Journal: bioRxiv

Article Title: L1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements

doi: 10.1101/2025.09.17.676864

Figure Lengend Snippet: A. A working model for DNA breakage resulting from L1 retrotranspostion. L1 retrotransposition occurs via target-primed reverse transcription. L1 ORF2p can nick genomic DNA at an EN target sequence (3’-AA/TTTT-5’) to liberate a polyT sequence that complementary bind the L1 RNA polyA sequence to form a primer-template structure. L1 ORF2p can then polymerize from the free 3’OH and use the L1 RNA as a template to generate de novo L1 cDNA that can be processed into a double-stranded insertion in the genome. Alternatively, DNA breakage can occur during the resolution of the L1 cDNA intermediate into an insertion generating recombinogenic DNA break ends. B. A schematic of the classic L1-GFP reporter assay. A pCEP4-Puro plasmid constitutively expresses a native human L1 sequence (L1RP) expressing ORF1p (RNA binding protein) and ORF2p (endonuclease and reverse transcriptase) containing a GFP reporter in the 3’ UTR that marks de novo L1 insertions. The GFP cassette is in the anti-sense direction relative to L1 sequence and is interrupted by an intron in the sense direction. The continuous GFP sequence is generated when the L1 transgene is transcribed and spliced but cannot be expressed and translated until the cassette is retrotransposed. Accordingly, GFP expression marks cells with de novo L1 insertions. C. Shown is the retrotransposition frequency of WT, reverse transcriptase mutant (RTmut, D702Y) RTmut or endonuclease mutant (ENmut, H230A) L1 measured by the L1-GFP reporter assay in U2OS cells. n = 6. P-value <0.0001, each compared to RTmut using one-way ANOVA with Dunnett’s test. D. Shown is a schematic of a new GFP reporter assay modeling the recombination between a L1-cDNA intermediate and a chromosomal break to generate a chromosomal rearrangement. Left: A pCEP4-Puro plasmid contains a modified version of the classic L1-GFP reporter construct, where the 3’ UTR contains an anti-sense 5’ GFP fragment (5’GFp) that is interrupted by a sense intron (L1-5’ GFp plasmid). Transcription, splicing and retrotransposition of the L1 RNA results in a canonical insertion containing an incomplete GFP cassette and does not induce GFP expression. Nonetheless, retrotransposition of the L1 RNA generates L1 insertion intermediates containing an 5’GFP fragment with the potential to be resolved in non-canonical events. Right: A 3’ GFP fragment containing 150 bp homology to the 5’ GFP fragment from L1-5’ GFp plasmid (3’ gFP 150bp Hom) was introduced chromosomally in U2OS cells using the FRT/ Flp recombinase system (150bpHom-3’ gFP U2OS cells). A chromosomal break can be induced by the CRISRP/Cas9 system at the edge of the 150bp homology of the 3’gFP 150bp Hom sequence (Cas9/sg150bp). Hence, L1 cDNA insertion intermediates containing a 5’ GFP fragment can recombine with a broken chromosome containing a 3’ GFP fragment forming a chromosomal rearrangement and restoring GFP expression. E. Shown are representative flow cytometry plots of 150bpHom-3’ gFP U2OS cells transfected with WT or RTmut L1-5’ GFp reporter plasmid, and Cas9/ sgCTRL or Cas9/ sg150bp expressing plasmid. GFP expression is only induced with transfection of the WT L1-5’ GFp reporter plasmid and the Cas9/150bp expressing plasmid. F. Shown is the GFP frequency induced in U2OS FRT 3’ gFP (150bp Hom) cells transfected with WT, RTmut or ENmut L1-5’ GFp reporter plasmid, and Cas9/sgCTRL or Cas9/sg150bp expressing plasmid. n = 6. P-value calculated using unpaired two-tailed Student’s t tests with the Holm-Sidak correction for multiple comparisons. G. Shown is the GFP frequency induced in HEK293 150bpHom-3’ gFP cells transfected with WT, RTmut or ENmut L1-5’ GFp reporter plasmid, and Cas9/sgCTRL or Cas9/sg150bp expressing plasmid. n = 3. P-value calculated using unpaired two-tailed Student’s t tests with the Holm-Sidak correction for multiple comparisons.

