CRISPR/Cas in Echinoderms

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.

Table of sgRNA sequences for S. purpuratus

This table shows the gene specific spacer RNA sequence that is 5' to the constant scaffold RNA sequence. The gRNA binds to the reverse complement of the DNA sequence indicated (other strand).

Name	Synonym	sgRNA sequence 5-3'	DNA sequence	Edits Yes/NO	References
nodall gRNA1	E1_123	GGCCUCAGGUCGCACCAGA	GGCCTCAGGTCGCACCAGA	NO	(Lin and Su 2016)
nodall gRNA2	E1_157	GGGCGUCCGGUGUGAUAAG	GGGCGTCCGGTGTGATAAG	Yes	(Lin and Su 2016)
nodall gRNA3	E1_272	GGAAGAAUGCCAAGCCAUUG	GGAAGAATGCCAAGCCATTG	Yes	(Lin and Su 2016)
nodall gRNA4	E2_63	GGACAGCUUCGUUGGCGGGC	GGACAGCTTCGTTGGCGGGC	Yes	(Lin and Su 2016)
nodall gRNA5	E2_777	GGUAGGCGUUGAACUGCUUU	GGTAGGCGTTGAACTGCTTT	Yes	(Lin and Su 2016)
nodall gRNA6	E2_885	GGUGUCCGUCUCUCGGGU	GGTGTCCGTCTCTCGGGT	Yes	(Lin and Su 2016)
LOC588806 gRNA1	Sp.PKS1.175(+)	GGUGGUGUCUUUGUCGGUAU	GTGGTGTCTTTGTCGGTAT	Yes	(Oulhen and Wessel 2016)
LOC588806 gRNA2	Sp.PKS1.547(-)	GGUGAGGGGUUUGAGGACAA	TGAGGGGTTTGAGGACAA	Yes	(Oulhen and Wessel 2016)
LOC588806 gRNA3	Sp.PKS1.806(-)	GGAGAGGGUUGUCUUUGGUG	GAGAGGGTTGTCTTTGGTG	Yes	(Oulhen and Wessel 2016)
gcml gRNA1	Sp.GCM.63(+)	GGCCGCCGGAGCUGCCGGGU	GCCGCCGGAGCTGCCGGGT	Yes	(Oulhen and Wessel 2016)
gcml gRNA2	Sp.GCM.390(+)	GGCUGUUCGGCCAGCCACUU	CTGTTCGGCCAGCCACTT	Yes	(Oulhen and Wessel 2016)
gcml gRNA3	Sp.GCM.541(+)	GGGAUCCUAUUCCAAUCGAA	GATCCTATTCCAATCGAA	NO	(Oulhen and Wessel 2016)
gcml gRNA4	Sp.GCM.944(-)	GGGAGUCCAGCCGUCCAUCU	GAGTCCAGCCGTCCATCT	Yes	(Oulhen and Wessel 2016)
nanos2l gRNA1	Sp nanos2.190	GGUGACUGGCUCGUCGAGAC	TGACTGGCTCGTCGAGAC	Yes	(Oulhen et al. 2017)
nanos2l gRNA2	Sp nanos2.250	GGGAUCUCAGCGAUGUUCAG	GATCTCAGCGATGTTCAG	NO	(Oulhen et al. 2017)
nanos2l gRNA3	Sp nanos2.295	GGAGGAAGGCGAGCCAACAA	GGAGGAAGGCGAGCCAACAA	Yes	(Oulhen et al. 2017)
nanos2l gRNA4	Sp nanos2.319	GGAGGUGGUGCUACGGGUGU	GAGGTGGTGCTACGGGTGT	Yes	(Oulhen et al. 2017)
dll gRNA1	Delta-1 (19D10)	GGGUGAACUGGUAGCACGG	GGGTGAACTGGTAGCACGG	Yes	(Mellott et al. 2017)
dll gRNA2	Delta-2 (20D11)	GGCUACACGUGCCUCUGUCC	GGCTACACGTGCCTCTGTCC	Yes	(Mellott et al. 2017)
dll gRNA3	Delta-3 (20D14)	GGACCGAAUCAGAUUCCGCG	GGACCGAATCAGATTCCGCG	nd	(Mellott et al. 2017)
alx1 gRNA1	Alx1-sgRNA1	GGAGAACAGGCGCGCCAAGUGG	AGAACAGGCGCGCCAAGTG	Yes	(Shevidi et al. 2017)
alx1 gRNA2	Alx1-sgRNA2	GGCGUGGGGGGCCUCAACCCGG	GCGTGGGGGGCCTCAACCCGG	Yes	(Shevidi et al. 2017)
LOC584189 gRNA1	Dsh-sgRNA1	GGGUAGCUUAGCCAGGCCGAUG	GGTAGCTTAGCCAGGCCGATG	Yes	(Shevidi et al. 2017)
LOC584189 gRNA2	Dsh-sgRNA2	GGCAUCGGUCCCCCUAGCCAGG	GGCATCGGTCCCCCTAGCCAGG	Yes	(Shevidi et al. 2017)
LOC588806 gRNA4	Pks-sgRNA1	GGGGAGGUUCUCAAGGAAGGUA	GGGAGGTTCTCAAGGAAGGTA	Yes	(Shevidi et al. 2017)
LOC588806 gRNA5	Pks-sgRNA2	GGGGGUUCUUUAAGAUCUCGCC	GTTCTTTAAGATCTCGCC	Yes	(Shevidi et al. 2017)
LOC588806 gRNA6	Pks-sgRNA3	GGGGUGGUGUCUUUGUCGGUAU	GTGGTGTCTTTGTCGGTAT	Yes	(Shevidi et al. 2017)
LOC588806 gRNA7	Pks-sgRNA4	GGGGGUAUUGCGCACAGUGUGU	GGTATTGCGCACAGTGTGT	Yes	(Shevidi et al. 2017)
LOC588806 gRNA8	Pks-sgRNA5	GGGGCGACACAGCUUGUGCCAG	CGACACAGCTTGTGCCAG	Yes	(Shevidi et al. 2017)
LOC588806 gRNA9	Pks-sgRNA6	GGGGUUGUCCUCAAACCCCUCA	TTGTCCTCAAACCCCTCA	Yes	(Shevidi et al. 2017)
LOC588806 gRNA10	Pks-sgRNA7	GGGGUAGCGCCAUCGCAGCCAA	GGTAGCGCCATCGCAGCCAA	Yes	(Shevidi et al. 2017)
LOC588806 gRNA11	Pks-sgRNA8	GGGGGCACUAUGUCGAAGCUCA	GCACTATGTCGAAGCTC	Yes	(Shevidi et al. 2017)
LOC588806 gRNA12	Pks-sgRNA9	GGGGGUGAUCCUCUGGAAGCAG	GGTGATCCTCTGGAAGCAG	Yes	(Shevidi et al. 2017)
LOC588806 gRNA13	Pks-sgRNA10	GGGGCACCAAAGACAACCCUCUC	GCACCAAAGACAACCCTCTC	Yes	(Shevidi et al. 2017)
LOC588806 gRNA14	Pks-sgRNA11	GGGGGCAACUUUGGACAUACCG	GCAACTTTGGACATACCG	Yes	(Shevidi et al. 2017)
LOC588806 gRNA15	Pks-sgRNA12	GGGGUACCAUCCCACCAACCAU	GTACCATCCCACCAACCAT	Yes	(Shevidi et al. 2017)
LOC588806 gRNA16	Pks-sgRNA13	GGGGAAGACAAGCACAUCAUUG	GAAGACAAGCACATCATTG	Yes	(Shevidi et al. 2017)
LOC588806 gRNA17	Pks-sgRNA14	GGGGAGGAGCUGACUCCAGAAC	AGGAGCTGACTCCAGAAC	Yes	(Shevidi et al. 2017)
LOC588806 gRNA18	Pks-sgRNA15	GGGGACAAGGAAGGACAGCCUA	GACAAGGAAGGACAGCCTA	Yes	(Shevidi et al. 2017)
LOC588806 gRNA19	Pks-sgRNA16	GGGGGACAGCUGUUGCCAAACAG	GACAGCTGTTGCCAAACAG	Yes	(Shevidi et al. 2017)

