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

Gabriel Martínez-Gálvez, Parnal Joshi, Iddo Friedberg, Armando Manduca, Stephen C. Ekker

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)67-78
Number of pages12
JournalNucleic acids research
Volume49
Issue number1
DOIs
StatePublished - Jan 11 2021

ASJC Scopus subject areas

  • Genetics

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