Date: 13th May 2020
Polygenic diseases are the most common genetic disorders, however, studying these disorders and correcting them is complex. It requires simultaneous and precise installation or replacement of multiple single nucleotides by genome editing. Now scientists have demonstrated the feasibility of multiplex base editing for polygenic disease modelling in primate zygotes.
The modelling of diseases in animals is crucial for making inroads into understanding the complexities of genetic diseases but also crucially for the development of therapies. One popular model, the cynomolgus monkey (Macaca fascicularis), is particularly well-suited in the biomedical arena as it has a close relationship to humans in both genetic and physiological aspects.
However, they have long reproduction cycles, meaning that the use of CRISPR-based approaches to produce polygenic monkeys carrying multiple single nucleotide variations (SNV) would require years of work – requiring the crossing of several mutant monkeys carrying a single SNV to create a polygenic model.
One way to overcome this limitation is to use multiplex genome editing to generate disease models in F0 animals, however, this approach contains potential limitations to either precise editing or multiplexing as each occurs through the cellular repair mechanism of non-homologous end joining (NHEJ) or homology-directed repair (HDR), respectively.
An alternative tactic to this therefore, would be to use base editing by cytidine deaminase or adenosine deaminase Cas9 fusion proteins (using Cas9 nickase or catalytically-dead Cas9), which converts cytosine (C) to thymine (T) or adenine (A) to guanine (G). However, it is currently unknown whether this type of base editing for targeted base conversions in cynomolgus monkey zygotes would actually work.
Now scientists led by Shihua Yang, Guoping Feng and Tomomi Aida in an international collaboration with the South China Agricultural University, the Guangdong Laboratory of Lingnan Modern Agriculture, China, the Massachusetts Institute of Technology and the Broad Institute of MIT and Harvard, US, have addressed this and used multiplexed CRISPR-based base editing of up to three target sites simultaneously in cynomolgus monkey embryos.
To do this the team first determined whether nucleotide base editing was possible in monkey embryos. They tested both C-to-T and A-to-G base editing independently, using a cas9 nickase fused to different base editors, guided to specific targets using sgRNAs (single guide RNAs).
They initially used a cytosine base editor (CBE), to introduce mutations in the gene fumarylacetoacetate hydrolase (FAH), which causes the genetic disorder, tyrosinaemia and 11 out of 16 embryos injected with RNA had genomic modifications at the target site in the FAH exon, with 10 of those having the desired outcome of an introduced stop codon.
Next they tested A-to-G base editing in the monkey embryos using an adenine base editor (ABE). Here they chose amyloid precursor protein (APP), which is mutated in familial early-onset Alzheimer’s disease and, on analysis, 6 out of 9 injected embryos had genomic modifications at the target site.
Encouraged by the results, the team then went on to simultaneously edit multiple loci by co-injection of sgRNAs targeting multiple genes with both base-editors. They built up a series of increasingly complex experiments trying double and triple editing, in one or multiple genes.
From this work, the group observed multiplexed base editing in up to three genes in whole embryos and in single cells of the embryo – blastomeres. In this final experiment – out of 8 injected embryos – all contained genomic modifications at 2 of the target genes (C-to-T conversion), with 5 of those also containing edits in the third gene (A-to-G conversions). Thus, demonstrating the viability of simultaneous, multiplexed C-to-T and A-to-G base editing. However, by genotyping single blastomeres, they did discover a significant amount of mosaicism within the multi-edited embryos.
The team, here have successfully showed the feasibility of multiplexed base editing in cynomolgus monkey embryos. Unbiased whole genome sequencing demonstrated this editing was highly specific.
However, they did find differences in the accuracy between the editors tested, with the adenosine base editor proving to show the highest conversion accuracy (here 100%) – a crucial factor for potential gene therapies as unwanted mutations can have huge deleterious and unanticipated effects.
In contrast though, this editor also exhibited relatively low and variable conversion efficiencies across genes and embryos. This suggested that further optimisations and improvements would need to be made to these editors before they can translate into the clinic.
One crucial next step for this technique to be considered for therapeutic applications is to address the mosaic nature of the gene edits. It is thought that by directly injecting the base editor as a recombinant protein –rather than the current system of injecting mRNA encoding the editor- at very early stages of embryo development, a decrease in the mosaicism between blastomeres could be achieved.
Whilst base editing is still considered a relatively new CRISPR-application, the work presented here represents an important milestone in the development of this tech for both creating animal models to study polygenic diseases but also in establishing its potential to restore of a significant number of human pathogenic SNVs.
CRISPR-based therapies have already started to enter the clinic and in these studies multiplexed gene editing is being tested. One such trial, designed to test the safety and feasibility of multiplexed CRISPR-Cas9 editing to engineer T cells in patients with refractory (unresponsive) cancer, has reported mixed but promising results. However, limited to just 3, very ill patients this work needs to be expanded before we can get a real sense of its worth. The question now poised to be answered is whether base editing can translate into potential therapies for some of the most devastating genetic disorders? It is hoped that this work will provide some of the basic underlying research to accelerate this translation.
Zhang, W., T. Aida, R. C. H. del Rosario, J. J. Wilde, C. Ding, X. Zhang, Z. Baloch, Y. Huang, Y. Tang, D. Li, H. Lu, Y. Zhou, M. Jiang, D. Xu, Z. Fang, Z. Zheng, Q. Huang, G. Feng and S. Yang (2020). “Multiplex precise base editing in cynomolgus monkeys.” Nature Communications 11(1): 2325.