- Letter to the Editor
- Open Access
Targeted gene disruption in Xenopus laevis using CRISPR/Cas9
Cell & Biosciencevolume 5, Article number: 15 (2015)
To test if the CRISPR/Cas9 system can mediate targeted gene disruption in Xenopus laevis, we targeted ptf1a/p48 and tyrosinase in this species and found that in addition to high sequence disruption efficiency, clear phenotypes were observed in G0 embryos. ptf1a/p48-targeted X. laevis embryos can mimic Xenopus tropicalis ptf1a/p48-null mutant tadpoles with respect to the loss of pdip expression. Simultaneous disruption of two X. laevis tyrosinase homeologs leads to almost full albinism. Our data indicate that CRISPR/Cas9 system is a simple and efficient tool for targeted gene disruption in X. laevis. It can be used for phenotype analysis in G0 embryos.
Since its introduction to modern biology in the 1950s, X. laevis has played a central role in most disciplines of biomedical research including developmental biology, biochemistry, and molecular biology . Its allotetraploid genome represents a common polyploidy in amphibians, which challenges genetic studies on this species . Fortunately, by the mid 1990s, X. tropicalis, the only diploid species in the Xenopus genus, was adopted as a genetically tractable complement to the classic model X. laevis. Together, the two frog species provide a unique and powerful system for addressing genome duplication/evolution, functional genomics, and human disease modelling at the post-genomic era .
Gene knockdown in X. laevis has been largely dependent on antisense Morpholino oligomers since 2000, which suffers from some off-target effects . A recent systematic analysis reveals poor phenotypic correlation between published Morpholino-induced morphants and mutant lines in zebrafish , further highlighting the off-target effects of Morpholino oligomers. Transcription activator-like effector nuclease (TALEN) was shown effective for targeted gene disruption in X. laevis G0 embryos [1,5-7]. With the establishment of efficient targeted gene disruption in X. tropicalis using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR/Cas9) system , it is worth addressing if this simple method applies to X. laevis.
CRISPR/Cas9 mediated disruption of ptf1a/p48 in X. laevis G0 embryos phenocopies X. tropicalis ptf1a/p48 mutants
The basic helix-loop-helix transcription factor Ptf1a/p48 is a key regulator early for pancreatic precursor specification and late for exocrine pancreas formation [9,10]. Our previous studies indicate that targeted disruption of X. tropicalis ptf1a/p48 in G0 embryos using either TALENs  or CRISPR/Cas9  leads to inhibition of pancreas formation as indicated by reduction of pancreas-specific marker gene pdip expression. These data are consistent with the X. laevis morphants  and Ptf1a/p48 knockout mice . Here, using our previously generated CRISPR/Cas9-targeted founder frogs, we established a X. tropicalis ptf1a/p48 mutant line. Unlike Ptf1a/p48-null mutant mice that can survive whole gestation  likely due to mother supply of blood/nutrition and die at birth, the free-swimming X. tropicalis ptf1a/p48 homozygous mutant tadpoles cannot develop to froglets and all died during feeding stages (around stages 47–48). Those collected at stage 43 showed complete absence of pdip expression (Figure 1A, C), while heterozygous tadpoles are indistinguishable from wild type siblings (Figure 1B, D), which is similar to the phenotype of Ptf1a/p48 knockout mice . The mutant phenotype obtained here also verified the reliability of previous observations on X. laevis morphants .
Given the allotetraploid genome of X. laevis, every gene might have two pairs of homeologs. For ptf1a/p48 allele, only one cDNA sequence can be found from current databases (GenBank: DQ007931.1). We designed two gRNAs targeting the first exon of this X. laevis ptf1a/p48 locus, namely ptf1a/p48-T1 and ptf1a/p48-T2 (Additional file 1: Table S1). First we injected Cas9 mRNA together with these two gRNAs either alone or in combination into animal pole of 1-cell stage X. laevis embryos and assessed their indel-inducing capacity via the PCR/sequencing protocol (Additional file 1: Table S2). Based on our experience with X. tropicalis embryos, we set the doses of both Cas9 mRNA and gRNAs at 300 pg/embryo, which were proven optimal as on one hand efficient targeted gene disruption was obtained (Figure 2) and on the other hand no morphological malformation was observed in injected embryos. Overall, the targeted sequence disruption efficiency is above 50% (Figure 2), which is comparable to that in X. tropicalis . It should be noted that slightly more than 1/3 of the disruption did not cause reading frame shift (Figure 2).
