Influence of homology arm length and structure on the efficiency of long transgene integration into a cleavage site induced by SpCas9 or AsCpf1

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Abstract

One of the promising new approaches to HIV infection treatment is the CRISPR/Cas-mediated knockout of the CCR5 receptor gene followed by the integration of an anti-HIV gene into the break site. Numerous studies have focused on the knockout of the CCR5 gene; however, the efficiency of subsequent targeted integration of long fragments remains poorly studied. To evaluate the efficiency of this approach, we used HT1080 cells and investigated the integration of a cassette expressing the EGFP gene into the CCR5 locus using two different nucleases (SpCas9 and AsCpf1) and various donor DNA constructs delivered by recombinant adeno-associated viral vectors (rAAV). For each nuclease, we designed five variants of donor DNA differing in the length (ranging from 150 to 1000 bp) or structure of the homology arms. The efficiency of transgene integration with 150 bp homology arms was the lowest for both nucleases and significantly differed from constructs with longer homology arms. Furthermore, it was shown that the presence of nuclease cleavage sites in the donor DNA flanking the cassette with homology arms did not affect the efficiency of transgene integration during AAV delivery. We demonstrated that the AsCpf1 nuclease provided higher efficiency of EGFP transgene integration than SpCas9, despite lower efficiency of CCR5 knockout. The maximum percentage of cells with integrated transgene was achieved using the AsCpf1 nuclease and an expression cassette with 600 bp homology arms, reaching 59 ± 6%.

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About the authors

J. А. Taran

Centre for Strategic Planning, of the Federal Medical and Biological Agency

Author for correspondence.
Email: taran.julia01@gmail.com
Russian Federation, Moscow

R. R. Mintaev

Centre for Strategic Planning, of the Federal Medical and Biological Agency

Email: taran.julia01@gmail.com
Russian Federation, Moscow

D. V. Glazkova

Centre for Strategic Planning, of the Federal Medical and Biological Agency

Email: taran.julia01@gmail.com
Russian Federation, Moscow

B. V. Belugin

Centre for Strategic Planning, of the Federal Medical and Biological Agency

Email: taran.julia01@gmail.com
Russian Federation, Moscow

E. V. Bogoslovskaya

Centre for Strategic Planning, of the Federal Medical and Biological Agency

Email: taran.julia01@gmail.com
Russian Federation, Moscow

G. A. Shipulin

Centre for Strategic Planning, of the Federal Medical and Biological Agency

Email: taran.julia01@gmail.com
Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. Knockout of the CCR5-EGFP gene in the HT1080-CCR5-EGFP cell line. a – Schematic diagram of the experiment to evaluate the knockout efficiency with SpCas9/L96 or AsCpf1/cL652 RNP complexes. b – Efficiency of CCR5-EGFP gene knockout with SpCas9/L96 or AsCpf1/cL652 RNP complexes (four replicates for each RNP complex). Mean values ​​and standard deviations are shown. Cells not subjected to electroporation were used as a negative control (K-).

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3. Fig. 2. Characteristics of AAV vectors encoding donor DNA. a – Scheme of rAAV vectors. CRISRP site – RNP cleavage site, ITR – inverted terminal repeats. b – Structure of the sequence integrated into the target region. EGFP – green fluorescent protein gene, pPGK – promoter, WPRE – post-transcriptional regulatory element, SV40 PA – transcription termination signal. c – Scheme of genomic DNA cleavage by AsCpf1 nuclease and insertion of donor DNA with the PAM mutation. d – Infectious titer of rAAV vectors. Average rAAV concentrations and standard deviations are shown.

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4. Fig. 3. Evaluation of the efficiency of EGFP transgene integration. a – Integration efficiency upon electroporation of HT1080 cells with SpCas9/L96 or AsCpf1/cL652 RNP complexes followed by transduction with one of 10 rAAVs. b – Transduction efficiency calculated from the proportion of the EGFP+ cell population 48 h after rAAV transduction without prior electroporation. The mean arithmetic values ​​and standard deviation are shown. “K–” – non-transduced HT1080 cells. c – Frequency of EGFP- transgene integration after cell electroporation with SpCas9/L96 or AsCpf1/cL652 RNPs and rAAV transduction with donor DNA. The characteristics of the donor DNA are given in the table below the diagram: the length of one homology arm is indicated in the Homology arm length line, the presence of CRISPR sites is indicated by the “+” sign. Mean values ​​and standard deviations are presented. Statistical significance of differences was assessed using one-way analysis of variance (ANOVA), followed by Tukey's test for paired comparisons between groups. Significance of differences between groups: *p-value 0.01 < 0.05, **p-value 0.001 < 0.01, ***p-value 0.0001 < 0.001, ****p-value < 0.0001.

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