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.
Animals ; Oryzias/genetics* ; Gene Editing/methods* ; CRISPR-Cas Systems ; Codon ; RNA, Guide, CRISPR-Cas Systems/genetics* ; Monophenol Monooxygenase/genetics* ; CRISPR-Associated Protein 9/genetics* ; RNA, Transfer/genetics* ; Staphylococcus aureus/genetics* ; PAX6 Transcription Factor/genetics*

CRISPR-Associated Protein 9 ; Gene Editing ; CRISPR-Cas Systems/genetics*

OBJECTIVE: In order to conduct high-throughput genome-wide translocation sequencing based on CRISPR/Cas9, Nalm6-cas9 monoclonal cell line expressing Cas9 protein was constructed by lentivirus transduction. METHODS: Lentiviral vectors LentiCas9-Blast, pSPAX2, and pMD2.G were used to co-transfect HEK293T cells to obtain recombinant lentivirus. After Nalm6 cells were infected with the recombinant lentivirus, the cells were screened by Blasticidin, and multiple monoclonal cell lines expressing Cas9 protein were obtained by limited dilution. Western blot was used to detect the expression level of Cas9 protein in monoclonal cell lines, and cell count analysis was used to detect the proliferation activity of monoclonal cell lines. LentiCRISPRV2GFP-Δcas9, LentiCRISPRV2GFP-Δcas9-AF4, LentiCRISPRV2GFP-Δ cas9-MLL plasmids were constructed, and transfected with pSPAX2 and pMD2.G, respectively. T vector cloning was used to detect the function of Cas9 protein in Nalm6-Cas9 monoclonal cell line infected with virus. RESULTS: Western blot showed that Nalm6-Cas9_1-6 monoclonal cell line had high expression of Cas9 protein. Cell count analysis showed that high expression of Cas9 protein in Nalm6-Cas9_1-6 monoclonal cell line did not affect cell proliferation activity. The Nalm6-Cas9_1-6 monoclonal cell line had high cleavage activity, and the editing efficiency of AF4 and MLL genes was more than 90% which was determined by T vector cloning. CONCLUSION Nalm6-Cas9_1-6 monoclonal cell line stably expressing highly active Cas9 protein was obtained, which provided a basis for exploring the translocation of MLL in therapy-related leukemias based on CRISPR/Cas9 genome-wide high-throughput genome-wide translocation sequencing.
CRISPR-Associated Protein 9/genetics* ; CRISPR-Cas Systems ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus/genetics* ; Plasmids

Streptococcus pyogenes Cas9 (SpCas9) has become a powerful genome editing tool, but has a limited range of recognizable protospacer adjacent motifs (PAMs) and shows off-target effects. To address these issues, we present a rational approach to optimize the xCas9 mutant derived from SpCas9 by directed evolution. Firstly, energy minimization with the Rosetta program was applied to optimize the three-dimensional structure of Cas9 to obtain the lowest energy conformation. Subsequently, combinatorial mutations were designed based on the mutations sites of xCas9 acquired during the directed evolution. Finally, optimal mutants were selected from the designed mutants by free energy ranking and subjected to experimental verification. A new mutant yCas9 (262A/324R/409N/480K/543D/694L/1219T) with multiple PAM recognition ability and low off-target effects was obtained and verified by DNA cleavage experiments. This mutant recognizes the NG, GAA and GAT PAMs and shows low off-target DNA cleavage activity guided by mismatched sgRNA, thus provides a gene editing tool with potential applications in biomedical field. Furthermore, we performed molecular dynamics simulations on the structures of SpCas9, xCas9 and yCas9 to reveal the mechanisms of their PAM recognition and off-target effects. These may provide theoretical guidance for further optimization and modification of CRISPR/Cas9 proteins.
CRISPR-Associated Protein 9/metabolism* ; CRISPR-Cas Systems/genetics* ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; RNA, Guide/genetics* ; Streptococcus pyogenes/metabolism*

Adenosine ; genetics ; Adenosine Deaminase ; genetics ; metabolism ; Animals ; CRISPR-Associated Protein 9 ; genetics ; metabolism ; CRISPR-Cas Systems ; genetics ; Embryo, Mammalian ; metabolism ; Gene Editing ; Genetic Variation ; genetics ; Mice ; Mice, Inbred C57BL ; Rats ; Rats, Sprague-Dawley

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.
APOBEC-1 Deaminase ; genetics ; metabolism ; Animals ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytidine ; genetics ; metabolism ; Embryo Transfer ; Embryo, Mammalian ; Endonucleases ; genetics ; metabolism ; Gene Editing ; methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; Mice, Inbred C57BL ; Microinjections ; Plasmids ; chemistry ; metabolism ; Point Mutation ; RNA, Guide ; genetics ; metabolism ; Thymidine ; genetics ; metabolism ; Zygote ; growth & development ; metabolism ; transplantation