CRISPR-LRS for mapping transgenes in the mouse genome

Microinjected transgenes, including bacterial artificial chromosomes (BACs), insert randomly in the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and the accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. Here, we introduce CRISPR-Cas9 long-read sequencing (CRISPR-LRS) to ascertain transgene integration locus and estimated copy number. This method revealed integration loci for both a BAC and Cre-driver line, and estimated the copy numbers for two other BAC mouse lines. CRISPR-LRS offers an easy approach to establish robust breeding practices and accurate phenotyping of most any transgenic mouse line.

Heterogeneity and genomic loci of ubiquitous Cre reporter transgenes in zebrafish

The most-common strategy for zebrafish Cre/lox-mediated lineage labeling experiments combines ubiquitously expressed, lox-based Switch reporter transgenes with tissue-specific Cre or 4-OH-Tamoxifen-inducible CreERT2 driver lines. Although numerous Cre driver lines have been produced, only a few broadly expressed Switch reporters exist in zebrafish and their generation by random transgene integration has been challenging due to position-effect sensitivity of the lox-flanked recombination cassettes. Here, we compare commonly used Switch reporter lines for their recombination efficiency and reporter expression pattern during zebrafish development. Using different experimental setups, we show that ubi:Switch and hsp70l:Switch outperform current generations of two additional Switch reporters due to favorable transgene integration sites. Our comparisons also document preferential Cre-dependent recombination of ubi:Switch and hsp70l:Switch in distinct zebrafish tissues at early developmental stages. To investigate what genomic features may influence Cre accessibility and lox recombination efficiency in highly functional Switch lines, we mapped these transgenes and charted chromatin dynamics at their integration sites. Our data documents the heterogeneity among lox-based Switch transgenes towards informing suitable transgene selection for lineage labeling experiments. Our work further proposes that ubi:Switch and hsp70l:Switch define genomic integration sites suitable for universal transgene or switch reporter knock-in in zebrafish.

Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells

Stable expression of transgenes is essential in both therapeutic and research applications. Traditionally, transgene integration has been accomplished via viral vectors in a semi-random fashion, but with inherent integration site biases linked to the type of virus used. The randomly integrated transgenes may undergo silencing and more concerningly, can also lead to dysregulation of endogenous genes. Gene dysregulation can lead to malignant transformation of cells and has unfortunately given rise to cases of leukaemia in gene therapy trials. Genomic safe harbour (GSH) loci have been proposed as safe sites for transgene integration. To date, a number of sites in the human genome have been used for directed integration; however none of these pass scrutiny as bona fide GSH. Here, we conducted a computational analysis to identify 25 putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

Transgene-free Genome Editing in Plants

Genome editing is widely used across plant species to generate and study the impact of functional mutations in crop improvement. However, transgene integration in plant genomes raises important legislative concerns regarding genetically modified organisms. Several strategies have been developed to remove or prevent the integration of gene editor constructs, which can be divided into three major categories: 1) elimination of transgenic sequences via genetic segregation; 2) transient editor expression from DNA vectors; and 3) DNA-independent editor delivery, including RNA or preassembled Cas9 protein-gRNA ribonucleoproteins (RNPs). Here, we summarize the main strategies employed to date and discuss the advantages and disadvantages of using these different tools. We hope that our work can provide important information concerning the value of alternative genome editing strategies to advance crop breeding.

Comprehensive Characterization of Transgene Integration in a Similar Intragenesis Rice Event Using Paired-end Whole-genome Sequencing Approach

Efficient, accurate molecular characterization of genetically modified (GM) organisms is challenging, especially for novel transgenic products of cisgenesis/intragenesis transferred with genes/elements of recipient species. Herein, GM rice event G281, involving transfer with native promoters and an RNA interference (RNAi) expression cassette in a process similar to intragenesis, was subjected to molecular characterization using paired-end whole genome sequencing (PE-WGS). The results showed that transgenes integrated at rice chromosome 3 locus 16,439,674 included a 36 bp deletion of rice genomic DNA, and the whole integration contained two copies of the complete transfer DNA (T-DNA) in a head-to-head arrangement. No unintended insertion or backbone sequence of the transformed plasmid were observed at the whole genome level. Molecular characterization of the G281 event will assist risk assessment and application for a commercial license. Additionally, the findings demonstrate the applicability of PE-WGS for molecular characterization of cisgenesis/intragenesis crops.

LIFE-Seq: A universal Large Integrated DNA Fragment Enrichment Sequencing strategy for transgene integration in genetically modified organisms

Molecular characterisation of genetically modified organisms (GMOs) yields basic information on exogenous DNA integration, including integration sites, entire inserted sequences and structures, flanking sequences and copy number, providing key data for biosafety assessment. However, there are few effective methods for deciphering transgene integration, especially for large DNA fragment integration with complex rearrangement, inversion, and tandem repeats. Herein, we developed a universal Large Integrated DNA Fragments Enrichment strategy combined with PacBio Sequencing (LIFE-Seq) for deciphering transgene integration in GMOs. Universal tilling DNA probes targeting transgenic elements and exogenous genes facilitate specific enrichment of large inserted DNA fragments associated with transgenes from plant genomes, followed by PacBio sequencing. LIFE-Seq were evaluated using six GM events and four crop species. Target DNA fragments averaging ∼6275 bp were enriched and sequenced, generating ∼26,352 high fidelity reads for each sample. Transgene integration structures were determined with high repeatability and sensitivity. Compared with whole-genome sequencing, LIFE-Seq achieved better data integrity and accuracy, greater universality, and lower cost, especially for transgenic crops with complex inserted DNA structures. LIFE-Seq could be applied in molecular characterisation of transgenic crops and animals, and complex DNA structure analysis in genetics research.

Transgenesis in the acoel Hofstenia miamia

The acoel worm Hofstenia miamia, which can replace tissue lost to injury via differentiation of a population of stem cells, has emerged as a new research organism for studying regeneration. To enhance the depth of mechanistic studies in this system, we devised a protocol for microinjection into embryonic cells that resulted in stable transgene integration into the genome and generated animals with tissue-specific fluorescent transgene expression in epidermis, gut, and muscle. We demonstrate that transgenic Hofstenia are amenable to the isolation of specific cell types, detailed investigations of regeneration, tracking of photoconverted molecules, and live imaging. Further, our stable transgenic lines revealed new insights into the biology of Hofstenia, unprecedented details of cell morphology and the organization of muscle as a cellular scaffold for other tissues. Our work positions Hofstenia as a powerful system with unparalleled tools for mechanistic investigations of development, whole-body regeneration, and stem cell biology.