CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii

Abstract

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms.

Figure 1. Map of CRISPR/Cas9 target sites in the MAA7, CpSRP43 and ChlM loci, and in vitro cleavage of target DNAs by sgRNAs and the Cas9 protein.

(a–c) Molecular maps of the MAA7 (a), CpSRP43 (b) and ChlM (c) loci with the CRISPR/Cas9 target sites indicated. CDS sequences are indicated as blue boxes, together with the sgRNA target sequences, the cut sites (arrowheads) and the associated PAM sites (5′-NGG-3′, underlined). Target sites of MAA7 were written in the 3′-to-5′ orientation, since the negative strand was targeted by sgRNAs. (d) The activities of the Cas9 protein and individual sgRNAs were tested in vitro. The Cas9-degraded products of the amplified fragments are indicated by arrowheads, with degraded products of 488 and 295 bp, 418 and 377 bp, and 736 and 274 bp identified for MAA7, CpSRP43 and ChlM, respectively.

Figure 2. Auxotrophic selection of MAA7 mutants and their sequence analyses.

(A) The isolated maa7 mutants were grown in the presence of 5-FI with tryptophan supplementation, and cell growth was examined by spot-test loading of 100, 500, and 2500 cells. The RNAi mutant, MAA7 RNAi #33, was used as a reference strain. (B) Small indels are produced by CRISPR/Cas9 at the Cas9 cut site of the MAA7 locus. Sequencing of genomic DNA fragments encompassing the cut site revealed eight small indels at the cut site (at the 3′ side of the arrow head). The protein sequences of the target region (the RPDAN motif) are listed on the right side, and the altered sequences are underlined. (C) Alignments of the N-terminal side of the TSB sequences from various organisms representing three kingdoms. GenBank IDs are included to facilitate identification. Mutated sequences are marked with arrowheads.

Figure 3. Complementation of MAA7 mutants with WT and MAA7 20-1 TSB sequences.

The MAA7 20-1 mutant was transformed with WT MAA7 gene for complementation (generating strain 20W). RNAi #33 (a reference strain with a RNAi knockdown mutation of MAA7) was transformed with the WT (generating RW) or 20-1 (generating R20) MAA7 genes. (A) Cell growth was analyzed in TAP medium supplemented with tryptophan. (B) The tolerance of each strain to 5-FI was analyzed by culturing cells in TAP medium containing both 5-FI and tryptophan. Error bars indicate standard deviations obtained from four independent experiments.

Figure 4. Phenotypic characterization of CpSRP43 mutants.

Cells were co-transformed with sgRNA, the Cas9 protein and the selection vector, and three colonies showing a stable lighter green color were isolated and designated CpSRP43 10-1, 10-2 and 10-3. The negative controls included a colony from the same screen that carried the normal green color (designated CpSRP43 20-1), and a colony from an experiment in which cells were transformed with only the selection vector (designated vec-2). The positive controls included CC-4561 (mt+) and CC-4562 (mt−), which are deletion mutants of CpSRP43 (also called tla3-cpsrp43). (A) The coloration of control colonies and those of CpSRP43 mutants induced by CRISPR/Cas9. Cells (2.50 × 10⁷ cells/mL) were loaded to a 24-well plate in identical volumes of TAP medium. (B) Changes in the chlorophyll contents of CpSRP43 mutants. The contents of chlorophyll a and b were measured at 72 hours of cultivation, normalized by the cell density. The ratios of chlorophyll a to chlorophyll b were calculated and plotted. Error bars indicate standard deviations obtained from four independent experiments. Significant differences compared to the WT were determined by the Student’s t test and are indicated by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001) (C) Immunoblots were probed with antibodies against the target protein (CpSRP43) and the β-subunit of ATP synthase (ATPβ). A band of the molecular mass expected for the former (43 kDa) is marked with an arrowhead.

Figure 5. NHEJ-mediated knock-in of the co-transformed vector at the CRISPR/Cas9 cut site in the CpSRP43 locus.

(a) PCR amplification of the CpSRP43 regions was performed using different elongation times. Amplification of fragments encompassing the cut site failed under a short elongation cycle (upper panel), but improved when we used a longer cycle (lower panel). (b) Various vector sequences were confirmed by PCR. The amplicon sizes of the fragments encompassing the hygromycin resistance region, region A, region B, region C, and the 18S rDNA were 387 bp, 509 bp, 1,662 bp, 1,089 bp and 380 bp, respectively. The specific primers used in this study are listed in Table S1. (c) Molecular map of the CpSRP43 locus and the integrated vector sequences. An insertion of 3 bp (GGA) was identified next to the 5′ side of the Cas9 cut site (left-half arrowhead). Considerable vector sequences (2,650 bp) were inserted at the cut site, followed by an additional 13 bp of unknown origin (GGGCGCCCGGACC) at the 3′ side of the cleaved site (right-half arrowhead). All of the CpSRP43 mutants (10-1, 10-2 and 10-3) contained the same sequences at the Cas9 cut site in the CpSRP43 locus. Abbreviations: TP, PsaD terminator; PB, beta2-tub promoter; IR, RBCS2 intron; TR, RBCS2 terminator; and PHR, HSP70A-RBCS2 promoter.

Figure 6. Phenotypic characterization of light green mutants induced by CRISPR/Cas9 at the ChlM locus.

(a) Coloration of 10 ChlM mutants (2.0 × 10⁸ cells/mL) loaded to a 24-well plate in liquid TAP medium. (b) Chlorophyll contents of the ChlM mutants. Data are expressed as ± SD (n = 4 replicates). Significant differences compared to the WT control were determined by the Student’s t test and are indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). (c) Semi-quantitative RT-PCR analysis was used to detect ChlM transcripts from the mutant lines. IDA5 was detected as a loading control.

Figure 7. NHEJ-mediated knock-in mutants of the co-transformed vector at the CRISPR/Cas9 cut site on the ChlM locus.

(A) Multiple PCR fragments encompassing the vector sequences. (B–G) Schematic molecular maps of the knock-in events in each ChlM mutant. The sizes of the inserted vector and PCR amplification are shown. Red and black letters indicated the deleted and inserted sequences, respectively. Abbreviations: TR, RBCS2 terminator; TP, PsaD terminator; PP, PsaD promoter; and PB, beta2-tub promoter.