Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

Abstract

Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as ∼36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy.One Sentence SummaryA new technology combining CRISPR-mediated genome editing and site-specific integrases enables efficient programmable gene integration at any targeted genomic locus without double-strand DNA breaks, leading to broad applications in basic science research, cell engineering, and gene therapy.

Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair

“…These results agree with the known substrate specificities of MutSα and MutSβ [16][17][18][19][20] . Given that one of the most promising applications of PE includes insertion, deletion or replacement of large sequences of DNA, for example to tag endogenous loci within the genome 10,41 , how these lesions are processed and how their insertion efficiency can be augmented, remains of substantial future interest.…”

Predicting efficiency of writing short sequences into the genome using prime editing

“…Small insertions can encode protein tags for purification and visualization, or allow manipulation of protein localization, half-life, and interaction profiles to control their function. Integrating sequences for transcription factor binding sites and splicing modulators provides control over gene expression, while introducing structural elements or recombinase sites can change DNA conformation and provide a substrate for large-scale engineering 1,2 . For therapeutic opportunities, over 16,000 small deletion variants have been causally linked to disease 3,4 , and could in principle be restored by inserting the missing sequence 5,6 .…”

Large-scale discovery of recombinases for integrating DNA into the human genome

“…In addition, LSRs or domains of LSRs could potentially be combined with programmable CRISPR targeting system to generate an approach that combines both site specificity and host-independent DNA recombination. While this manuscript was in preparation, such an approach was described with prime editors (Ioannidi et al 2021;Anzalone et al 2021). An exciting future direction would be to combine prime editors with the efficient and specific landing pad LSRs described here to most efficiently integrate large cargos into programmed locations.…”