PUBLICATION

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

Authors
Martínez-Gálvez, G., Joshi, P., Friedberg, I., Manduca, A., Ekker, S.C.
ID
ZDB-PUB-210123-2
Date
2021
Source
Nucleic acids research   49: 67-78 (Journal)
Registered Authors
Ekker, Stephen C.
Keywords
none
MeSH Terms
  • Algorithms*
  • Animals
  • Bacterial Proteins/metabolism
  • Caspase 9/metabolism
  • DNA Breaks, Double-Stranded*
  • DNA End-Joining Repair*
  • Datasets as Topic
  • Embryonic Stem Cells/metabolism
  • Frameshift Mutation*
  • Gene Editing/methods*
  • Genetic Therapy/methods*
  • Genomics/methods*
  • Humans
  • INDEL Mutation*
  • Loss of Function Mutation*
  • Mice
  • ROC Curve
  • Reverse Genetics/methods*
  • Streptococcus pyogenes/enzymology
  • Zebrafish/genetics
PubMed
33305328 Full text @ Nucleic Acids Res.
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.
Genes / Markers
Figures
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Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping