PCR reactions with various combinations of primers are given in S3 Table. The wavy line in Class 4 indicates a different flanking sequence from the original Ty1 element. (D) CHEF gel analysis of rearranged chromosome III in MD745-21F. The size of chromosome III was altered as a result of a crossover-associated gene conversion. MD745 is the control strain.

Fig 4. Depictions of various classes of LOH events along one chromosome as analyzed by DNA sequencing or microarrays.

The Y-axis shows the ratio of coverage (RC), the number of “reads” for each SNP divided by the average “reads” of the two types of SNPs for the whole genome. Thus, a ratio of 0.5 indicates heterozygosity for a SNP. The RCs for each W303-1A- or YJM789-specific SNP are shown by red and blue circles, respectively. The open black circles indicate centromeres. The X-axis represents the SGD (Saccharomyces Genome Database) coordinate of the chromosome of the reference strain (S288c) numbered from the left to the right telomere. (A) Terminal LOH (T-LOH) event, reflecting a crossover or BIR. (B) Interstitial LOH (I-LOH) resulting from gene conversion or a double-crossover (less likely). (C) Terminal deletion (T-DEL). (D) Terminal duplication (T-DUP). (E) Interstitial deletion (I-DEL). (F) Interstitial duplication (I-DUP). (G) Double T-DEL or I-DUP that contains a centromere. (H) Coupled intrachromosomal T-DEL and T-DUP; isochromosome.

Fig 5. Locations of CRISPR/Cas9-induced genomic alterations relative to the positions of Ty elements.

The locations of Ty1 and Ty2 elements are shown as short vertical black or red lines, respectively, and the centromeres are shown as black ovals. Two partial Ty elements with the CRISPR/Cas9 target (one at 803 kb on IV and one at 215 kb on XII) are also shown as black lines. The breakpoints for various types of events are indicated by horizontal lines of different colors. The color code for genomic alterations is: I-LOH (red), T-LOH (gray), I-DEL (black), I-DUP (green), T-DEL (yellow), T-DUP (purple). Most I-LOH and T-LOH events are not associated with Ty elements at their breakpoints, whereas the breakpoints of I-DEL, T-DEL, I-DUP, and T-DUP events are associated with Ty elements.

Fig 6. Analysis of the translocation in MD741-7.

(A) CHEF gel showing a novel chromosome band at 820 kb. This chromosome hybridizes to probes derived from the right end of chromosome V and the left end of chromosome XIII. (B) Sequencing data showing a T-DUP on chromosome V and a T-DEL on chromosome XIII; both breakpoints are at Ty1 elements (Dataset S2.1 in S2 Data). (C) Production of the translocation by crossing-over between Ty elements (shown as yellow triangles). The translocation could also result from a BIR event (Fig 6B). (D) Nanopore sequencing of a chromosome V-XIII translocation (depiction by Ribbon software). The breakpoints of the translocation are 493,149 on V (close to a Crick-oriented Ty at 493 kb, S2 Data) and 748,223 (close to a Crick-oriented Ty at 748 kb, S2 Data) on XIII.

Fig 7. Breakpoints within Ty elements as determined by Nanopore sequencing.

Since Ty elements often have polymorphisms that distinguish different elements, we can sometimes determine the breakpoints within the Ty elements involved in homologous recombination. These Ty-Ty recombination events sometimes involve elements located on non-homologous chromosomes (giving rise to translocations; A, E, H, and K) or elements located on the same chromosome (giving rise to I-DEL, I-DUP or other rearrangements; B, C, D, F, G, I, and J). The position of the target guide RNA is indicated by an arrow marked “DSB” and different colors represent sequences derived from different elements. In some of the recombination events (C, G, I, and J), the breakpoint between the contributions of the different Ty elements to the recombinant element is near the DSB site. In most of the other events, the transition between the different Ty elements does not coincide exactly with the DSB site. In these cases, it is likely that the repair of the DSB was associated with a gene conversion event. Lastly, one of the isolates (K) was associated with a tripartite recombination event between Ty elements located on three different chromosomes as illustrated in S24 and S25 Figs.