Simultaneous precise editing of multiple genes in human cells

Nucleic Acids Research
Stephan RiesenbergSvante Pääbo

Abstract

When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.

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Citations

Sep 10, 2020·International Journal of Molecular Sciences·Han YangNingshao Xia
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Datasets Mentioned

BETA
PRJNA543747

Methods Mentioned

BETA
transfection
PCR
Illumina sequencing
electrophoresis
genotyping

Clinical Trials Mentioned

NCT03116971
NCT03770689

Software Mentioned

SAMtools
UCSC genome browser
Cytena
T HaplotypeCaller
fastq
Integrated Genome Viewer ( IGV )
GATK IndelRealigner
CRISPResso
leeHom
GenomeAnalysisTK

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