Justin Lim

A prospective randomized controlled trial comparing open and percutaneous repair of closed ruptured Achilles tendons was performed over a period of 30 months. Sixty-six patients from seven district general hospitals were entered into the study with 33 patients randomized into each group. A modification of the technique described by Ma and Griffith was used in the percutaneous group and a Kessler suture supplemented with interrupted sutures was used in the open group. Patients were followed up for a minimum of six months. The mean age was 38.5 years (26 to 53 years). Forty patients were male and 26 female. After the rupturing event but prior to surgery, it was noted that seven patients had paresthesia in the territory of the sural nerve. The mean duration of immobilization was 12.4 weeks (10 to 14). The complications in the open group included seven wound infections (21%), two adhesions (6%) and two cases of re-rupture (6%). In the percutaneous group there were three cases of wound puckering (9%), one re-rupture (3%) and one case with persistent paresthesia in the sural nerve territory (3%). The difference in infective wound complications between the two groups was statistically significant (Fisher's exact test P = 0.01). Percutaneous repair is advocated on the basis of the low rate of complications and improved cosmetic appearance.

Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic homolog of prokaryotic TnpB proteins, have been detected in genomes of eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. Here, we characterize Fanzors as RNA-programmable DNA endonucleases, using biochemical and cellular evidence. We found diverse Fanzors that frequently associate with various eukaryotic transposases. Reconstruction of Fanzors evolution revealed multiple radiations of RuvC-containing TnpB homologs in eukaryotes. Fanzor genes captured introns and proteins acquired nuclear localization signals, indicating extensive, long-term adaptation to functioning in eukaryotic cells. Fanzor nucleases contain a rearranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that Fanzors can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.

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 Summary A 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.