Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts

Abstract

We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated "Edit Plasmids," were constructed with two expression cassettes, one for the expression of Cas9, codon-optimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and Chlamydomonas chloroplasts. We also showed that Edit Plasmids persist and replicate in mitochondria autonomously for several dozens of generations in the presence of the wild-type genomes. Finally, we did not find insertions and/or deletions at one of the Cas9 cleavage sites in Chloroplasts, which are otherwise hallmarks of Cas9/gRNA-mediated non-homologous end joining (NHEJ) repair events in nuclear DNA. This is consistent with previous reports of the lack of NHEJ repair system in most bacteria, which are believed to be ancestors of organelles. This is the first demonstration of CRISPR-mediated genome editing in both mitochondria and chloroplasts in two distantly related organisms. The Edit Plasmid approach is expected to open the door to engineer organelle genomes of a wide range of organisms in a precise fashion.

Keywords: CRISPR; Cas9/gRNA; Chlamydomonas; Chloroplast; DNA replacement; Edit Plasmid; Genome editing; Mitochondria; Organelle; Yeast.

Figure 1. Schematic representation of the Edit Plasmids for Chlamydomonas chloroplasts.

See ‘Materials and Methods’ for details. (A) Overall structures of Edit Plasmids; (B) Structure of the Cas9 expression cassette; (C) Structure of the gRNA expression cassette; and (D) Structure of the donor DNA. Scales are provided for B–D.

Figure 2. Replacement of the chloroplast genome with donor DNA by Cas9/gRNA.

(A) Schematic view of the psaA-E3 genomic region targeted by two gRNAs (vertical arrows) and the donor DNA composed of codon-optimized GFPc gene that is provided by the Edit Plasmid. The recognition sites of primers used for the amplification of the junction regions are indicated (C1–C4). (B) PCR amplification of the junction region of the replaced DNA. Pooled DNAs extracted from independent colonies was used as templates with primers C2 and C4. Total number of colonies for each Edit Plasmids was 20 for YP13, 17 for YP14, 10 for YP21, 10 for YP22, 16 for YP23 and 24. Template for Lane 14 was untransformed wild-type cells. C2/C4 amplicon was 852 bp long. M: 1 kb plus molecular weight marker (New England Biolabs, Ipswich, MA, USA). (C) De-convolution of junction-PCR positive pools of YP13 transformants. A total of 12 events of the positive pools from Lanes 2 and 3 of (B) were analyzed by two primer sets C2/C4 and C1/C3 to amplify the left and right junction regions, respectively. Events #2 and #9 carried replaced DNA. C1/C3 amplicon was 712 bp long. (D) The sequence obtained from PCR amplification of the replacement DNA locus in Chlamydomonas plastid DNA modified by the Edit Plasmid approach. Underlined sequences: wild-type chloroplast genomic sequence that are not present on the Edit Plasmid. Sequences in bold: homologous regions (HR1c and HR2c) present in the donor DNA on the Edit Plasmid. Sequences in bold underlined: modified gRNA target sites present in the donor DNA. Sequences with double underlines: Silent mutations at the 3′ side of guide RNA sites to preclude re-cleavage by Cas9/sgRNA. (D) The sequence obtained from PCR amplification of the replacement DNA locus in Chlamydomonas plastid DNA modified by the Edit Plasmid approach. Underlined sequences: wild-type chloroplast genomic sequence that are not present on the Edit Plasmid. Sequences in bold: homologous regions (HR1c and HR2c) present in the donor DNA on the Edit Plasmid. Sequences in bold underlined: modified gRNA target sites present in the donor DNA. Sequences with dotted underlines: silent mutations at the 3′ side of guide RNA sites to preclude re-cleavage by Cas9/sgRNA.

Figure 3. Detection of SNPs at Cas9/sgRNA2c cleavage site in Chlamydomonas chloroplasts.

DNA Sequences (5′–3′) near the cleavage site in cloned amplicons lacking the AvaII site are aligned with the wild-type parent DNA shown on the top line. Deduced amino acid sequence is shown under each DNA sequence. The 20-nucleotide target sequence for the Cas9/sgRNA complex is indicated in blue. The PAM site (in green), the AvaII recognition site (in bold blue), and SNPs and the resulting amino acid changes (in red) are also labeled.

Figure 4. Schematic representation of the Edit Plasmids for yeast mitochondria.

See “Materials and Methods” for details. (A) Overall structures of Edit Plasmids; (B) Structure of the Cas9 expression cassette; (C) Structure of the gRNA expression cassette; and (D) Structure of the donor DNA. Scales are provided for B–D.

Figure 5. Replacement of the mitochondrial genome with donor DNA by Cas9/gRNA.

(A) Schematic view of the COX1 genomic region targeted by two gRNAs (arrows) and the donor DNA with GFP gene that is provided by the Edit Plasmid. The recognition sites of primers used for the amplification of the junction regions are indicated (C, F, 11 and 12). (B) PCR analysis of the junction regions of the integrated donor DNA. Left: 5′ region amplified with C/12 primer set; right: 3′ region amplified with F/11 primer set. Lanes 1–5: Five control lines with HS6 Edit Plasmid without Cas9 activity; lanes 6–10; Five lines with HS8 Edit Plasmid with Cas9 activity. (C) Wild-type CUY563 strain; (M) 1 kb plus molecular weight marker (New England Biolabs, Ipswich, MA, USA). Arrows: the size expected from the deduced sequence with integrated donor DNA (C/12 amplicon: 870 bp; F/11 amplicon: 907 bp). DNA fragments separated in lanes 6 and 10 for the both amplicons were isolated for sequence confirmation (see text). (C) The sequence obtained from PCR amplification of the replacement DNA locus in transformed yeast mitochondrial DNA modified by the Edit Plasmid approach. Underlined sequences: wild-type mitochondrial genomic sequences that are not present on the Edit Plasmid. Sequences in bold: short homologous regions present in the donor DNA (HR1 and HR2) adjacent to gRNA target sites. Sequences with dotted underlining: modified gRNA target sites present in the donor DNA (altered nucleotides are shown in bold). The codon-optimized GFP coding region is presented in italics. Sequences presented in lower case correspond to primers C and F that were used for amplification of the replacement DNA locus. Homologous recombination leading to DNA replacement occurred without causing any sequence changes either in the replacement DNA nor in the surrounding wild-type mitochondrial DNA.

Figure 6. Characterization of Edit Plasmids rescued from yeast mitochondrial transgenic cells.

(A and B) Restriction analysis of rescued Edit Plasmids. Lane 1, the original Edit Plasmid DNA, Lane 2–6, rescued Edit Plasmids from yeast cells. Plasmids were digested with NcoI and ClaI (A) or NcoI and PstI (B), and separated by 1.0% agarose gel electrophoresis. (C) Semi-quantitative PCR assay of the Edit Plasmid DNA (HS8) after the cross with the wild-type ρ⁺ strain. Lane 1, first overnight culture; lane 2, second; lane 3, third; and lane 4, fourth overnight culture. (M) 1 kb plus molecular weight marker (New England Biolabs, Ipswich, MA, USA).