Fig 1.
Improvement of the M. polymorpha CRISPR/Cas9 system by codon optimization.
(A) Diagrams of the vectors used. pMpGE006 contains a cassette for the expression of Atco-Cas9 fused with an NLS under the control of MpEFpro. pMpGWB301_ARF1_1 contains a cassette for the expression of the gRNA ARF1_1 under the control of MpU6-1pro [25]. (B) Photograph of auxin-insensitive co-transformants of the two vectors described in (A). Agrobacterium-co-cultured sporelings that corresponded to one eighth of sporangium were plated on a medium in containing 3 μM NAA and two vector selection substances, 10 mg/L hygromycin and 0.5 μM chlorsulfuron. Diameter of the circle dish shows 10 cm. (C) Direct sequencing analysis of the target locus of MpARF1 in auxin-selected T1 co-transformants. Inserted or substituted bases are colored in magenta. The target guide sequence of ARF1_1 is shown in bold face with the PAM sequence in blue. (D and E) Proportions of genome editing patterns (D) and sequences of the target site (E) in T1 co-transformants, which were obtained with no auxin selection. Sixteen independent transformants were analyzed for the target-site sequence by direct sequencing. “Monoclonal” and “Mosaic” indicate direct sequencing read patterns with mutated sequence peaks only and those with mixed sequence peaks, respectively. “WT” indicates read patterns identical to the original target sequence.
Fig 2.
All-in-one vector systems for genome editing in M. polymorpha.
(A) Designs of Gateway-based all-in-one binary vectors and entry plasmids for gRNA cloning. pMpGE010 and pMpGE011 contain a cassette for the expression of Atco-Cas9 fused with an NLS under the control of MpEFpro, a Gateway cassette, and a cassette for the expression of hygromycin phosphotransferase (HPT) in pMpGE010 and mutated acetolactate synthase (mALS) in pMpGE011. pMpGE_En01 contains recognition sites for two restriction enzymes, SacI and PstI, upstream of a gRNA backbone for the insertion of a guide sequence by In-Fusion/Gibson cloning, which automatically places a G nucleotide for transcription initiation by RNA polymerase III (extra initial G). Expression of single guide RNAs is controlled by a 2 kbp fragment of MpU6-1pro. pMpGE_En02 and pMpGE_En03 contain two BsaI recognition sites upstream of the gRNA backbone for the insertion of a guide sequence by ligation without or with an “extra initial G,” whose expression is under the control of a 500 bp MpU6-1pro fragment. For all the entry vectors, the gRNA cassette is flanked by the attL1 and attL2 sequences and is thus transferrable to the Gateway cassette in pMpGE010 or pMpGWB011 by the LR reaction. (B) Designs of all-in-one binary vectors for direct gRNA cloning. pMpGE013 (HPT marker) and pMpGE014 (mALS marker) contain the Atco-Cas9-NLS expression cassette, a unique AarI site in the upstream of the gRNA backbone for insertion of a guide sequence by ligation with an “extra initial G,” whose expression is under the control of a 500 bp MpU6-1pro fragment.
Fig 3.
High-efficiency genome editing by the gateway-based system in M. polymorpha.
Sporelings were transformed with pMpGE010 harboring ARF1_1 gRNA (A and B) or NOP1_1 gRNA (C-E). Randomly selected transformants were genotyped. Proportions of genome editing patterns (A and C) and sequences of the target sites (B and D) in 32 T1 transformants are shown as in Fig 1. Panel E shows photographs of a wild-type plant (Tak-1) and a T1 transformant that exhibited the typical NOP1-defective “transparent” phenotype (Mpnop1ge).
Fig 4.
Effects of guide sequence lengths.
(A) Classification of phenotypes in MpNOP1 genome editing lines by the appearance patterns of transparent portions. Class I, entirely transparent (Mutant); class II, mosaic of transparent and non-transparent sectors (Mosaic); class III, entirely non-transparent (wild-type, WT). Scale bars = 1 cm. (B) Proportions of mutant phenotype classes in T1 plants transformed with MpNOP1-targeting gRNAs (NOP1_2) of different lengths. The shortest gRNA guide sequence tested was 16 nt. The numbers of T1 plants inspected are shown on the right-hand side.
