June 11, 2015

Genetic modification of mice embyros approaching 100% efficiency using CRISPR CAS9 gene therapy

Mice embryos can be genetically modified with CRISPR gene therapy. The procedure is approaching 100% efficiency already.

Prof Perry said the technique could one day be performed during fertility treatment.

Dr Tony Perry believes genetic diseases like cystic fibrosis could be prevented before conception

Invitro fertilization (IVF) treatments are currently about 1.5% of births in the USA. There is estimated demand of 4.8 million IVF in China. This based on 90 million Chinese men and women with fertility problems who have the ability to pay the USD 80 thousand to USD 120 thousand cost the typical surrogacy program in the United States, and not many more can afford to pay the n times USD 12.5 thousand per IVF cycle average cost charged by the typical US-based fertility clinic.

If the cost of IVF drops to $300 per treatment, the success rate goes up to 60% and the income levels increase then over the next ten years the IVF amounts in China and around the world should rapidly increase.

* IVF costs are decreasing by orders of magnitude
* IVF success rates are increasing from 30% to 60%
* the genetic manipulation and genetic screening and embryo selection capabilities are increasing

(A) Schematic of 1-step (upper) and sequential methods of Cas9-mediated mII editing. mII, metaphase II. (B) Paired Hoffman modulation (upper) and eGFP expression (eGFP) images of E4.0 blastocysts produced by 1-step injection of wt mII oocytes with 129-eGFP sperm from hemizygotes, with concentrations of injected Cas9 cRNA and eGFP gRNA indicated below. An asterisk indicates a presumptively phenotypic mosaic. Bar, 100 μm. (C) Numerical representation of embryo development and green fluorescence following injection of sperm from 129-eGFP hemizygous males. Percentages are of blastocyst development on embryonic day 4 (E4.0) (open) and of blastocysts that fluoresced green (filled) indicating 129-eGFP transgene expression in 1-step (green) or sequential (red) methods. Starting embryo numbers and injected concentrations of Cas9 cRNA and eGFP gRNA (gRNA) are shown beneath. (D) Paired Hoffman modulation (upper) and eGFP expression (eGFP) images as for (B) except that the sequential method of injection (Fig. 1A) was used. (E) Histograms as for (C) except that one of either the injected sperm (left) or oocyte carried a Nanog-eGFP knock-in allele. Red highlights provide at-a-glance indication of RNA concentrations in (B) to (E)

Scientific Reports - Asymmetric parental genome engineering by Cas9 during mouse meiotic exit

Mammalian genomes can be edited by injecting pronuclear embryos with Cas9 cRNA and guide RNA (gRNA) but it is unknown whether editing can also occur during the onset of embryonic development, prior to pronuclear embryogenesis. We here report Cas9-mediated editing during sperm-induced meiotic exit and the initiation of development. Injection of unfertilized, mouse metaphase II (mII) oocytes with Cas9 cRNA, gRNA and sperm enabled efficient editing of transgenic and native alleles. Pre-loading oocytes with Cas9 increased sensitivity to gRNA ~100-fold. Paternal allelic editing occurred as an early event: single embryo genome analysis revealed editing within 3 h of sperm injection, coinciding with sperm chromatin decondensation during the gamete-to-embryo transition but prior to pronucleus formation. Maternal alleles underwent editing after the first round of DNA replication, resulting in mosaicism. Asymmetric editing of maternal and paternal alleles suggests a novel strategy for discriminatory targeting of parental genomes.

There is steady progress towards determining the genetic basis of intelligence

8 pages of supplemental material

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