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Expanding the CRISPR/Cas genome-editing scope in Xenopus tropicalis
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Expanding the CRISPR/Cas genome-editing scope in Xenopus tropicalis
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Journal Article
Expanding the CRISPR/Cas genome-editing scope in Xenopus tropicalis
2022
Overview
Background
The true diploid frog,
Xenopus tropicalis
(
X. tropicalis
) is an excellent genetic model organism. To date, the CRISPR/Cas-mediated genome editing methods established in this species are mostly based on SpCas9 that requires the stringent NGG protospacer-adjacent motif (PAM) for target recognition, which limits its genome editing scope. Thus, it is highly desirable to circumvent this limitation.
Results
Through one-cell stage injection of Cas/gRNAs into
X. tropicalis
embryos, we evaluated the mutagenic efficiency of 8 different Cas variants using T7EI assay, Sanger DNA sequencing, or deep sequencing. Our data indicate that SaCas9 and KKH SaCas9 are highly effective in frogs, which could be used for direct phenotyping in G0 embryos. In contrast, VQR Cas9, xCas9 3.7, SpG Cas9, and SpRY Cas9 were ineffective in
X. tropicalis
embryos and no activity was detected for iSpyMac Cas9. We also found that LbCas12a/crRNA RNP complexes with paired crRNAs efficiently induced small fragment deletions in
X. tropicalis
embryos.
Conclusion
SaCas9 and KKH SaCas9 are robust genome editing tools in
X. tropicalis
embryos. LbCas12a/crRNA RNP complexes are useful for inducing DNA fragment deletions in frog embryos. These tools expand the CRISPR/Cas genome editing scope in
X. tropicalis
and increase the flexibility for various genome editing applications in frogs
.
