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Targeted gene disruption in Xenopus laevis using CRISPR/Cas9.

Wang F, Shi Z, Cui Y, Guo X, Shi YB, Chen Y - Cell Biosci (2015)

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Anhui University, Hefei, 230601 China ; CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

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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- mutant tadpoles with respect to the loss of pdip expression... 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... 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)... 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... 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 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... 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.

No MeSH data available.


CRISPR/Cas9 is effective in targeting X. laevis tyrosinase. DNA sequencing data reveal the indel-inducing efficiencies of tyra-T and tyrb-T in X. laevis embryos. For all panels, the wild-type sequence is shown at the top with the target site highlighted in yellow and the PAM sequence in blue. Red dashes indicate deletions (−) and lowercase letters in red indicate insertions (+). The left two panels show data from tyra-T and tyrb-T co-injection and right two panels indicate individual injection results.
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Fig4: CRISPR/Cas9 is effective in targeting X. laevis tyrosinase. DNA sequencing data reveal the indel-inducing efficiencies of tyra-T and tyrb-T in X. laevis embryos. For all panels, the wild-type sequence is shown at the top with the target site highlighted in yellow and the PAM sequence in blue. Red dashes indicate deletions (−) and lowercase letters in red indicate insertions (+). The left two panels show data from tyra-T and tyrb-T co-injection and right two panels indicate individual injection results.

Mentions: 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 [7]. 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 [6]. 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 [8]. 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.Figure 4


Targeted gene disruption in Xenopus laevis using CRISPR/Cas9.

Wang F, Shi Z, Cui Y, Guo X, Shi YB, Chen Y - Cell Biosci (2015)

CRISPR/Cas9 is effective in targeting X. laevis tyrosinase. DNA sequencing data reveal the indel-inducing efficiencies of tyra-T and tyrb-T in X. laevis embryos. For all panels, the wild-type sequence is shown at the top with the target site highlighted in yellow and the PAM sequence in blue. Red dashes indicate deletions (−) and lowercase letters in red indicate insertions (+). The left two panels show data from tyra-T and tyrb-T co-injection and right two panels indicate individual injection results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4403895&req=5

Fig4: CRISPR/Cas9 is effective in targeting X. laevis tyrosinase. DNA sequencing data reveal the indel-inducing efficiencies of tyra-T and tyrb-T in X. laevis embryos. For all panels, the wild-type sequence is shown at the top with the target site highlighted in yellow and the PAM sequence in blue. Red dashes indicate deletions (−) and lowercase letters in red indicate insertions (+). The left two panels show data from tyra-T and tyrb-T co-injection and right two panels indicate individual injection results.
Mentions: 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 [7]. 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 [6]. 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 [8]. 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.Figure 4

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Anhui University, Hefei, 230601 China ; CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

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- mutant tadpoles with respect to the loss of pdip expression... 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... 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)... 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... 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 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... 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.

No MeSH data available.