Efficient genome editing in medaka (Oryzias latipes) using a codon-optimized SaCas9 system

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, belonging to the type II CRISPR/Cas system, is an effective gene-editing tool widely used in different organisms, but the size of Streptococcus pyogenes Cas9 (SpCas9) is quite large (4.3 kb), which is not convenient for vector delivery. In this study, we used a codon-optimized Staphylococcus aureus Cas9 (SaCas9) system to edit the tyrosinase (tyr), oculocutaneous albinism II (oca2), and paired box 6.1 (pax6.1) genes in the fish model medaka(Oryzias latipes), in which the size of SaCas9 (3.3 kb) is much smaller and the necessary protospacer-adjacent motif (PAM) sequence is 5'-NNGRRT-3'. We also used a transfer RNA (tRNA)‍-single-guide RNA (sgRNA) system to express the functional sgRNA by transcription eitherin vivo or in vitro, and the combination of SaCas9 and tRNA-sgRNA was used to edit the tyr gene in the medaka genome. The SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems were shown to edit the medaka genome effectively, while the PAM sequence is an essential part for the efficiency of editing. Besides, tRNA can improve the flexibility of the system by enabling the sgRNA to be controlled by a common promoter such as cytomegalovirus. Moreover, the all-in-one cassette cytomegalovirus (CMV)‍-SaCas9-tRNA-sgRNA-tRNA is functional in medaka gene editing. Taken together, the codon-optimized SaCas9 system provides an alternative and smaller tool to edit the medaka genome and potentially other fish genomes.

Keywords: Gene editing; Medaka; Oculocutaneous albinism II (oca2); Paired box 6.1 (pax6.1); Staphylococcus aureus Cas9 (SaCas9); Transfer RNA(tRNA); Tyrosinase (tyr).

Fig. 1. Syntheses of mSaCas9 DNA, single-guide RNA (sgRNA) scaffold, and sgRNA. (a) Map of Staphylococcus aureus Cas9 (SaCas9) expression vector. SaCas9 uses NNGR(A/T)RT as the protospacer-adjacent motif (PAM) sequence, and its length is smaller than that of Streptococcus pyogenes Cas9 (SpCas9). (b) Primers were designed and annealed to synthesize mSaCas9 sgRNA scaffold, and spacers carrying the T7 promoter were synthesized using mSaCas9 sgRNA scaffold as a template. (c) Construction of the transfer RNA (tRNA)-sgRNA expression vector. The target fragment sgRNA was ligated into the linearized vector pCS2-tRNA by infusion to obtain a recombinant plasmid pCS2+tRNA-mSatyr-sgRNA including a fragment of tRNA and sgRNA. (d) Construction of the tRNA-sgRNA-tRNA expression vector. The target fragment in-RNA was ligated into the linearized vector pCS2+tRNA-mSatyr-sgRNA by infusion to obtain a recombinant plasmid pCS2+tRNA-mSatyr-sgRNA+tRNA including a fragment of two tRNAs and one sgRNA. CMV: cytomegalovirus; PCR: polymerase chain reaction.

Fig. 2. Analyses of R4445 and F4553 knockout in tyrosinase (tyr) gene. (a) The sequences of R4445 and F4553 are underlined in blue, while protospacer-adjacent motif (PAM) sequences are underlined in green. (b) A significant loss of pigmentation was observed in the mutation group (MT, 56/135) compared to wild type (WT, 0/192). (c) The PCR products from heterozygous mutant embryos (1, 2) and wild-type embryos (WT) were treated with T7 endonuclease (T7EI). (d) Monoclonal sequencing of different bands. There were several base mutations in the single or dual single-guide RNA (sgRNA) knockout sites. PCR: polymerase chain reaction; M: marker.

Fig. 3. Analyses of F9011 and F9254 knockout in oculocutaneous albinism II (oca2) gene. (a) The sequences of F9011 and F9254 are underlined in blue, while protospacer-adjacent motif (PAM) sequences are underlined in green. (b) Compared with wild-type embryos (WT, 0/192), the heterozygous mutations showed a significant pigment deletion in the mutation group (MT, 40/41). (c) Monoclonal sequencing of PCR fragments. The PCR products from mutant (MT1/MT2) and WT embryos were treated with T7 endonuclease (T7EI). Different types of mutations were found in the same embryo. PCR: polymerase chain reaction; M: marker.

