Epgfp and CRISPR/Cas in Echinoderms: Difference between pages
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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'' ([https://www.echinobase.org/literature/article.do?method=display&articleId=44372 Lin and Su 2016]), ''polyketide synthase 1'', ''gcml'' ([https://www.echinobase.org/literature/article.do?method=display&articleId=48855 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. | |||
'''Single nucleotide edits''' | |||
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 ([https://www.echinobase.org/literature/article.do?method=display&articleId=45725 Shevidi et al. 2017]). | |||
'''Reviews''' | |||
Reviews of the methods are available ([https://www.echinobase.org/literature/article.do?method=display&articleId=45567 Cui et al. 2017]; [https://www.echinobase.org/literature/article.do?method=display&articleId=47096 Lin et al. 2019]). | |||
'''Editing other echinoderm species''' | |||
Editing technology has also been used in ''Hemicentrotus pulcherrimus'' ([https://www.echinobase.org/literature/article.do?method=display&articleId=47348 Liu et al. 2019]; [https://www.echinobase.org/literature/article.do?method=display&articleId=48597 Wessel et al. 2020]) and ''Temnopleurus reevesii'' ([https://www.echinobase.org/literature/article.do?method=display&articleId=48853 Yaguchi et al. 2020]). | |||
'''Design overview''' | |||
CRISPR systems in nature are composed of the Cas9 nuclease and two RNAs, the CRISPR RNA (crRNA) that binds to a complementary DNA sequence and binds to the transactivating RNA (tracrRNA) that also binds to a specific Cas9 protein. For ease of use the crRNA and tracrRNA have been combined into a single guide RNA (sgRNA) molecule for use with the ''Streptococcus pyogene''s Cas9 (SpCas9). The sgRNA has the target gene '''spacer''' sequence and the '''scaffold''' sequence that interacts with the Cas9 protein. The design of the gRNA target gene specific spacer sequence can be performed using online tools such as CRISPRscan. Briefly the software will scan for the NGG protospacer adjacent motif (PAM) sequence then evaluate the 20 nucleotides that are 5' of the PAM site for their suitability as a spacer sequence. Favorable spacer sequences are more than 50% GC, 20 nucleotides in length and do not have "off-target" binding sites. Additionally, if using T7 RNA polymerase to produce the sgRNA then two '''5' GGs''' should be considered, editing occurred with an 80% frequency with GG-, 75% NG-, 60% GN- and 37.5% if NN- ([https://www.echinobase.org/literature/article.do?method=display&articleId=48856 Thomas et al. 2014]). | |||
'''Method overview''' | |||
Published methods have used microinjection of RNA into embryos. | |||
The capped mRNA for ''Streptococcus pyogenes'' Cas9 with nuclear localization signals was produced using either linearized [https://www.addgene.org/47929/ pCS2-nCas9n] or [https://www.addgene.org/51307/ pCS2-3XFLAG-NLS-SpCas9-NLS] as the template for the MEGAscript SP6 Transcription Kit or the mMESSAGE mMACHINE SP6 Transcription Kit. The RNA was then purified. | |||
To make the gRNAs several approaches have been used. The [https://www.addgene.org/46759/ pT7-gRNA] was designed to clone the gene specific spacer/target sequence into BsmBI restriction sites. The vector contains the T7 promoter and the gRNA scaffold followed by a restriction site for linearization prior to RNA production. More recently the pT7-gRNA plasmid has been used as template for a primer containing the T7 promoter, spacer sequence and an overlap sequence to prime the PCR and add the scaffold. This overlap approach has also been used with a synthesized scaffold oligo for PCR (eg. 5’ AAAAGCACCG ACTCGGTGCC ACTTTTTCAA GTTGATAACG GACTAGCCTT ATTTTAACTT GCTATTTCTA GCTCTAAAAC 3' where the overlap sequence is underlined) . The sgRNA is then produced using the MEGAshortscript T7 Transcription Kit and RNA is purified. | |||
For microinjection the 500-1000 ng/ul Cas9 mRNA and 150-400 ng/ul sgRNA are mixed (literature varies). The NLS-Cas9-NLS protein is approximately 4.4X the mass of gRNAs so sgRNAs are in excess. If 50pl is injected this is on the order of 10^7 molecules of Cas9 mRNA and 10^8 molecules of sgRNA. | |||
Revision as of 11:16, 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).
Design overview
CRISPR systems in nature are composed of the Cas9 nuclease and two RNAs, the CRISPR RNA (crRNA) that binds to a complementary DNA sequence and binds to the transactivating RNA (tracrRNA) that also binds to a specific Cas9 protein. For ease of use the crRNA and tracrRNA have been combined into a single guide RNA (sgRNA) molecule for use with the Streptococcus pyogenes Cas9 (SpCas9). The sgRNA has the target gene spacer sequence and the scaffold sequence that interacts with the Cas9 protein. The design of the gRNA target gene specific spacer sequence can be performed using online tools such as CRISPRscan. Briefly the software will scan for the NGG protospacer adjacent motif (PAM) sequence then evaluate the 20 nucleotides that are 5' of the PAM site for their suitability as a spacer sequence. Favorable spacer sequences are more than 50% GC, 20 nucleotides in length and do not have "off-target" binding sites. Additionally, if using T7 RNA polymerase to produce the sgRNA then two 5' GGs should be considered, editing occurred with an 80% frequency with GG-, 75% NG-, 60% GN- and 37.5% if NN- (Thomas et al. 2014).
Method overview
Published methods have used microinjection of RNA into embryos.
The capped mRNA for Streptococcus pyogenes Cas9 with nuclear localization signals was produced using either linearized pCS2-nCas9n or pCS2-3XFLAG-NLS-SpCas9-NLS as the template for the MEGAscript SP6 Transcription Kit or the mMESSAGE mMACHINE SP6 Transcription Kit. The RNA was then purified.
To make the gRNAs several approaches have been used. The pT7-gRNA was designed to clone the gene specific spacer/target sequence into BsmBI restriction sites. The vector contains the T7 promoter and the gRNA scaffold followed by a restriction site for linearization prior to RNA production. More recently the pT7-gRNA plasmid has been used as template for a primer containing the T7 promoter, spacer sequence and an overlap sequence to prime the PCR and add the scaffold. This overlap approach has also been used with a synthesized scaffold oligo for PCR (eg. 5’ AAAAGCACCG ACTCGGTGCC ACTTTTTCAA GTTGATAACG GACTAGCCTT ATTTTAACTT GCTATTTCTA GCTCTAAAAC 3' where the overlap sequence is underlined) . The sgRNA is then produced using the MEGAshortscript T7 Transcription Kit and RNA is purified.
For microinjection the 500-1000 ng/ul Cas9 mRNA and 150-400 ng/ul sgRNA are mixed (literature varies). The NLS-Cas9-NLS protein is approximately 4.4X the mass of gRNAs so sgRNAs are in excess. If 50pl is injected this is on the order of 10^7 molecules of Cas9 mRNA and 10^8 molecules of sgRNA.