Table of sgRNA sequences for Temnopleurus reevesii

This table shows the gene specific spacer RNA sequence that is 5' to the constant scaffold RNA sequence. The gRNA binds to the reverse complement of the DNA sequence indicated (other strand).

Name	Synonym	sgRNA sequence 5-3'	DNA sequence	Edits Yes/NO	References
TBD	Pks1#1sgRNA	GGGGUCGAUGUGAGGGUUUG	GGGGTCGATGTGAGGGTTTG	Yes	(Yaguchi et al. 2020)
TBD	Pks1#2sgRNA	GGACAGGUACUCGUCAUGC	GGACAGGTACTCGTCATGC	Yes	(Yaguchi et al. 2020)
TBD	Pks1#3sgRNA	GGGAUCAACCCUGAUACCCU	GGGATCAACCCTGATACCCT	NO	(Yaguchi et al. 2020)
TBD	Pks1#4sgRNA	GGUUAACUAUGAGGCCUUUG	GGTTAACTATGAGGCCTTTG	Yes	(Yaguchi et al. 2020)
TBD	Pks1#5sgRNA	GGUCUGAUCGAUGACCUGGA	GGTCTGATCGATGACCTGGA	Yes	(Yaguchi et al. 2020)

Table of sgRNA sequences for Hemicentrotus pulcherrimus

This table shows the gene specific spacer RNA sequence that is 5' to the constant scaffold RNA sequence. The gRNA binds to the reverse complement of the DNA sequence indicated (other strand).

Name	Synonym	sgRNA sequence 5-3'	DNA sequence	Edits Yes/NO	References
TBD	HpPks1 sgRNA#1	GUGGUGUCUUUGUCGGUAU	GTGGTGTCTTTGTCGGTAT	NO	(Liu et al. 2019)
TBD	sgRNA#2	ACUGAGGGGUUUGAGGACAA	ACTGAGGGGTTTGAGGACAA	Yes	(Liu et al. 2019)
TBD	sgRNA#3	UGAGAGGGUUGUCACGUGUG	TGAGAGGGTTGTCACGTGTG	Yes	(Liu et al. 2019)
TBD	HpPKS 82-105	GGAGCAAUAAAACCAGCUGG	GGAGCAATAAAACCAGCTGG	Pool Yes	(Wessel et al. 2020)
TBD	HpPKS 101-123	GGGGAACUCGCCAUGCUUG	GGGGAACTCGCCATGCTTG	Pool Yes	(Wessel et al. 2020)
TBD	HpPKS 134-157	GGGAACCUUCCAGAAGUCAU	GGGAACCTTCCAGAAGTCAT	Pool Yes	(Wessel et al. 2020)
TBD	HpPKS 297-320	GGGGCGAGAUCUUGAAGAAC	GGGGCGAGATCTTGAAGAAC	Pool Yes	(Wessel et al. 2020)
TBD	HpPKS 421-444	GGUGGUGUCUUUGUCGGUAU	GGTGGTGTCTTTGTCGGTAT	Pool Yes	(Wessel et al. 2020)
TBD	HpPKS 485-508	GGCGGUCAGGGUGUACGCAU	GGCGGTCAGGGTGTACGCAT	Pool Yes	(Wessel et al. 2020)
TBD	HpFMO3 26-49	GGGAGCUGGUGUGAGUGGCU	GGGAGCTGGTGTGAGTGGCT	Pool Yes	(Wessel et al. 2020)
TBD	HpFMO3 98-121	GGGGGACGAGAAAGUUGACG	GGGGGACGAGAAAGTTGACG	Pool Yes	(Wessel et al. 2020)
TBD	HpFMO3 152-175	GGGAGUUGGUAAUCAGGGCG	GGGAGTTGGTAATCAGGGCG	Pool Yes	(Wessel et al. 2020)
TBD	HpFMO3 204-227	GGAGGACAGUCCUUGGGGAA	GGAGGACAGTCCTTGGGGAA	Pool Yes	(Wessel et al. 2020)
TBD	HpFMO3 333-356	GGCGAAGACUAUGACAUCAC	GGCGAAGACTATGACATCAC	Pool Yes	(Wessel et al. 2020)
TBD	HpFMO3 366-389	GGGACCACGGAAGGACCAGG	GGGACCACGGAAGGACCAGG	Yes	(Wessel et al. 2020)
TBD	Hp GCM 23-46	GGGCGUGGUUAUCAGUCGCC	GGGCGTGGTTATCAGTCGCCG	Pool Yes	(Wessel et al. 2020)
TBD	Hp GCM 75-98	GGAGCCUCGGAUUCAGCAAC	GGAGCCTCGGATTCAGCAAC	Pool Yes	(Wessel et al. 2020)
TBD	Hp GCM 138-161	GGGGGUGGUAGAAGUGGCAG	GGGGGTGGTAGAAGTGGCAG	Yes	(Wessel et al. 2020)
TBD	Hp GCM 263-286	GGACGAGGUCGAGAGUUUGG	GGACGAGGTCGAGAGTTTGG	Pool Yes	(Wessel et al. 2020)
TBD	Hp GCM 302-325	GGCCGCCGGAGCUGCCGGAU	GGCCGCCGGAGCTGCCGGAT	Pool Yes	(Wessel et al. 2020)
TBD	Hp GCM 351-374	GGGCAGGAGAAGGGAACUAG	GGGCAGGAGAAGGGAACTAG	Pool Yes	(Wessel et al. 2020)