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Dev Biol
2022 Mar 01;483:66-75. doi: 10.1016/j.ydbio.2021.12.015.
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An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.
Godden AM
,
Antonaci M
,
Ward NJ
,
van der Lee M
,
Abu-Daya A
,
Guille M
,
Wheeler GN
.
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In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively 'drop out' a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO's both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.
Fig. 2. CRISPR-Cas9 approach for knocking out miRNAs in X. tropicalis and validation strategies. (A-A’) Schematic showing the approach taken with use of two sgRNAs for miR-196a (A) and miR-219 (A’). (B) Q-RT-PCR validation of miR-KO, with individual data points from biological repeats shown in orange. MiR-196a KO showed a 67% reduction in expression (B), and miR-219 KO showed a 93% reduction in expression following CRISPR-Cas9 treatment (B’). Embryos were injected at the 1 ​cell stage, bar charts show mean_+/− S.E.M. Experiments were conducted with biological and technical triplicate. (C–D) PCR/nested PCR validation of gDNA miRNA regions from embryos injected into one cell at 2 ​cell stage of development, with KOs showing an extra smaller band in the fourth lane of each gel. (C’-D’) Sanger sequencing validation of miRNA KOs and CRISPR events. Cas9+GFP control samples were also harvested, genomic DNA extracted, PCR amplified and subcloned. Purple text highlights primers used for cloning, red text shows miRNA stem-loop. Yellow highlight shows a mis-match, and red highlight with scissor icons show where CRISPR events occurred, grey text shows sgRNA. WT and Cas9 sequences show miRNA WT sequence, whereas Cas9+sgRNAs show 3 repeats of mutated sequences, with significantly shorter sequences. (E) Phenotype analysis of miRNA KO embryos, representative embryos are shown. Embryos were co-injected with CRISPR-reagents a GFP capped RNA tracer into one cell at a two cell stage of development. Embryos are imaged on left and right sides. WT had no injection, Cas9 protein was co-injected with GFP cRNA and miR-KO were pairs of sgRNAs, Cas9 protein and GFP cRNA tracer. The fluorescent side of miR-196a KO embryo red arrows indicate a pigment phenotype, and for miR-219 KO, the red arrows indicate a strong craniofacial phenotype, with smaller eyes, branchial arch and flattened face features. (E’) Bar charts show count data of yes/no phenotypes for miR-196a KO (pigment loss) and miR-219 KO (craniofacial disfigurement), with chi-squared tests for statistical significance. There was a significant difference between and Cas9 and miR-196a KO groups p ​= ​2.22 ​× ​10-7 and between Cas9 and miR-219 KO p ​= ​1.1 ​× ​10-10. Embryo phenotypes were blind counted on three biological repeats on embryos from different Xenopus parents.
Fig. 3. Analysis of key NC, neural plate and hatching gland markers after miRNA KO and KD. (A) Whole mount in situ hybridisation profiles on neurula stage Xenopus embryos of Sox10, Snail2, Pax3 and Xhe2. CRISPR-Cas9 was carried out in X. tropicalis embryos with GFP cRNA as a tracer and morpholino-KD was carried out in X. laevis embryos with lacZ cRNA as a tracer. Embryos for whole mount in situ hybridisation were injected with tracer at 4-cell stage of development into the right dorsal blastomere. Panel A, a-f show Sox10 expression following CRISPR and MO experiments. Panel A g-l show Snail2 expression following CRISPR and MO experiments. Panel A m-r show Pax3 expression following CRISPR and MO experiments. Panel A, s-x shows Xhe2 expression following CRISPR and MO experiments. Overall phenotypes show a reduction of NC and altered neural plate and hatching gland profiles. (B) Phenotype analysis for individual markers. a, c, e and g show count data of yes/no phenotype prescence. Chi-squared statistical tests were carried out on three biological repeats of whole mount in situ hybridisation on embryos from different frogs. b, d f and h, Q-RT-PCR results of mRNA expression analysis following CRISPR KO and MO KD. Normalised to U6 expression, RNA was pooled from 5 individual neurula embryos for one biological sample, Q-RT-PCR was carried out with biological and technical triplicates. The Q-RT-PCR data supports phenotypes shown in (A). Panel B, a-b show Sox10 expression following CRISPR KO and MO KD. Panel B, c-d show Snail2 expression following CRISPR KO and MO KD. Panel B, e-f show Pax3 expression following CRISPR KO and MO KD. Panel B, g-h show Xhe2 expression following CRISPR KO and MO KD experiments. Panel B, a,c,e and g show phenotype count data. Panel B, b,d,f and h show q-RT-PCR expression of mRNAs following miRNA KO and KD. Abbreviations for phenotype and q-RT-PCR bar charts in (B): miR-KO refers to CRISPR miRNA KO and miR-KD refers to MO KD of miRNA. Phenotypes for miRNA KD/KO: Sox10 phenotype is a reduction in expression, Snail2 is a reduction/shift in profile, Pax3 phenotype is a shift/reduced profile for miR-196a and an expansion for miR-219 experiments, finally, Xhe2 is an increased profile for miR-196a and a reduced profile for miR-219 experiments respectively. Statistical significance: Sox10 Cas9 vs miR-196a KO p ​= ​4.02 ​× ​10-8, Cas9 vs miR-219 KO p ​= ​1.04 ​× ​10-5, Snail2 Cas9 vs miR-196a KO p ​= ​6.15 ​× ​10-9, Cas9 vs miR-219 KO p ​= ​4.07 ​× ​10-7, Pax3 Cas9 vs miR-196a KO p ​= ​7.19 ​× ​10-7, Cas9 vs miR-219 KO p ​= ​2.29 ​× ​10-8, Xhe2 Cas9 vs miR-196a KO p ​= ​7.19 ​× ​10-7, Cas9 vs miR-219 KO p ​= ​2.29 ​× ​10-8.
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