CREEPY: CRISPR-mediated editing of synthetic episomes in yeast

Abstract

Use of synthetic genomics to design and build 'big' DNA has revolutionized our ability to answer fundamental biological questions by employing a bottom-up approach. Saccharomyces cerevisiae, or budding yeast, has become the major platform to assemble large synthetic constructs thanks to its powerful homologous recombination machinery and the availability of well-established molecular biology techniques. However, introducing designer variations to episomal assemblies with high efficiency and fidelity remains challenging. Here we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, a method for rapid engineering of large synthetic episomal DNA constructs. We demonstrate that CRISPR editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes. We optimize CREEPY for efficient and precise multiplex editing of >100 kb yeast episomes, providing an expanded toolkit for synthetic genomics.

Graphical Abstract

Figure 1.

CRISPR/Cas9 constructs used in this study. (A) The two-plasmid system. Cas9 plasmid is pre-transformed into yeast, followed by a second transformation of the sgRNA plasmid. (B) The single-plasmid system. Cas9 and sgRNA are co-expressed from a single plasmid, with a CEN/ARS (top) or a 2μ backbone (bottom).

Figure 2.

Genomic editing in yeast with one- or two-plasmid systems. (A) Editing efficiency of genomic ADE2. Error bars represent mean ± SD of three replicates. ns, not significant as calculated by an unpaired t-test. (B) Survival rate of yeast transformed with Cas9/sgRNA alone without donor DNA. Error bars represent mean ± SD of three replicates. P< 0.0001 (****), as calculated by an unpaired t-test. (C) Spot assay on SC–Ura plates with BY4741 strains transformed with Cas9 expressed from CEN/ARS or 2μ plasmids. CEN-URA3 and 2μ-URA3 were used as empty plasmid controls. (D) Doubling time of the same yeast strains used in the spot assay, extrapolated from growth liquid rates (Supplementary Figure S2). Error bars represent mean ± SD of three replicates. P< 0.001 (***), as calculated by an unpaired t-test. (E) Method to test the Cas9 toxicity and plasmid stability. Single colonies pre-transformed with either Cas9 or empty plasmids were inoculated in non-selective YPD media, then plated as single colonies and replicated onto SC–Ura selective plates. The proportion of Ura^– colonies is calculated. Three single colonies were tested as biological triplicates for each group. (F) Proportion of Ura^– colonies. Error bars represent mean ± SD, n = 3 and P< 0.0001 (****), as analyzed with unpaired t test.

Figure 3.

Episomal editing in yeast targeting at mSox2 YAV. (A) Structure of episomal mSox2 YAV. BAC, bacteria artificial chromosome backbone. (B) Episomal editing strategy. Cas9 creates a DSB at CTCF8 which can be repaired with a donor DNA containing CTCF8Δ. (C) Editing efficiency with the single-plasmid (CEN or 2μ backbone) or two-plasmid (CEN/2μ) systems, as determined by genotyping using deletion-specific primers (Supplementary Figure S1). Error bars represent mean ± SD of three replicates. P< 0.05 (*), as calculated by an unpaired t-test. (D) Survival rate of colonies transformed with Cas9/gRNA targeting CTCF8 without donor DNA templates. Error bars represent mean ± SD of three replicates. P< 0.005 (**), as calculated by an unpaired t-test. (E) WGS read coverage of colonies with failed or successful edits aligned to the mSox2 YAV. (F) Unintended deletion boundaries mapped to the indicated mouse genome repetitive elements (see also Supplementary Table S6). (G) Editing strategy of a genome-integrated mSox2 CTCF8 site inserted between YFL021C and YFL021W. (H) Editing efficiency of a genome-integrated mSox2 CTCF8. Error bars represent mean ± SD of three replicates (n = 32 for each group). (I) Survival rate of colonies for genomic and episomal YAV editing without donor DNA templates. Error bars represent mean ± SD of three replicates. P< 0.0001 (****), as calculated by an unpaired t-test. (J) Episomal YAV editing with an SpHIS5 marker adjacent to CTCF8 site. His⁺ or His^–, prototrophic or auxotrophic for histidine, respectively. (K) Ratio of His^– colonies surviving episomal YAV editing (shown in J). Error bars represent mean ± SD of three replicates. P< 0.0001 (****), as calculated by an unpaired t-test. Representative images of transformation plates are depicted in Supplementary Figure S5).

Figure 4.

Multiplex editing of three CTCF sites in mSox2 YAV. (A) Single-plasmid system for multiplex episomal editing. The tRNA-gRNA array was built using Golden Gate assembly with BsmBI. (B) Episomal editing efficiency for at least one or all three designer deletions. Error bars represent mean ± SD (n = 3). (C) Episomal editing efficiency calculated for each designer deletion. Error bars represent mean ± SD (n = 3). (D) Distributions of successful edits of CTCF13Δ, CTCF17Δ and CTCF25Δ. (E) WGS read coverage from colonies obtained in multiplex episomal editing experiments aligned to the mSox2 YAV. (F) Genomic boundaries of unintended deletions from multiple episomal editing experiments mapped to the mouse genome and annotated with relevant repetitive elements mapped at the sites of deletion (see also Supplementary Table S7).

Figure 5.

Internal deletion mechanism and multiplex editing with five targets. (A) Junctions from the internal deletions in colony 13. More junction sequences are shown in Supplementary Figure S9. (B) Survival rate with Cas9 and gRNA.CTCF17, in wild-type or rad52Δ0 background. (C) WGS read coverage from three colonies obtained in CTCF17 cleavage experiments in rad52Δ0 strain background, aligned to the original mSox2 YAV. (D) CREEPY construct used for multiplex editing with five targets. (E) Editing efficiency with five targets. Colonies (n = 96) were first screened for CTCF17Δ (left). Positive colonies (n = 10, green) were subsequently selected for further screening of all other targets. The raw PCR screening gel image is shown in Supplementary Figure S12.