Robust activation of microhomology-mediated end joining for precision gene editing applications

Abstract

One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated F0 mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.

MMEJ-based Precision Gene Editing for applications in Gene Therapy and Functional Genomics

“…We and others (15,16,19) have shown that DSBs directed at sites likely to be repaired via MMEJ significantly increase the homogeneity of the resulting repair outcomes in zebrafish. Despite there being no known clear biochemical mechanism as to how MMEJ repairs DSBs, we and others (20)(21)(22)(23) have published software tools that predict the occurrence of MMEJ based on the sequence context surrounding any given target DSB site.…”

“…This deletion pattern is then useful as a heuristic to identify probable MMEJbased repairs from a mixed-repair pool. The ability to generate predictable genotypes (14), sometimes even resulting in an identical allele in over half of the editing outcomes (15), makes MMEJ an attractive alternative to NHEJ for precision genome engineering (16)(17)(18)(19).…”

“…In particular, we have published MENTHU (22), which screens genetic sequences for DSBs likely to generate consistent repair outcomes (with 50% or more of a DSB repair profile displaying the same genotype) as a result of MMEJ, aka PreMAs (predominant MMEJ alleles) ( Figure 1A and 1B). As opposed to conventional targeting designs, using MENTHUrecommended SpCas9 and TALEN cut sites resulted in PreMAs more often, facilitating subsequent zebrafish mutant screenings by decreasing the sequence variability of the resulting allele pool (15). In parallel, the tools ForeCasT (20) and inDelphi (23) were simultaneously and independently developed to predict the probability of occurrence of individual repair outcomes after Cas9-mediated DSBs on mammalian cells, and were shortly followed by Lindel (21).…”

Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

“…In the early zebrafish embryo, microhomology mediated end joining (MMEJ) is an active DNA repair pathway that generates predictable alleles after nuclease targeting (Thyme and Schier 2016) (He et al 2015) (Ata et al 2018). MMEJ-based gene editing has been described in zebrafish and mammalian cells, though the homology arms used for these events may be more similar in length to those employed in Single Strand Annealing (SSA) (Aida et al 2016) (Hisano et al 2015) (Nakade et al 2014) (Bhargava, Onyango, and Stark 2016).…”

Microhomology-mediated CRISPR/Cas9-based method for genome editing in fission yeast

“…In organisms such as silkworm and zebrafish, the MMEJ-mediated integration of 417 fluorescent marker genes with TALEN and CRISPR/Cas9 system has been successful 418 (Nakade et al 2014;Ata et al 2018). Therefore, we attempted to generate knock-in yeast 419 cells using the MMEJ-mediated CRISPR/Cas9 system.…”