a) A single guide RNA (sgRNA) recognizes a genomic region followed by 5’-NGG-3’ PAM sequence, which recruits the Cas9 DNA endonuclease. This introduces a double-stranded break that is repaired by:
i) Non-Homologous End Joining (NHEJ), an error prone pathway that can result in the creation of Indels that can disrupt the gene.
ii) Or by homology directed repair (HDR) in the presence of a donor construct.
b) Clients will provide us with specific Guide RNA that we anneal with constant Tracer RNA and then mixed with Cas9 enzyme that we provide.
c) Guide RNA and Cas9 enzyme is introduced directly to the zygote by electroporation.
d) Electroporated zygote is transferred to the Embryo Recipient Pseudo-pregnant mouse.
e) After 3 weeks, weaned pups will be delivered to the client for genotyping.
f) Conditional Alleles:
i) Insertion of LoxP sites to flank an exon (exons) of interest is possible using CRISPR technology, but can be problematic due to low event probability.
ii) The most common method involves electroporation of two separate sgRNAs, targeted to sequences flanking an exon of interest and two ssODN donors, each containing a LoxP site flanked by short (40–80 bases) homology arms.
iii) Both LoxP sites must be inserted into the same allele. Efficiency tends to be low, and a variety of mutations (including the full unconditional knockout, single LoxP insertions, double LoxP insertions located in trans, deletions resulting from non-homologous end-joining –NHEJ- and insertion of mutated LoxP sites) are commonly detected.
iv) Alternative strategies using the Strandase enzyme kit could be used.
v) Strandase enzyme can digest a sence or antisence DNA strand that is produced by simple PCR, performed with phosphorylated primer.
vi) This technology allows us to perform Homology Directed Repair (HDR) with up to 5 kb donors, resulting in reduced tendency to randomly integrate into the genome and improved gene editing efficiency.
i) CRISPR mice have been reported to have far fewer off-targets then cell lines
h) Guide RNAs, Tracer RNAs, Cas9 enzyme and HDR oligos:
i) We work with molecules produced by IDT.
ii) Guide RNAs and HDR oligos are provided by client, whereas Tracer RNAs and Cas9 enzyme are provided by us.
i) Testing new Guide RNAs and HDR donors:
i) Before starting the project proper, we can electroporate a number of embryos (about 50), and leave them to develop in vitro to blastocyst stage.
ii) The embryos can then be analyzed for the presence of the mutation, and for indels at the cut sites.
iii) If indels persist in a satisfying quantity (percentage) we will complete the project and transfer the electroporated embryos.
j) GEMM facility guarantees.