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Cell Biosci 2022 Jul 08;121:104. doi: 10.1186/s13578-022-00841-3.
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Expanding the CRISPR/Cas genome-editing scope in Xenopus tropicalis.

Shi Z , Jiang H , Liu G , Shi S , Zhang X , Chen Y .

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.

PubMed ID: 35804405
Article link: Cell Biosci
Grant support: [+]

Species referenced: Xenopus tropicalis
Genes referenced: apc ash1l bace2 best1 bmp4 ctcf kcnj2 nfyb pam psmd6 pten ptf1a rad21 sftpb slc45a2 tbx5 tyr
GO keywords: CRISPR-cas system

Article Images: [+] show captions
References [+] :
Aslan, High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 <i>Xenopus</i>. 2017, Pubmed, Xenbase