Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish

Abstract

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.

Figure 1.

Heritable chromosomal deletions and inversions in zebrafish in the region of sema3fb mediated by two pairs of TALENs. (A) The annotated structure of the zebrafish sema3fb gene and the locations and sequences of the TALEN target sites. The binding sites for the four TALEN monomers of TALEN pairs E4 and E18 are highlighted with different colors. (B) Graphic views of the target sites and PCR primers in the wild-type (wt) chromosome or the chromosomes with deletions or inversions in the sema3fb locus. The primers (E4S, E4A, E18S and E18A) are marked with similar colors as the corresponding TALEN-binding sites. (C and D) PCR results of the genomic DNA from embryos injected with two (E4 + E18), one (E4 or E18) or none (WT) of these pairs of TALENs, with the sequencing results of the PCR products from the embryos injected with the two pairs of TALENs (F₀) and their progeny (F₁) carrying deletions (C) or inversions (D).

Figure 2.

Heritable chromosomal deletions in zebrafish in the region of miRNA genes and miRNA gene clusters mediated by two pairs of TALENs. (A) The structure of the four loci of two zebrafish miRNA genes dre-mir-126a and dre-mir-126b and two miRNA gene clusters located on Chr. 1 and Chr. 9, with the approximate locations and distances of TALEN target sites and primers for PCR detection. (B) PCR results of the genomic DNA from injected (two TALENs) and un-injected (WT) embryos. For the loci of dre-mir-126a and dre-mir-126b, the PCR products of both wild-type sequences (white arrowheads) and sequences containing deletions (black arrowheads) can be seen. For the loci of the two miRNA gene clusters (the two lower panels), only the sequences carrying deletions (black arrowheads) can be amplified with the PCR program used in this experiment. Only one combination of TALEN pairs is shown here for each locus. Results of other combinations of TALENs for the same locus can be found in Supplementary Figures S2 and S4.

Figure 3.

Chromosomal deletions in zebrafish in the region of an miRNA gene and an miRNA gene cluster mediated by Cas9 and gRNA pairs. (A) Sequences of the TALEN and Cas9/gRNA target sites around the miRNA gene dre-mir-126a. The protospacer target sequences of the Cas9/gRNA are highlighted with different colors; the 3-nt sequence of PAM (-NGG, or shown as its reversed sequence CCN-) is shown in red. The binding sites of TALEN monomers are underlined. (B) Chromosomal deletions induced by Cas9 and two gRNAs targeting this locus. The result from one of the four combinations of gRNA pairs used to generate deletions in this region is shown here, and the results for other gRNA combinations can be found in Supplementary Figure S2E. Deletion induced by two pairs of TALENs is shown in the last lane for comparison (as a positive control). The PCR products of wild-type sequences (white arrowhead), sequences with deletions induced by Cas9/gRNAs (blue arrowhead) or TALENs (black arrowhead) are indicated. (C) Sequences of the TALEN and Cas9/gRNA target sites around the miRNA gene cluster on Chr. 9 (from dre-mir-17a-2 to dre-mir-92a-2). The target sites of Cas9/gRNA and TALENs are indicated as shown in (A). (D) Chromosomal deletions induced by Cas9 and two gRNAs targeting this locus. The result from one of the four combinations of gRNA pairs used to generate deletions in this region is shown here, and the results for other gRNA combinations can be found in Supplementary Figure S5C. Deletions induced by two pairs of TALENs are shown in the last lane for comparison (as a positive control). The PCR products of wild-type sequences (white arrowhead) and sequences with deletions induced by Cas9/gRNAs or TALENs (black arrowhead) are indicated. A relatively longer extension time for PCR was used in this experiment to simultaneously amplify the wild-type allele.