Abstract
Common polygenic diseases result from compounded risk contributed by multiple genetic variants, meaning that simultaneous correction or introduction of single nucleotide variants is required for disease modeling and gene therapy. Here, we show precise, efficient, and simultaneous multiplex base editing of up to three target sites across 11 genes/loci in cynomolgus monkey embryos using CRISPR-based cytidine- and adenine-base editors. Unbiased whole genome sequencing demonstrates high specificity of base editing in monkey embryos. Our data demonstrate feasibility of multiplex base editing for polygenic disease modeling in primate zygotes.
Highlights
Common polygenic diseases result from compounded risk contributed by multiple genetic variants, meaning that simultaneous correction or introduction of single nucleotide variants is required for disease modeling and gene therapy
It is still difficult to control the output of the editing because DNA double-strand breaks (DSBs) induced by CRISPR are primarily repaired through non-homologous endjoining (NHEJ), which can lead to the formation of small insertions or deletions, rather than precise base correction by homologous recombination (HR) in the presence of a donor DNA template
The low number of de novo single nucleotide variations (SNVs) and indels in our mutant, together with our low estimate of de novo BE3 off-targets (3.6 SNVs) suggest that we could not detect clear guide RNA-independent C-to-T conversion by BE3 beyond the range of germline de novo SNVs per generation in cynomolgus macaque. These results suggest that base editing is highly specific in cynomolgus monkey embryos, due to the limitation of the experimental design, we could not distinguish BE3-induced offtarget mutation from naturally occurring de novo mutation. Because it does not require the induction of double-strand breaks, base editing has great potential for precise therapeutic gene correction and disease modeling
Summary
Single C-to-T base editing in monkey embryos. First, we tested whether targeted base editing works in cynomolgus monkey embryos using BE3, a cytosine base editor (CBE) consisting of the Cas[9] nickase fused to rAPOBEC1 and a uracil glycosylase inhibitor[5]. We found that 6 out of 9 injected embryos had genomic modifications at the target site in the APP exon 2 locus (Table 1) These were all A-to-G conversions with allele frequencies that ranged between 20% and 88.6% for at least at one A (Fig. 1d, i, Supplementary Fig. 1b). Other blastomeres were single edited with A-to-G conversion (7.7% at HBB and 23.1% at TP53), and non-edited wild-type allele (Fig. 2h) These results indicate the feasibility of BE3- or ABE-mediated multiple targeted base editing at up to three target genes/sites in a single blastomere of cynomolgus monkey embryos at the same time, and highlight the necessity of further improvements of efficiency and accuracy of base editing. The three genes are associated with Kallmann Syndrome[23], Fanconi anemia[24], and FAH Locus Chr[7] F1
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