Presumably after a day spent in a local tavern instead of writing and editing verse, the famous Scottish poet Robert Burns noted that “The best laid schemes of mice and men, Often go awry”. However, a new study of genome editing in the embryos of mice and men led by Kathy K. Niakan (Francis Crick Institute, London, UK) went far from “awry” and indeed, their successful strategy now provides important new insight into embryo development!
In their new Nature article, Fogarty et al. targeted the OCT4 (or POU5F1) locus in mouse and human embryos via CRISPR-Cas9 editing to understand the role of this pluripotency factor during the development the one-cell zygote through to the blastocyst stage embryo. The combination of high-efficiency on-target editing and undetectable off-target editing highlights CRISPR-Cas9 as the perfect strategy to make best use of material as scarce (and as important) as early human embryos.
So how did the “best laid schemes” of these well-read researchers pan out?
- By employing engineered human embryonic stem cells (hESCs), the team first screened for OCT4-specific single-guide RNAs (sgRNAs)
- hESCs constitutively expressed Cas9 and contained an inducible sgRNA cassette
- Induction of one sgRNA prompted a 2-bp deletion that generated a frameshift mutation and a premature stop codon, leading to the desired downregulation of OCT4 expression
- Targeted deep sequencing revealed no unwanted editing at putative off-target sites
- Live embryo imaging following microinjection of gene-editing components into mouse fertilized zygotes demonstrated that a lack of Oct4 still permitted blastocyst formation, but not the outgrowth of the inner cell mass when cultured under mouse ESC derivation conditions
- At the transcriptional level, Oct4 loss reduced the expression of some differentiation-associated genes but did not affect pluripotency genes
- These findings suggest a role for Oct4 in embryo maintenance in mouse
- CRISPR-Cas9 genome editing of the OCT4 locus in human embryos employed in vitro fertilized (IVF) zygotes donated by patients undergoing infertility treatment
- Following microinjection of the sgRNA–Cas9 ribonucleoprotein complex, the authors observed high on-target editing efficiency and low off-target activity, which confirmed sgRNA specificity
- However, in contrast to the mouse study, OCT4 loss reduced both viability and quality of blastocysts, suggesting a role for OCT4 in blastocyst establishment
- Loss of OCT4 in human embryos not only downregulated differentiation-associated genes but also inhibited pluripotency gene expression, thus disrupting embryonic development
Overall, these data suggests that OCT4 has an unexpected earlier function in humans when compared to mice. “We were surprised to see just how crucial this gene is for human embryo development, but we need to continue our work to confirm its role” notes Dr Norah Fogarty first author of the study, who adds that “Other research methods, including studies in mice, suggested a later and more focused role for OCT4, so our results highlight the need for human embryo research.”
While this sensational research has relevance to both stem cell therapeutics and IVF treatments, their findings also highlight the limitations of animal models such as the mouse. Future research will take heart from the observed efficiency of the CRISPR-Cas9 genome editing, although the team suggests that even higher editing efficiency may be attainable through the co-injection of gene-editing components with sperm during the creation of the fertilized zygote.
Lay aside your Burns and put down your Steinbeck; this new study “Of Mice and Men” is the only tale worth reading today; see it all at Nature, Sept 2017.