Article Snippet: To generate the L1-5’GFp reporter plasmid (pCMD66A) and the L1-3’gFP reporter plasmids (pCMD172A and pCMD173AA), GFP fragments were synthesized via Genewiz or Twist Bioscience to replace the canonical GFP-AI reporter cassette in the 3’ UTR of the L1-GFP reporter plasmid (pCMD66B).

Techniques: Reverse Transcription, Sequencing, Reporter Assay, Plasmid Preparation, Expressing, RNA Binding Assay, Generated, Mutagenesis, Modification, Construct, Flow Cytometry, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: L1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements

doi: 10.1101/2025.09.17.676864

Figure Lengend Snippet: A. GFP+ cells harbor the predicted recombination product. Shown are PCR amplification products from untransfected cells and sorted GFP+ cells derived from U2OS 150bpHom-3’ gFP cells transfected with WT L1 5’ GFp plasmid and Cas9/sg150bp expressing plasmid using primers hPGKFw (L1 5’GFp plasmid) and GFPrev (3’ gFP 150bp Hom). Amplification of GAPDH locus was used as a control. B. Schematic of the Cas9 targeted nanopore sequencing strategy to map the upstream and the downstream sequences of the GFP loci of sorted GFP+ cells. Genomic DNA was isolated from sorted GFP+ cells derived from U2OS FRT 3’ gFP (150bp Hom) cells after transfection with WT L1 5’ GFp plasmid and Cas9/sg150bp expressing plasmid. Genomic DNA was first mechanically fragmented to 30 kb length and dephosphorylated. Dephosphorylated genomic DNA was then incubated with Cas9 ribonucleoproteins using sgRNAs targeted to the 3’gFP sequence at the FRT locus. Cleaved genomic DNA ends are then ligated with Oxford Nanopore Technologies (ONT) sequencing adapters. These DNA fragments were sequenced on a MinION flow cell. Long reads were first mapped to a GFP reference to select GFP-containing long reads for subsequent mapping to the human genome (T2T) to determine their genomic integration site. C. Shown are several ONT long reads containing the 3’ GFP fragment that were mapped to the human genome (T2T) revealing the chromosomal FRT locus in U2OS cells is in chromosome 11 (7784 +). D. Shown are ONT long reads containing the 5’ GFP fragment and the recombination junction mapping to chromosome 14 (101156465 +) and showing evidence of L1 retrotransposition at the genomic site of integration: inserted at (5’-GGTT/CA-3’) and containing an polyA sequence. E. A circos plot showing the genomic locations of L1 insertion intermediates that recombined with the Cas9-mediated DNA double-strand break generated at edge of the 150 bp homology of the 3’gFP (150 bp Hom) locus in chromosome 11 to restore GFP expression via a chromosomal translocation. Each translocation is represented by an arch originating from the p arm of chromosome 11 and terminating at the location of each L1 insertion intermediate.

Article Snippet: To generate the L1-5’GFp reporter plasmid (pCMD66A) and the L1-3’gFP reporter plasmids (pCMD172A and pCMD173AA), GFP fragments were synthesized via Genewiz or Twist Bioscience to replace the canonical GFP-AI reporter cassette in the 3’ UTR of the L1-GFP reporter plasmid (pCMD66B).