Next, we evaluated the pancreas development in injected embryos by whole mount in situ hybridization with a pdip probe. Similar to the observation in ptf1a/p48-targeted X. tropicalis G0 embryos , severe inhibition of pdip expression was observed in all three injection groups (Figure 3). For reasons unknown, in spite of their lower sequence disruption efficiency in comparison to the individually injected embryos (Figure 2), ptf1a/p48-T1 and -T2 co-injected embryos displayed stronger pancreatic phenotype with quite a few of them showing almost complete absence of pdip expression reminiscent of X. tropicalis ptf1a/p48-null mutants (Figure 3K, N and Figure 1A, C). Taken together, these data indicate that CRISPR/Cas9 system is effective in X. laevis. For ptf1a/p48, one gRNA is sufficient to cause an obvious pancreatic phenotype in X. laevis G0 embryos.
Simultaneous disruption of two X. laevis tyrosinase homeologs leads to albinism
To further test if CRISPR/Cas9 is a robust tool for gene targeting in X. laevis, we chose to target tyrosinase. There are two homeologs for X. laevis tyrosinase, tyra and tyrb . A previous study showed that one pair of TALENs targeting a highly conserved region of the two homeologs was sufficient to induce albinism in X. laevis . As no suitable gRNA targeting sites can be found in this conserved region for both homeologs, we have to design two gRNAs (tyra-T and tyrb-T) to target tyra and tyrb, respectively (Additional file 1: Table S1). Again, we injected Cas9 mRNA (300 pg/embryo) with these two gRNAs either alone or in combination (300 pg/embryo for each gRNA) into the animal pole of X. laevis fertilized eggs. PCR/sequencing data indicate that both gRNAs are very effective with targeted sequence disruption efficiencies from 87.5% up to 100% (Figure 4). The tyra-T and tyrb-T co-injected tadpoles showed severe reduction of pigmentation (Figure 5B, E), with 37% (37/100) of them showing almost full albinism (Figure 5A, D), which is stronger than TALENs-induced phenotype in X. laevis [6,7] and CRISPR/Cas9-induced phenotype in X. tropicalis . An independent injection led to similar results. In contrast, although the sequence disruption efficiency remained high (Figure 4), individual injection of either tyra-T or tyrb-T did not cause any obvious alteration on embryonic pigmentation (Figure 5H, I, K, L), indicating the functional redundancy of tyra and tyrb. In sum, these data further confirm that CRISPR/Cas9 system is effective in X. laevis. Distinct homeologs of X. laevis genes can be easily disrupted by application of two gRNAs.
Due to Morpholino’s potential off-target effects, a recent study recommends mutant phenotypes as the standard metric to define gene function in zebrafish . Our previous study did not detect any CRISPR/Cas9-induced off-target effects in X. tropicalis . The issue of CRISPR/Cas9’s off-target effects in X. laevis remains to be defined. Nevertheless, our data demonstrate that CRISPR/Cas9 is equally a superb tool for targeted gene disruption in X. laevis as in X. tropicalis. It can be used for an immediate phenotype evaluation in X. laevis G0 embryos. It should be noted that the allotetraploid genome and longer generation time (1–2 years) of X. laevis makes it impractical to do genetic research. For establishment of stable knockout lines, we recommend to use the diploid frog X. tropicalis that has much shorter generation time (4–6 months).
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This work was supported in part by National Basic Research Program of China (2015CB942803), the National Natural Science Foundation of China (31471367, 31271554, and 31301192), Shenzhen Key Laboratory of Cell Microenvironment (ZDSYS20140509142721429), and the Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. This research was also supported in part by the Intramural Research Program of NICHD, NIH.
The authors declare that they have no competing interests.
FW, YS, and YChen designed the work and analyzed the data. FW, ZS, YCui, and XG carried out the experiments. YChen and FW wrote the manuscript. All authors read and approved the final manuscript.
These two authors Fengqin Wang and Zhaoying Shi contributed equally to this work.
Oligonucleotides used to construct gRNA expression templates. Table S2. PCR primers used to amplify the targeted loci.