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The Choice of a Donor Molecule in Genome Editing Experiments in Animal Cells

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Abstract—

Genome editing is a powerful tool that allows study of the properties of genes or changes to be made to the genetic sequence. Programmable nucleases that can induce double-strand breaks in the genomic sequence of interest have been developed over the past few decades. After initiation of a double-strand break (DSB) in DNA, the DSB can be repaired by the NHEJ (non-homologous end joining), which leads to various errors and gene knockout. Other repair options, HDR (homology directed repair) or SSTR (single-strand template repair), allow researchers to make desired changes in the gene. HDR occurs in the presence of a donor template, in natural conditions the donor template is a sister chromatid. The efficiency of HDR and SSTR is significantly lower than the efficiency of NHEJ in genome editing. Double-stranded, single-stranded and long single-stranded DNAs are used to increase efficiency and to make desired changes in genomic DNA. In this review, we discuss donor molecules that are used for DSB repair using HDR or SSTR during genome editing, their application, and modifications to increase the efficiency of HDR and SSTR.

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This study was conducted within the state assignment set out by the Ministry of Science and Higher Education of the Russian Federation for the Research Centre for Medical Genetics.

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  1. Research Centre for Medical Genetics, 115522, Moscow, Russia

    O. V. Volodina & S. A. Smirnikhina

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  1. O. V. Volodina
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Correspondence to S. A. Smirnikhina.

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Translated by M. Novikova

Abbreviations: ZFN, zinc finger nuclease; TALEN, transcription activator-like effector nuclease; CRISPR, clustered regularly interspaced short palindromic repeats; PAM, protospacer adjacent motif; a-NHEJ, alternative non-homologous end joining; lssDNA, long single-strand DNA; dsDNA, double-strand DNA; DSB, double-strand break; с-NHEJ, classic non-homologous end joining; MMEJ, microhomology-mediated end joining; HDR, homology directed repair; NHEJ, non-homologous end joining; ssODNs, single-strand oligodeoxyribonucleotides; SSTR, single-strand template repair; SDSA, synthesis-dependent strand annealing.

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Volodina, O.V., Smirnikhina, S.A. The Choice of a Donor Molecule in Genome Editing Experiments in Animal Cells. Mol Biol 56, 372–381 (2022). https://doi.org/10.1134/S002689332203013X

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