A versatile platform for locus-scale genome rewriting and verification

Abstract

Routine rewriting of loci associated with human traits and diseases would facilitate their functional analysis. However, existing DNA integration approaches are limited in terms of scalability and portability across genomic loci and cellular contexts. We describe Big-IN, a versatile platform for targeted integration of large DNAs into mammalian cells. CRISPR/Cas9-mediated targeting of a landing pad enables subsequent recombinase-mediated delivery of variant payloads and efficient positive/negative selection for correct clones in mammalian stem cells. We demonstrate integration of constructs up to 143 kb, and an approach for one-step scarless delivery. We developed a staged pipeline combining PCR genotyping and targeted capture sequencing for economical and comprehensive verification of engineered stem cells. Our approach should enable combinatorial interrogation of genomic functional elements and systematic locus-scale analysis of genome function.

Synthetic genomic reconstitution reveals principles of mammalianHoxcluster regulation

“…We used capture sequencing to validate that SynHoxA mESC clones contained the entire assemblon at Hprt. Nick-translation of our BACs yielded biotinylated probes specific to the synthetic ectopic locus, that were used to enrich for DNA from the assemblon, facilitating high-coverage sequencing at low cost (52). In all cases, we eliminated clones that contained deletions or rearrangements of SynHoxA sequence.…”

“…(Figure 2C). mESC clones that passed the sequencing pipeline were further validated for precise on-target single-copy integration and absence of any off-target integration by analyzing reads spanning junctions between the synthetic construct and mouse genome (52). Importantly, we only detected reads spanning the expected junctions at the landing pad and no off-target integrations (Figure 2B).…”

“…mESC gDNA was purified from 1-5 million cells using the QIAamp DNA mini kit (Qiagen 51306) according to the manufacturer's protocol. Illumina libraries were prepared as previously described (52). 1 µg of DNA was sheared to ∼500 to 900 bp in a 96-well microplate using the Covaris LE220 (450 W, 10% Duty Factor, 200 cycles per burst, and 90-s treatment time).…”

“…Targeted sequencing using in-solution hybridization capture (Capture-seq) was performed on SynHoxA-delivered mESCs as previously described (52). Biotinylated baits for capture sequencing were prepared from assemblon BACs using nick translation.…”

“…The custom genome sequences, annotations and bowtie2 references can be found at: https://genome.med.nyu.edu/public/boekelab/SynHox_genomes/ Parental mESCs WGS analysis WGS data were analyzed as previously described (52). Reads were demultiplexed with Illumina bcl2fastq v2.20 requiring a perfect match to indexing BC sequences.…”