Find and cut-and-transfer (FiCAT) mammalian genome engineering

Pallarès-Masmitjà, Maria; Ivančić, Dimitrije; Mir-Pedrol, Júlia; Jaraba-Wallace, Jessica; Tagliani, Tommaso; Oliva, Baldomero; Rahmeh, Amal; Sánchez-Mejías, Avencia; Güell, Marc

doi:10.1038/s41467-021-27183-x

Cited by 34 publications

(27 citation statements)

References 33 publications

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“…To assess potential of INSERT-seq for analysing emerging precise gene delivery vectors, we determined integration sites in a Cas9-PiggyBac chimeric fusion previously reported [ 39 ]. We detected insertion at the Cas9 induced double-strand-break in the TRAC locus, which was nevertheless very low compared to total integration with on-target fraction being 0.016 and off-target fraction being 0.98 (Fig.…”

Section: Resultsmentioning

confidence: 99%

INSERT-seq enables high-resolution mapping of genomically integrated DNA using Nanopore sequencing

et al. 2022

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Comprehensive characterisation of genome engineering technologies is relevant for their development and safe use in human gene therapy. Short-read based methods can overlook insertion events in repetitive regions. We develop INSERT-seq, a method that combines targeted amplification of integrated DNA, UMI-based correction of PCR bias and Oxford Nanopore long-read sequencing for robust analysis of DNA integration. The experimental pipeline improves the number of mappable insertions at repetitive regions by 4.8–7.3% and larger repeats are processed with a computational peak calling pipeline. INSERT-seq is a simple, cheap and robust method to quantitatively characterise DNA integration in diverse ex vivo and in vivo samples.

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Section: Resultsmentioning

confidence: 99%

INSERT-seq enables high-resolution mapping of genomically integrated DNA using Nanopore sequencing

et al. 2022

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“…We first constructed a C-terminal fusion of S. pyogenes Cas9 and PDRM9 and tested recombination efficiency with a split GFP reporter system previously described (Fig. 1A; (Pallarès-Masmitjà et al 2021)), both as episomal DNA (Fig. 1B) and in cells containing the split GFP reporter in the genome (Fig.…”

Section: Resultsmentioning

confidence: 99%

Synergic homology directed recombination by PRDM9 meiotic factor

Sanvicente-García

Gonzalez-Bermudez

Batlle

et al. 2022

Preprint

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Genome editing requires precision to broadly move on to industrial and clinical applications. For this reason, homologous directed repair (HDR) is one of the preferred methods for small edits, other than knock-outs. However, HDR has low efficiency. Current investigations to enhance HDR have mainly gone in the direction of finding non-homologous end joining (NHEJ) inhibitors. NHEJ is crucial for cellular integrity, then the inhibition of this pathway is detrimental for the correct survival of living entities. In other studies, a second opportunity is given to HDR by targeting the byproducts of NHEJ, using an extra gRNA. In this study, we propose the use of a meiotic factor, PRDM9, to directly enhance homology recombination. Through the exploration of combinatorial factors and donor design, we have established an optimized protocol for HDR. PRDM9-Cas9 fusion combined with CtIP improves HDR/NHEJ ratio. In addition, we have validated this combinatorial approach for small edits through a traffic light reporter system, as well as for longer edits with a split-GFP reporter system.

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“…To assess potential of INSERT-seq for analysing emerging precise gene delivery vectors, we determined integration sites in a Cas9-PiggyBac chimeric fusion previously reported [39]. We detected insertion at the Cas9 induced double-strand-break in the TRAC locus, which was nevertheless very low compared to total integration with on-target fraction being 0.016 and offtarget fraction being 0.98 (Fig.…”

Section: Assessment Of a Cas9-piggybac Chimeric Transposasementioning

confidence: 88%

INSERT-seq enables high resolution mapping of genomically integrated DNA using nanopore sequencing

Ivančić

Mir-Pedrol

Jaraba-Wallace

et al. 2022

Preprint

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Comprehensive characterization of genome engineering with viral vectors, transposons, CRISPR/Cas mediated DNA integration and other DNA editors remains relevant for their development and safe use in human gene therapy. Currently, described methods for measuring DNA integration in edited cells rely on short read based technologies. Due to the repetitive nature of the human genome, short read based methods can potentially overlook insertion events in repetitive regions. We modelled the impact of read length in resolving insertion sites, which suggested a significant drop in insertion site detection with shorter read length. Based on that, we developed a method that combines targeted amplification of integrated DNA, UMI-based correction of PCR bias and Oxford Nanopore long-read sequencing for robust analysis of DNA integration in a genome. This method, called INSERT-seq, is capable of detecting events occurring at a frequency of up to 0.1%. INSERT-seq presents a complete handling of all insertions independently of repeat size. The experimental pipeline improves the number mappable insertions at repetitive regions by 7.3% and repeats larger than the long read sequencing size are processed computationally to perform a peak calling in a repeat database. INSERT-seq is a simple, cheap and robust method to quantitatively characterise DNA integration in diverse ex-vivo and in-vivo samples.

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Find and cut-and-transfer (FiCAT) mammalian genome engineering

Cited by 34 publications

References 33 publications

INSERT-seq enables high-resolution mapping of genomically integrated DNA using Nanopore sequencing

INSERT-seq enables high-resolution mapping of genomically integrated DNA using Nanopore sequencing

Synergic homology directed recombination by PRDM9 meiotic factor

INSERT-seq enables high resolution mapping of genomically integrated DNA using nanopore sequencing

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