January 1, 2016;
Efficient genome editing of genes involved in neural crest development using the CRISPR/Cas9 system in Xenopus embryos.
BACKGROUND: The RNA guided CRISPR/Cas9 nucleases have been proven to be effective for gene disruption in various animal models including Xenopus tropicalis. The neural crest (NC) is a transient cell population during embryonic development and contributes to a large variety of tissues. Currently, loss-of-function studies on NC development in X. tropicalis are largely based on morpholino antisense oligonucleotide. It is worthwhile establishing targeted gene knockout X. tropicails line using CRISPR/Cas9 system to study NC development.
METHODS: We utilized CRISPR/Cas9 to disrupt genes that are involved in NC formation in X. tropicalis embryos. A single sgRNA and Cas9 mRNA synthesized in vitro, were co-injected into X. tropicalis embryos at one-cell stage to induce single gene disruption. We also induced duplex mutations, large segmental deletions and inversions in X. tropicalis by injecting Cas9 and a pair of sgRNAs. The specificity of CRISPR/Cas9 was assessed in X. tropicalis embryos and the Cas9 nickase was used to reduce the off-target cleavages. Finally, we crossed the G0 mosaic frogs with targeted mutations to wild type frogs and obtained the germline transmission.
RESULTS: Total 16 target sites in 15 genes were targeted by CRISPR/Cas9 and resulted in successful indel mutations at 14 loci with disruption efficiencies in a range from 9.3 to 57.8 %. Furthermore, we demonstrated the feasibility of generation of duplex mutations, large segmental deletions and inversions by using Cas9 and a pair of sgRNAs. We observed that CRISPR/Cas9 displays obvious off-target effects at some loci in X. tropicalis embryos. Such off-target cleavages was reduced by using the D10A Cas9 nickase. Finally, the Cas9 induced indel mutations were efficiently passed to G1 offspring.
CONCLUSION: Our study proved that CRISPR/Cas9 could mediate targeted gene mutation in X. tropicalis with high efficiency. This study expands the application of CRISPR/Cas9 platform in X. tropicalis and set a basis for studying NC development using genetic approach.
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Single gene disruption targeting genes involved in NC development in X. tropicalis embryos. a Schematic diagram of sgRNA-guided Cas9 nuclease interacting with the DNA target site. The red letters indicate the target site, the green letters indicate the PAM and the blue letters indicate the sequence of tracer RNA. b Schematic drawing of Cas9 target site (protospacer). The protospacer adjacent motif (PAM) 3′ to the protospacer is highlighted in red. The seed region which consists of 12 nucleotides immediate 5′ to PAM is shown in blue. c T7E1 assay of somatic mutations induced by pax3, snai1, ctnnb, sox9, or Ap2α sgRNAs and Cas9 nuclease. d Somatic mutation rate calculated from T7E1 assay in (c). e DNA sequences targeted by sgRNAs at the indicated gene loci and representative somatic mutations induced in X. tropicalis embryos. PAM is underlined and highlighted in green. Deleted sequences are highlighted in gray with red dashes while insertions are indicated by lowercase letters in blue. The parentheses enclosed the number of deleted or inserted base pairs, while the square brackets show the frequencies of the mutation in the sequenced samples
Duplex indel mutations in single embryo. a, d T7E1 assay of duplex indel mutations in the loci of pax3 and zic1 (a) or snail1 and snail2 (d) in five single embryos. b, e Quantification of the T7E1 assay in (a) or (d). c, f Representative sequencing data confirmed the indel mutations induced at the two target loci in one single embryo
Segmental deletions and inversions induced by Cas9 and a pair of sgRNAs in X. tropicalis embryos. a Schematic diagram showing the structure of X. tropicalis pax3 and snail1 genes, and the sgRNA targeting sequences and their locations in the two genes. b Schematic diagram illustrating detection of segmental deletion and inversion at targeted loci with PCR. c, d PCR assay and subsequent sequencing detected segmental deletions and inversion at pax3 locus (c) and snail1 locus (d). The embryos injected with Cas9 and indicated sgRNAs were collected at 2 days after fertilization. Five embryos were pooled for DNA extraction. T1, embryos injected with Cas9 mRNA and sgRNA T1; T2, embryos injected with Cas9 mRNA and sgRNA T2; T1 + T2, embryos injected with mixture of Cas9 mRNA, sgRNA T1 and sgRNA T2. F forward primer; R reverse primer
Off-target effects of CRISPR/Cas9 in X. tropicalis evaluated via T7E1 assay. a Representative gel image of T7E1 digested off-target amplicons. The percentage under panels indicated the frequencies of off-target effects. b Quantification of the cleavage frequencies of all off-target sites. No clear and specific bands after T7E1 assay were generated at sox9-579 and lrig3-E306 loci. The cutting efficiencies at sox9-579 and lrig3-E306 sites were calculated by sequencing results. Red dash line indicated 10 % in indels efficiency
Double-nicking induced efficient genome editing with lower off-target effects. a Two sgRNA targeting sites in sox9. Letters in blue is the target site 1 while letters in red is the target site 2. The red arrows point the predicted cleavage sites, and the PAMs are underlined. b Sequencing data of the indels induced by D10A and the sgRNA pairs. Letters in bold is the wild type sequence, red dashes indicate deletions. c T7E1 assay of genome editing induce by wide-type Cas9 or by D10A. d Quantification of the T7E1 assay shown in (c). e Mutagenesis at a representative off-target locus (sox9-600) induced by Cas9 and sox9 T1gRNA or by D10A and sox9 T1 and T2 sgRNAs. PCR was performed to amplify the targeted sox9-600 locus. Amplicons harboring targeted gene fragments were TA-cloned into pMD-18T. The indel mutagenesis rates were calculated by direct sequencing. (f) Comparison of the off-target efficiency induced by D10A with double nicking versus wide type Cas9 with sox9 sgRNA
Snail precedes slug in the genetic cascade required for the specification and migration of the Xenopus neural crest.