The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing.

Scientific Reports
Jae Hoon LeeHan-Woong Lee

Abstract

Engineered nucleases are widely used for creating frameshift or nonsense mutations in the target genes to eliminate gene functions. The resulting mRNAs carrying premature termination codons can be eliminated by nonsense-mediated mRNA decay. However, it is unclear how effective this process would be in vivo. Here, we found that the nonsense-mediated decay was unable to remove the mutant mRNAs in twelve out of sixteen homozygous mutant mice with frameshift mutations generated using engineered nucleases, which is far beyond what we expected. The frameshift mutant proteins translated by a single nucleotide deletion within the coding region were also detected in the p53 mutant mice. Furthermore, we showed that targeting the exons present downstream of the exons with a start codon or distant from ATG is relatively effective for eliminating mutant mRNAs in vivo, whereas the exons with a start codon are targeted to express the mutant mRNAs. Of the sixteen mutant mice generated, only four mutant mice targeting the downstream exons exhibited over 80% clearance of mutant mRNAs. Since the abnormal products, either mutant RNAs or mutant proteins, expressed by the target alleles might obscure the outcome of genome editing, these findings wil...Continue Reading

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Citations

Nov 21, 2020·Plant Biotechnology Journal·Yongchao TangKaijun Zhao

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Methods Mentioned

BETA
gene knockout
PCR
electrophoresis
genotyping
in vitro transcription

Software Mentioned

CFX manager

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