Bac vector library

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. 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)