Massively parallel DNA target capture using long adapter single stranded oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long‐Adapter Single‐Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase‐sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre‐LASSO probes) and a plasmid vector that supplies the linker sequence for the mature LASSO probe through Cre‐LoxP intramolecular recombination. This strategy generates high quality LASSO probes libraries (≈46% of correct probes). We performed NGS analysis of the post‐capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 Escherichia coli ORFs spanning from 400‐ to 5000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260‐fold. Here, we show that LASSO probes obtained in this manner, were able to capture full‐length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long‐read sequencing libraries and for massively multiplexed cloning of human sequences. Graphical and Lay Summary LASSO probes are highly specific in that they are essentially the DNA equivalent of an antibody in molecular design. The probe consists of a single strand DNA oligonucleotide with two variable arms that are complementary to a specific DNA target. These complementary regions on the LASSO probes are designed with the knowledge of a genome sequence, to have specificity and high affinity. In this work, we tested the capture robustness and efficiency of improved LASSO probes that were assembled with a novel methodology that relies on Cre‐recombination. With an exponentially growing list of new genomes from all kingdoms of life, this method can enable the rapid and inexpensive production of large collections of genes. Such genetic libraries can enable the discovery of novel biological functions that can be exploited in different fields such as drug development, biofermentation, and biofuel industry.