Table 1.
Off target analysis of T1 transformants.
Fig 5.
De novo mutations found in the G1 generation.
(A) Delayed generation of new mutants from non-mutated sectors. G1 gemmae formed on a transparent or non-transparent sector of a class-II Mpnop1ge plant, described in Fig 4, were grown. Arrowhead shows a transparent sector due to a de novo mutation. Scale bar = 1 cm. (B) Proportions of genotypes in G1 populations derived from three independent class-II Mpnop1ge T1 lines (#7, #11, and #13). “Inherited mutation” and “de novo mutation” indicate mutations identical to or different from those identified in individual T1 lines, respectively. (C) Target-site sequences in lines with “Inherited mutation” and “de novo mutation” by direct sequencing analysis. Inserted or substituted bases are colored in magenta. The target guide sequence of NOP1_1 is shown in bold face with the PAM sequence in blue.
Fig 6.
Induction of large deletions using co-transformation of two genome editing vectors.
(A) Design of gRNAs to the MpNOP1 locus to dissect efficiencies of induction of large deletions. Grey boxes and lines indicate exons and introns. The dotted line indicates the downstream region of MpNOP1. gRNA positions are shown in red lines. Primer sets for PCR-based genotyping are also shown. (B) Representative images of electrophoresis of PCR-based genotyping. The gRNAs and primer sets used in the genotyping are shown. Expected sizes of the PCR products from the wild-type genome are colored in blue. Expected sizes of the PCR products from the genome in which inductions of large deletion occurred are colored in magenta. PCR products from non-specific amplifications are indicated by asterisks. (C) Summary table of the co-transformation experiment using pMpGE013 and pMpGE014. The ratio in the “Large deletion” row shows the number of T1 plants harboring the expected large deletion out of all the T1 plants inspected.
Fig 7.
Generation of conditional knockout mutants of an essential gene.
(A) Gene model of the MpMPK1 gene and the gRNA target site. Grey boxes and lines indicate exons and introns. “ATG” and “Stop” denote the predicated initiation and termination codons. PAM, target, and intron sequences are shown in blue, in bold, and by small letters, respectively. Note that the third base (g) of the PAM sequence is in intron 1 and that the first base of exon 2 is not G. (B) Targeted mutagenesis rate of the endogenous MpMPK1 locus by CRISPR/Cas9. Bar graphs show proportions of clonal frameshift (magenta), clonal in-frame (green), mosaic (yellow), and no mutations (grey) that were identified in plants transformed with pMpGE010 containing the gRNA shown in (A) only (Exp. 1) or together with MpMPK1 cDNA-containing pMpGWB337 (Exp. 2) or pMpGWB337tdTN (Exp. 3). (C) Target-site sequences of plants obtained in Exp. 3. Sequences of wild type (WT) and transgenic lines (#1 to #8 except #4) are shown as in (A). Inserted or deleted bases are colored in magenta with their numbers in parentheses. In line #4, a 2 bp insertion and a large 970 bp deletion that covers the predicted initiation codon were identified (magenta). (D) Growth defect manifested by conditional deletion of the MpMPK1 cDNA. Gemmae of transgenic line #4 were (+) or were not (–) subjected to heat shock (HS) and dexamethasone (DEX) treatment on day 0 and day 1 and grown at 22°C for 2 weeks (top) before observation by fluorescent microscopy for tdTomato-NLS (bottom). Scale bars = 5 mm (top); 1 mm (bottom). (E) Confirmation of deletion events by genomic PCR. From HS/DEX-treated plants (line #4), tdTomato-positive, disorganized tissues ("KO") and tdTomato-negative, thallus-looking sectors [such as the arrow in (D); "non"] were collected from two different individuals and analyzed by genomic PCR using primer sets 1 and 2 shown in the schematic illustration of the construct used (predicted product sizes are indicated) and primer set 3 shown in panel (C).