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Deploying MMEJ using MENdel in precision gene editing applications for gene therapy and functional genomics.

ABSTRACT: Gene-editing experiments commonly elicit the error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. We compared three DSB repair prediction algorithms - MENTHU, inDelphi, and Lindel - in identifying MMEJ-repaired, homogeneous genotypes (PreMAs) in an independent dataset of 5,885 distinct Cas9-mediated mouse embryonic stem cell DSB repair events. MENTHU correctly identified 46% of all PreMAs available, a ?2- and ?60-fold sensitivity increase compared to inDelphi and Lindel, respectively. In contrast, only Lindel correctly predicted predominant single-base insertions. We report the new algorithm MENdel, a combination of MENTHU and Lindel, that achieves the most predictive coverage of homogeneous out-of-frame mutations in this large dataset. We then estimated the frequency of Cas9-targetable homogeneous frameshift-inducing DSBs in vertebrate coding regions for gene discovery using MENdel. 47 out of 54 genes (87%) contained at least one early frameshift-inducing DSB and 49 out of 54 (91%) did so when also considering Cas12a-mediated deletions. We suggest that the use of MENdel helps researchers use MMEJ at scale for reverse genetics screenings and with sufficient intra-gene density rates to be viable for nearly all loss-of-function based gene editing therapeutic applications.

SUBMITTER: Martinez-Galvez G

PROVIDER: S-EPMC7797032 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Deploying MMEJ using MENdel in precision gene editing applications for gene therapy and functional genomics.

Martínez-Gálvez Gabriel G Joshi Parnal P Friedberg Iddo I Manduca Armando A Ekker Stephen C SC

Nucleic acids research 20210101 1

Gene-editing experiments commonly elicit the error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. We compared three DSB repair prediction algorithms - MENTHU, inDelphi, and Lindel - in identifying MMEJ-repaired, homogeneous genotypes (PreMAs) in an independent dataset of 5,885 distinct Cas9-mediated mouse embryonic stem cell DSB ...[more]

PMID: 33305328

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Project description: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.

Dataset Information

Deploying MMEJ using MENdel in precision gene editing applications for gene therapy and functional genomics.

Publications

Deploying MMEJ using MENdel in precision gene editing applications for gene therapy and functional genomics.

Similar Datasets

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