Fig. 4. Analyses of R14069 and R14135 knockout results in paired box 6.1 (pax6.1) gene. (a) The sequences of R14069 and R14135 are underlined in blue, while protospacer-adjacent motif (PAM) sequences are underlined in green. (b) Compared with wild-type embryos (WT, 0/192), the heterozygous mutant embryos (MT, 23/73) showed significant ocular malformation. (c) The PCR products from MT and WT embryos were treated with T7 endonuclease (T7EI). (d) Monoclonal sequencing of PCR fragments. There were several types of mutations only in the R14135 knockout site. PCR: polymerase chain reaction; M: marker.

Fig. 5. Analyses of R4414+R4468 and NR4443+NR4475 knockout results intyrosinase (tyr)gene. (a). The sequences of R4414 and R4468 are underlined in blue, and those of NR4443 and NR4475 are underlined in red. The protospacer-adjacent motif (PAM) sequences are underlined in green. (b) The heterozygous mutants showed significant pigment deletion in the R4414+R4468 group (R, 29/51) but not in the NR4443+NR4475 group (NR, 0/39). (c) Monoclonal sequencing of PCR products. The PCR products from mutation (R), NR, and wild type (WT) groups were treated with T7 endonuclease (T7EI). There were several base mutations in the single-guide RNA (sgRNA) knockout sites in the R group but not in the NR or WT groups. PCR: polymerase chain reaction; M: marker.

Fig. 6. Analyses of transfer RNA (tRNA)-single-guide RNA (sgRNA) in RNA or DNA content in tyr-sgRNA-tRNA knockout results. (a) The sequence of R4445 is underlined in blue, while that of the protospacer-adjacent motif (PAM) is underlined in green. (b) The heterozygous mutants showed a significant pigment deletion in SP6-pCS2+tRNA-tyr-sgRNA (7/23), SP6-pCS2+tRNA-tyr-sgRNA-tRNA (43/100), and pCS2+tRNA-mSatyr-sgRNA+tRNA DNA groups (12/152). (c) Monoclonal sequencing of PCR products. The PCR products from mutants (1‒3) and wild type (WT) were treated with T7 endonuclease (T7EI). There were several types of mutations in the R4445 knockout site in different groups. tyr: tyrosinase; PCR: polymerase chain reaction; M: marker.

Fig. 7. mSaCas9-transfer RNA (tRNA)-single-guide RNA (sgRNA) in tyrosinase (tyr) gene knockout results. (a) The sequence of R4445 is underlined in blue, while that of the protospacer-adjacent motif (PAM) is underlined in green. (b) An illustration of the all-in-one vector cytomegalovirus (CMV)‍-SaCas9-tRNA-sgRNA-tRNA. The heterozygous mutants showed a significant pigment deletion in the mSaCas9-tRNA-tyr sgRNA-tRNA (49/50) and mSaCas9-tRNA-tyr sgRNA-tRNA+tyr sgRNA groups (44/51). (c) The PCR products from mutants (1, 2) and wild type (WT) were treated with T7 endonuclease (T7EI), and multiple peaks were detected in the sequencing of the PCR products. PCR: polymerase chain reaction; M: marker.

Fig. 8. Adult fish of the tyrosinase (tyr) mutant generated by the Staphylococcus aureus Cas9 (SaCas9) or Streptococcus pyogenes Cas9 (SpCas9) system. (a) Wild-type (WT) male medaka (♂). (b) WT female medaka (♀). (c) The male homozygote of the SaCas9 knockout (♂). (d) The female homozygote of the SaCas9 knockout (♀). (e) The male homozygote of the SpCas9 knockout (♂). (f) The female homozygote of the SpCas9 knockout (♀). (g) Monoclonal sequencing of PCR products in WT and homozygous medaka. There were mutations in different single-guide RNA (sgRNA) regions in the SpCas9 and SaCas9 strains. PCR: polymerase chain reaction.