Difference between revisions of "CRISPR/Cas in Echinoderms"

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To date CRISPR/Cas9 has been used to introduce insertion and deletion mutations (indels) into ''S. purpuratus nodall'' ([https://www.echinobase.org/literature/article.do?method=display&articleId=44372 Lin and Su 2016]), ''polyketide synthase 1'', ''gcml'' (Oulhen and Wessel 2016), ''nanos2l'' ([https://www.echinobase.org/literature/article.do?method=display&articleId=45207 Oulhen et al. 2017]) and ''dll1'' (''delta'') ([https://www.echinobase.org/literature/article.do?method=display&articleId=45720 Mellott et al. 2017]) genes. Attempts to mutate ''foxy'' ([https://www.echinobase.org/literature/article.do?method=display&articleId=47178 Oulhen et al. 2019]) were unsuccessful. A number of different methods were used for gRNA synthesis (several using pT7-gRNA)  and NLS-SpCas9-NLS (pCS2-nCas9n (zebrafish codon-optimized), or pCS2-3xFLAG-NLS-SpCas9-NLS (codon optimized for human with a 3XFLAG-tag) were used in these studies (see below for details). The gRNAs and mRNAs were microinjected into fertilized eggs.
 
To date CRISPR/Cas9 has been used to introduce insertion and deletion mutations (indels) into ''S. purpuratus nodall'' ([https://www.echinobase.org/literature/article.do?method=display&articleId=44372 Lin and Su 2016]), ''polyketide synthase 1'', ''gcml'' (Oulhen and Wessel 2016), ''nanos2l'' ([https://www.echinobase.org/literature/article.do?method=display&articleId=45207 Oulhen et al. 2017]) and ''dll1'' (''delta'') ([https://www.echinobase.org/literature/article.do?method=display&articleId=45720 Mellott et al. 2017]) genes. Attempts to mutate ''foxy'' ([https://www.echinobase.org/literature/article.do?method=display&articleId=47178 Oulhen et al. 2019]) were unsuccessful. A number of different methods were used for gRNA synthesis (several using pT7-gRNA)  and NLS-SpCas9-NLS (pCS2-nCas9n (zebrafish codon-optimized), or pCS2-3xFLAG-NLS-SpCas9-NLS (codon optimized for human with a 3XFLAG-tag) were used in these studies (see below for details). The gRNAs and mRNAs were microinjected into fertilized eggs.
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'''Single nucleotide edits'''
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Additional studies fused a deaminase to two mutants of SpCas9 for achieving targeted, single nucleotide edits to ''[https://www.echinobase.org/gene/showgene.do?method=display&geneId=23094247& alx1]'', ''[https://www.echinobase.org/gene/showgene.do?method=display&geneId=23083023 segment polarity protein dishevelled homolog DVL-3]'' (''Dsh'') and ''[https://www.echinobase.org/gene/showgene.do?method=display&geneId=23139056 polyketide synthase 1]'' (''Pks1'') to produce STOP codons (Shevidi et al. 2017).
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'''Reviews'''
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Reviews of the methods are available (Cui et al. 2017; Lin et al. 2019).
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'''Editing other echinoderm species'''
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Editing technology has also been used in ''Hemicentrotus pulcherrimus'' (Liu et al. 2019; Wessel et al. 2020) and ''Temnopleurus reevesii'' (Yaguchi et al. 2020).

Revision as of 16:45, 1 December 2020

Welcome to the Echinobase CRISPR/Cas resource. A brief literature and method review is followed by tables of gRNA spacer sequences.

Updated December 2020


S. purpuratus genome editing to create insertions and deletions

To date CRISPR/Cas9 has been used to introduce insertion and deletion mutations (indels) into S. purpuratus nodall (Lin and Su 2016), polyketide synthase 1, gcml (Oulhen and Wessel 2016), nanos2l (Oulhen et al. 2017) and dll1 (delta) (Mellott et al. 2017) genes. Attempts to mutate foxy (Oulhen et al. 2019) were unsuccessful. A number of different methods were used for gRNA synthesis (several using pT7-gRNA) and NLS-SpCas9-NLS (pCS2-nCas9n (zebrafish codon-optimized), or pCS2-3xFLAG-NLS-SpCas9-NLS (codon optimized for human with a 3XFLAG-tag) were used in these studies (see below for details). The gRNAs and mRNAs were microinjected into fertilized eggs.


Single nucleotide edits

Additional studies fused a deaminase to two mutants of SpCas9 for achieving targeted, single nucleotide edits to alx1, segment polarity protein dishevelled homolog DVL-3 (Dsh) and polyketide synthase 1 (Pks1) to produce STOP codons (Shevidi et al. 2017).


Reviews

Reviews of the methods are available (Cui et al. 2017; Lin et al. 2019).


Editing other echinoderm species

Editing technology has also been used in Hemicentrotus pulcherrimus (Liu et al. 2019; Wessel et al. 2020) and Temnopleurus reevesii (Yaguchi et al. 2020).