Techniques: Amplification, Derivative Assay, Transfection, Plasmid Preparation, Expressing, Control, Nanopore Sequencing, Isolation, Incubation, Sequencing, Generated, Translocation Assay

Journal: bioRxiv

Article Title: L1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements

doi: 10.1101/2025.09.17.676864

Figure Lengend Snippet: A . Shown is a schematic of GFP recombination reporters assaying the influence of the length of homologous sequences between a L1-cDNA intermediate and a chromosomal break for the formation of chromosomal rearrangements. Left: The L1-5’ GFp plasmid is transfected into cells to generate L1 insertion intermediates containing a 5’GFP fragment with the potential to be resolved in non-canonical events such as rearrangements. Right: 3’ GFP fragments containing varying degree of homology to the 5’ GFP fragment from L1-5’ GFp plasmid were introduced chromosomally in U2OS cells using the FRT/ Flp recombinase system: 3’gFP 150bp Hom, 3’gFP 50bp Hom, and 3’gFP 0bp Hom. Each of these 3’GFP fragments was engineered to contain a specific sgRNA target sequence to induce a Cas9-mediated chromosomal break at the edge of the homology. Hence, we can express the L1-5’GFp plasmid and the respective Cas9/sgRNA plasmid in each 3’gFP reporter cell line to monitor the influence of the homology between L1 cDNA insertion intermediates containing a 5’ GFP fragment and a broken chromosome containing a 3’ GFP fragment on the formation of a chromosomal rearrangement by assaying GFP expression. B. Shown is the GFP frequency induced in each U2OS 3’gFP reporter cell line transfected with WT, RTmut or ENmut L1-5’ GFp reporter plasmid, and the respective Cas9/sgRNA expressing plasmid or the control Cas9/sgCTRL expressing plasmid. n = 6 or 3. P-value calculated by comparing WT and ENmut versus RTmut in each condition using a two-way ANOVA with the Dunnett correction for multiple comparisons.

Article Snippet: To generate the L1-5’GFp reporter plasmid (pCMD66A) and the L1-3’gFP reporter plasmids (pCMD172A and pCMD173AA), GFP fragments were synthesized via Genewiz or Twist Bioscience to replace the canonical GFP-AI reporter cassette in the 3’ UTR of the L1-GFP reporter plasmid (pCMD66B).

Techniques: Plasmid Preparation, Transfection, Sequencing, Expressing, Control

Journal: bioRxiv

Article Title: L1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements

doi: 10.1101/2025.09.17.676864

Figure Lengend Snippet: A. A working model for how two independent L1 retrotransposition events on distinct chromosomes can cause a chromosomal translocation. B. Shown is a novel GFP-based reporter assay to ascertain recombination between two independent L1 retrotransposition intermediates generating a chromosomal rearrangement. Left: The L1-5’ GFp plasmid is transfected into cells to generate L1 insertion intermediates containing a 5’GFP fragment with the potential to be resolved in non-canonical events such as rearrangements. Right: A pCEP4-Puro plasmid contains a modified version of the classic L1-GFP reporter construct, where the 3’ UTR contains a sense 3’ GFP fragment (3’gFP) that is interrupted by a sense intron (L1-3’ gFP plasmid). Transcription, splicing and retrotransposition of the L1 RNA results in a canonical insertion encompassing an incomplete GFP cassette. Both reporter constructs the L1-5’ GFp plasmid and L1-3’ gFP plasmid can be expressed in cells to generate L1 reverse transcribed cDNA intermediates that share homologous sequences within the GFP fragment which can recombine to generate a GFP-expressing rearrangement. C. Shown are representative flow cytometry plots of parental U2OS cells transfected with EV, WT or RTmut L1-5’ GFp reporter plasmid, and the WT L1-3’gFP plasmid, which 3’GFP fragment contains 150bp of homology to the 5’ GFP fragment to L1-5’GFp plasmid. GFP expression is only induced with transfection of the WT L1-5’ GFp reporter plasmid and the WT L1-3’ gFP reporter plasmid. D. Shown are the frequencies of GFP+ cells induced in parental U2OS cells transfected with EV, WT, RTmut or ENmut L1 5’GFp plasmid with WT L1-3’gFP (150 bp Hom) or WT L1-3’gFP (50 bp Hom). n=5 P-value calculated using one-way ANOVA test comparing to WT L1 5’ GFp with Hom-Sidak correction for multiple comparisons.

