Echinobase curators have collected the gRNA sequences on the wiki.
Gene editing technologies are important research tools and Echinobase is supporting these efforts by cataloguing the gRNA sequences that have been published. Tools are being developed on Echinobase to align the gRNA to the genome. As a temporary resource we have listed gRNA methods and sequences onto the EchinoWiki. A brief description of the content is provided below, see the CRISPR/Cas wiki page for more details.
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 genes. A number of different methods were used for gRNA synthesis and NLS-SpCas9-NLS (zebrafish codon-optimized), or codon optimized for human with a 3XFLAG-tag, were used. 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.
Reviews of the methods are available.
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-.
Published methods have used microinjection of RNA into embryos
The capped mRNA for Streptococcus pyogenes Cas9 with nuclear localization signals was produced using linearized plasmid as the template for a transcription Kit. The RNA was then purified.
To make the gRNAs several approaches have been used; cloning and PCR or overlap extension PCR followed by T7 transcription.
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
The 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).
Last Updated: 2020-12-21