Steven presenting CRISPR cell paper
One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering
Background
- CRIPR repeat sequences separated by spacers. Spacers are different, match different bacterial phage sequences.
- components: Cas1, Cas2, Csn2 (involved in encorporating the sequences)
- Cas9 involved in fighting against future infections
- CRISPR locus transcribed, digested into short fragments that have part spacer and part repeat.
- Cas9 binds cleaved CRISPR fragment and a tracrRNA bnds repeat associates with complex.
- why doesn’t this system cut the endogenous CRISPR locus? requires protospacer motif and PAM sequence in phage (very short simple motif (e.g. GGG).
Re-engineering
- fuse crRNA and tracrRNA into single transcript, sufficient to directing cutting behavior of cas9.
- published by 3 labs earlier this year (e.g. Cong et al Science 2013).
- cut genome, non-homologous end-joining (used for gene deletion/disruption). Even single breaks are often incorrectly repaired, result in indels (insertions and deletions).
- have to chose target sequences near PAM sequences. (need only GG or CC in this system).
Previous knockout mice (2007 Nobel)
- disect embryonic cells, plasmid with homology arms + positive selection marker and negative selection marker
- grow individual colonies. Select colonies inject cells back into embryo
- emplant embryo to foster mother, create hybrid chymeric mouse.
- use coat-color to select high frequency of donor cell incorporation.
- mate chimeric mouse to wt mouse, hope that germline has also been transformed.
- next generation get complete heterozygotes
- 3 wks gestation + 1 mo development. Min length .5 years to generate gene knockout.
This work
- work with single cell zygote. inject multiple guide sequences with cas9. grow zygote into a blastcyst, emplant into mother.
- inject with DNA with homology arms, these get incorporated into the disrupted gene, get specific base-pair mutation.
- 2 genes, in 80% offspring all 4 alleles modified (targeted mutation, NJE).
- with 1 gene
- with targeted mutation, NHEJ still happens so only 10% have both targeted changes.
- key advantage: easy to inject RNA/DNA don’t need to get some complicated zinc-finger protein.
follow up
- mutant cas9 creates just a nick, not a DSB, enhancers HDR, avoids