Increasing the Level of Knock-In of the MT-C34-Encoding Construct into the CXCR4 Locus by Modifying Donor DNA with Cas9 Target Sites

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Abstract

For successful application of genome editing technology using CRISPR/Cas9 system in clinical practice, it is necessary to achieve high efficiency of knock-in, the insertion of a genetic construct into a given locus in the genome of a target cell. One approach to increasing knock-in efficiency involves modifying donor DNA with the same targets for Cas9 (Cas9 targeting sequence, CTS) that are used for induction of double-strand breaks in the cell genome (the “double-cut donor” method). Another approach is based on introducing truncated targets for Cas9 (truncated CTS, tCTS), including a PAM site and 16 nucleotides proximal to it, into the donor DNA. Presumably, tCTS sites do not induce cleavage of the donor plasmid, but can support its transport into the nucleus by Cas9. However, the exact mechanisms for the increase in knock-in levels with both types of donor DNA modifications are unknown. Here, we evaluated the effect of these modifications on the knock-in efficiency of the MTC34 genetic construct encoding the HIV-1 fusion inhibitor, MT-C34 peptide, into the CXCR4 locus of the CEM/R5 T cell line. When full-length CTS sites were introduced into the donor plasmid DNA, the knock-in level increased twofold, regardless of the number of CTSs or their position relative to the donor sequence. Modifications of donor plasmids with tCTS sites did not affect knock-in levels. It was found that in vitro both types of sites were efficiently cleaved by Cas9. In order to study the mechanism of action of these modifications in detail, it is necessary to evaluate their cleavage in vitro and in vivo.

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About the authors

M. V. Shepelev

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences

Email: natalya.a.kruglova@yandex.ru
Russian Federation, Moscow, 119334

D. S. Komkov

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences; Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev

Email: natalya.a.kruglova@yandex.ru
Russian Federation, Moscow, 119334; Beer-Sheva, 8410501 Israel

D. S. Golubev

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences

Email: natalya.a.kruglova@yandex.ru
Russian Federation, Moscow, 119334

S. E. Borovikova

Institute of Gene Biology, Russian Academy of Sciences

Email: natalya.a.kruglova@yandex.ru
Russian Federation, Moscow, 119334

D. V. Mazurov

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences; Division of Infectious Diseases and International Medicine, Department of Medicine, University of Minnesota

Email: natalya.a.kruglova@yandex.ru
Russian Federation, Moscow, 119334; Minneapolis, 55455 USA

N. A. Kruglova

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences; Division of Infectious Diseases and International Medicine, Department of Medicine, University of Minnesota

Author for correspondence.
Email: natalya.a.kruglova@yandex.ru
Russian Federation, Moscow, 119334; Minneapolis, 55455 USA

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Supplementary files

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2. Fig. 1. Scheme of the donor construct for the MTC34 knockin in exon 2 of the CXCR4 locus (a) and its modifications (b, c). Designations: 5ʹ-HA – 5ʹ-homology arm; P2A – ribosome skipping signal; LS – leader sequence; GPI – sequence for modification of peptide with GPI anchor; pA ​​– polyadenylation signal; 3ʹ-HA – 3ʹ-homology arm. b – Schematic representation of plasmids carrying CTS at the 5ʹ- or 3ʹ-end of donor DNA (before 5ʹ-HA or after 3ʹ-HA, respectively); c – CTS and tCTS sequences. 0MM – there are no noncomplementary bases in the protospacer; 4MM, 6MM and 8MM – at the 5ʹ-end of the protospacer there are 4, 6 or 8 non-complementary bases, respectively. The PAM site is shown in bold, the protospacer is underlined, the non-complementary bases in its composition are underlined with a dotted line. Here: CTS is the Cas9 target sequence corresponding to the protospacer together with the PAM; tCTS is the Cas9 target sequence corresponding to the protospacer with non-complementary bases.

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3. Fig. 2. Effect of CTS modifications of donor DNA on the level of MTC34 knockin (a), CXCR4 knockout (b) and the knockin/knockout ratio (c) in CEM/R5 cells. The cells were electroporated with pcDNA3.3-hCas9, pKS-gRNA-X4ex2 plasmids and one of the variants of the donor plasmid pKS-don_MT-C34. On day 5, the level of CXCR4 knockout (KO) and MTC34 knockin (KI) was assessed by the expression of the corresponding CXCR4 protein and MT-C34 peptide on the cell surface using flow cytofluorimetry. KI/KO – knockin to knockout ratio; control (‒) – donor plasmid pKS-don_MT-C34 without modifications; (0, 4, 6, 8) MM – number of non-complementary bases in the CTS protospacer. Mean values ​​± standard deviation (SD) and individual results of three independent experiments are shown. *p < 0.01, **p < 0.0001.

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4. Fig. 3. Cleavage of CTS sites by Cas9 in vitro. Donor plasmids with CTS sites containing 0, 4, 6, or 8 noncomplementary bases, as well as an unmodified donor plasmid (‒CTS) were incubated in vitro with a complex of Cas9 and gRNA against CXCR4 (gRNA-X4) (a) or control (gRNA-Scr) (b), and the level of plasmid cleavage was assessed by electrophoresis in 1% TAE-agarose gel. Legend: r (relaxed) – relaxed form of plasmid, sc (super-coiled) – supercoiled form, d – fragment of ~1,400 bp with the donor sequence flanked by CTS or tCTS sites. Donor plasmids with CTS sites containing 0 (c) or 4 (d) noncomplementary bases were incubated in vitro with increasing amounts of RNP, and the level of plasmid cleavage was assessed by electrophoresis in 1% TAE-agarose gel. RNP/plasmid is the molar ratio of RNP to plasmid DNA. M are DNA length markers: 10,000, 8,000, 6,000, 5,000, 4,000, 3,500, 3,000, 2,500, 2,000, 1,500, 1,000, 750 bp (#SM1163; Thermo Fisher Scientific, USA).

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