Efficient Generation of Pathogenic A-to-G Mutations in Human Tripronuclear Embryos via ABE-Mediated Base Editing

Abstract

Base editing systems show their power in modeling and correcting the pathogenic mutations of genetic diseases. Previous studies have already demonstrated the editing efficiency of BE3-mediated C-to-T conversion in human embryos. However, the precision and efficiency of a recently developed adenine base editor (ABE), which converts A-to-G editing in human embryos, remain to be addressed. Here we selected reported pathogenic mutations to characterize the ABE in human tripronuclear embryos. We found effective A-to-G editing occurred at the desirable sites using the ABE system. Furthermore, ABE-mediated A-to-G editing in the single blastomere of the edited embryos exhibited high product purity. By deep sequencing and whole-genome sequencing, A or T mutations didn’t increase significantly, and no off-target or insertion or deletion (indel) mutations were detected in these edited embryos, indicating the ABE-mediated base editing in human embryos is precise and controllable. For some sites, since a different editing pattern was obtained from the cells and the embryos targeted with the same single guide RNA (sgRNA), it suggests that ABE-mediated editing might have different specificity in vivo . Taken together, we efficiently generated pathogenic A-to-G mutations in human tripronuclear embryos via ABE-mediated base editing.

CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells

“…With these differences in mind, we wanted to investigate what may cause the disparities in editing efficiency among the different approaches. Previous works established that base editing efficiency is context dependent, with particular preference dictated by the nucleotide preceding the target base 35 – 37 . These works also sought to change the context dependencies of the preceding nucleotide by employing cytidine deaminase paralogs, orthologs, and engineered variants to change the context dependencies of base editors.…”

“…To determine these dependencies, we performed an meta-analysis for both BE4 and ABE7.10 with data across multiple cell types, genes, and delivery methods from the literature 19 , 35 , 36 , 38 and data generated by our group (6 papers, 102 guides, 447 edits in total). We chose to focus our analysis on editing efficiency as a function of the position in the protospacer and the identity of the preceding base.…”

Methods and applications of CRISPR/Cas system for genome editing in stem cells

“…BE shows widespread versatility across plants, rodents, fish, and human embryos, and it has been applied in modeling and correcting human disease [[80], [81], [82], [83], [84], [85], [86], [87], [88]]. Of note, genetic disorder with pathogenic mutation has been corrected in human embryos by BE, demonstrating its potential in precise gene therapy [89].…”

Highly efficient generation of sheep with a defined FecBB mutation via adenine base editing

“…The resulting ABEmax editor can alter single nucleotide polymorphisms (SNPs) with substantially increased efficiency and low genomewide off-target activity in a variety of mammalian cell types [18]. xCas93.7 replaced the SpCas9 of ABE7.10 to obtain xCas9-ABE, which leads to higher base editing efficiencies than ABE7.10 in HEK293T cells [19]. Although cytosine base editors often produce cell populations with mixed base editing populations, ABE do not prominently show A to non-G substitutions at the target sites.…”