Article Snippet: To generate the L1-5’GFp reporter plasmid (pCMD66A) and the L1-3’gFP reporter plasmids (pCMD172A and pCMD173AA), GFP fragments were synthesized via Genewiz or Twist Bioscience to replace the canonical GFP-AI reporter cassette in the 3’ UTR of the L1-GFP reporter plasmid (pCMD66B).

Techniques: Translocation Assay, Reporter Assay, Plasmid Preparation, Transfection, Modification, Construct, Reverse Transcription, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: L1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements

doi: 10.1101/2025.09.17.676864

Figure Lengend Snippet: A. Sorted GFP+ cells harbor the predicted GFP recombination product. Shown are PCR amplification products from sorted GFP+ cells derived from parental U2OS cells transfected with L1-5’ GFp plasmid and with L1-3’ gFP plasmid, as well as untransfected cells, using primers hPGKFw (L1-5’GFp plasmid) and bGHRev (L1-3’gFP plasmid). Amplification of the GAPDH locus was used as a control. B. Representative Nanopore long reads from GFP+ cells that align to the predicted GFP recombination sequence. Three types of aligned reads emerge: Type 1) Rearrangements include reads that span the complete recombination reference sequence and are flanked by L1 TPRT evidence such as inserted polyA sequences at the genomic junctions. Type 2) L1 canonical insertions by L1-5’GFp reporter plasmid include reads that only map to the 5’ side of the recombination reference sequence. Type 3) L1 canonical insertions by L1-3’GFp reporter plasmid include reads that only map to the 3’ side of the recombination reference sequence. C. Shown are schematics of the chromosomal rearrangements inferred by reading through the recombined GFP cassette: two segmental deletion rearrangements, a translocation predicted to generate a stable derivative chromosome, and a translocation predicted to form an unstable dicentric chromosome. Shown are the locations of the L1 retrotransposition events in each chromosome and the target sequences that resembled the EN cleavage motif providing evidence of L1 TPRT activity. D. Landscape of L1 insertions in GFP+ cells induced by the L1-5’ GFp reporter plasmid and the L1-3’ gFP reporter plasmid. Shown are the number of L1 insertions, including full-length insertions, 5’ truncations and 5’ inversions. Also, shown are the length of target site duplications of L1 insertions and the length of L1 insertions generated by L1-5’GFp and L1-3’gFP. Finally, also shown are the logo sequence motif of the genomic sequence where L1 insertions were integrated at the 3’ end, reconstituting the known EN cleavage motif sequence: 5’-TT/AAAA-3’.

Article Snippet: To generate the L1-5’GFp reporter plasmid (pCMD66A) and the L1-3’gFP reporter plasmids (pCMD172A and pCMD173AA), GFP fragments were synthesized via Genewiz or Twist Bioscience to replace the canonical GFP-AI reporter cassette in the 3’ UTR of the L1-GFP reporter plasmid (pCMD66B).

Techniques: Amplification, Derivative Assay, Transfection, Plasmid Preparation, Control, Sequencing, Translocation Assay, Activity Assay, Generated

Journal: bioRxiv

Article Title: L1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements

doi: 10.1101/2025.09.17.676864

Figure Lengend Snippet: A. BRCA1 and CtIP mediate homologous recombination. Shown is a schematic of the homology-directed repair (HDR) DR-GFP reporter assay chromosomally integrated in U2OS cells. Also, shown are the frequency of GFP+ cells after induction of the DR-GFP reporter assay in cells treated with siCTRL, siBRCA1 or siCtIP. n=3. P-value calculated using one-way ANOVA with Dunnett’s test for multiple corrections. B. BRCA1 and CtIP suppress L1 retrotransposition. Shown is a schematic of the Tet-On ORFeusHs-GFP reporter integrated on HeLa cells, which upon doxycycline treatment the L1-GFP reporter transcript is expressed to generate de novo L1 insertions marked by GFP expression. Shown is the frequency of L1 retrotransposition in cells treated with siCTRL, siBRCA1 or siCtIP. n=3. P-value calculated using one-way ANOVA with Dunnett’s test for multiple corrections. C. BRCA1 and CtIP mediates recombination between L1 cDNA intermediates and chromosomal breaks with homologous sequences for genome rearrangements. Shown are the frequencies of GFP+ cells in 150bpHom-3’gFP U2OS cells treated with siCTRL, siBRCA or siCtIP and co-transfected with WT L1-5’GFp reporter plasmid and the Cas9/sg150bp plasmid. n=7 for siCTRL and n=5 for siBRCA1 and siCtIP. P-value calculated using one-way ANOVA with Dunnett’s test for multiple corrections. D. BRCA1, but not CtIP, appears to promote recombination between L1 cDNA intermediates that share homologous sequences. Parental U2OS cells were treated with siCTRL, siBRCA, or siCtIP and co-transfected with the L1-5’GFp plasmid and the L1-3’gFP reporter plasmid. n=3. P-value calculated using one-way ANOVA with Dunnett’s test for multiple corrections.

