Allele specific chromosome removal by crispr/cas9 to correct trisomy 18

Trisomies have significant clinical consequences including failed embryo development, pregnancy loss and rare cases of live birth resulting in significant congenital defects. Previous experiments have demonstrated chromosome changes in diploid embryos by introducing CRISPR/Cas9. The objective of this experiment is to determine if CRISPR/Cas9 can be used to target and eliminate an allele specific chromosome in trisomy 18 embryos. CRISPR/Cas9 was transfected into a trisomy 18 embryonic stem cell line targeting SNPs specific to only 1 chromosome copy. Guide RNAs were designed to target 4 mid-arm locations across chromosome 18 with an extra maternally inherited SNP, to attempt to remove the chromosome. The guide RNAs were transfected in groups individually, and together. After learning linkage information, transfection was performed targeting 1 maternal haplotype at a time. The guide RNA efficiency and specificity were tested in embryonic stem cells prior to attempting chromosome removal in embryos with the same genome. Cells in each transfection group were analyzed by Amplicon deep sequencing and SNP array analysis to assess efficiency and specificity of guide RNA cutting and for trisomy correction. Somatic cell nuclear transfer was then performed with embryos with the same genome. Cas9-RNPs were injected into the embryos at the fertilization stage, cultured to the cleavage stage and blastomeres were analyzed for resultant chromosomal changes. 4 guide RNAs were evaluated for specificity and activity in a stem cell model, each targeting an extra maternally inherited SNP. Indel frequency, as analyzed by Amplicon deep sequencing of the individual guide RNAs was 49%, 0%, 17%, and 5% for guide RNAs targeting four different SNPs. All indels took place at the targeted SNP giving specificity of 100% for each guide RNA.Colonies grown from single cells in each group were analyzed by SNP array analysis. 9 of 16 colonies (56.3%) demonstrated chromosome loss, including evidence of spontaneous loss, not induced by CRISPR/Cas9. Next, stem cells were differentiated to fibroblasts to perform nuclear substitution in human oocytes and generate embryos with an identical trisomy 18 to the stem cell experiments. 6 embryos were injected after activation using guide RNAs targeting 2 maternally inherited SNPs, 1 on the mid-P arm and 1 on the mid-Q arm. A total of 12 blastomeres were analyzed at the cleavage stage. 5 blastomeres (40%) demonstrates copy number changes originating at the Cas9 cut sites, with 3 copy number changes noted at the P arm cut site and 2 copy number changes noted at the Q arm cut site. In trisomic embryos generated by nuclear transfer, there is demonstration of specific chromosome loss originating at the target alleles. CRISPR/Cas9 induced chromosome loss could not be analyzed in the stem cell model because of spontaneous trisomy correction.