Article Snippet: To generate the L1-5’GFp reporter plasmid (pCMD66A) and the L1-3’gFP reporter plasmids (pCMD172A and pCMD173AA), GFP fragments were synthesized via Genewiz or Twist Bioscience to replace the canonical GFP-AI reporter cassette in the 3’ UTR of the L1-GFP reporter plasmid (pCMD66B).

Techniques: Homologous Recombination, Reporter Assay, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Genetic Toolbox for Photorhabdus and Xenorhabdus : pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling

doi: 10.1101/2024.01.07.574529

Figure Lengend Snippet: A ) Schematic representation of spacer sequence and homology regions selection; schematic cloning of synthetic dsDNA fragments harboring the repair templates and spacer sequence and the vanillic acid inducible promoter sequence (P van ) into BsaI digested pAR20; schematic representation of the genome editing approach mediated by pAR20; the sequence between the homology arms is replaced by the promoter sequence; detailed genotype of pAR20 can be found in Table S1. HR-L/R = homologues region L (left) or R (right), dsDNA = synthetic double strand DNA, DR = FnCpf1 specific direct repeat, red or green diamond = spacer sequence A or B, crossing dashed lines representing crossover event after CRISPR induced double strand break. B ) Phenotypic comparison of induced and non-induced mutants; DMSO extract of induced mutant; biosynthesis of 2. Figure was taken from BioRender.com and modified.

Article Snippet: Target sequence and homology repair templates, ordered as synthetic dsDNA gene fragments, and were obtained from Twist Bioscience and Integrated DNA Technologies, respectively.

Techniques: Sequencing, Selection, Cloning, CRISPR, Comparison, Mutagenesis, Modification

Journal: Journal of Translational Medicine

Article Title: GeneBits: ultra-sensitive tumour-informed ctDNA monitoring of treatment response and relapse in cancer patients

doi: 10.1186/s12967-025-06993-3

Figure Lengend Snippet: Coverage and error metrics after UMI-assisted consensus calling. Deduplicated read depths and error rates for different commercial cell-free DNA reference standards (Twist Std v2, Horizon Std, OncoSpan Std), tested with the IDT and Twist library preparation and hybridization capture workflows. (A, B) Error rates when using only consensus reads formed by 1x, 2x, 3x, or ≥ 4x UMI families (PCR duplicates) using the IDT (A) or Twist (B) workflows. (C, D) Depth of coverage when using only consensus reads formed by ≥ 1x, ≥ 2x, ≥ 3x, or ≥ 4x UMI-families (PCR duplicates) using the IDT (C) or Twist (D) workflows. (E) Comparing error rates between three workflows using only duplex-consensus reads formed by ≥ 4x UMI-families. (F) Comparing the limit of detection (LOD) between three workflows using only duplex-consensus reads formed by ≥ 4x UMI-families. (Lower is better for error rates and LOD, higher is better for depth of coverage)

Article Snippet: The Twist Library Preparation MF Kit (Twist Bioscience, cat. # 104176) referred to as “Twist” or “Twist (old)” was used for most analyses.

Techniques: Hybridization