Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing.

Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.

Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference.

TadA-derived cytosine base editors (TadCBEs) enable programmable C•G-to-T•A editing while retaining the small size, high on-target activity, and low off-target activity of TadA deaminases. Existing TadCBEs, however, exhibit residual A•T-to-G•C editing at certain positions and lower editing efficiencies at some sequence contexts and with non-SpCas9 targeting domains. To address these limitations, we use phage-assisted evolution to evolve CBE6s from a TadA-mediated dual cytosine and adenine base editor, discovering mutations at N46 and Y73 in TadA that prevent A•T-to-G•C editing and improve C•G-to-T•A editing with expanded sequence-context compatibility, respectively.

Evaluation of appendectomy as a potential risk factor for immune checkpoint inhibitor-associated enterocolitis.

The relationship between appendectomy and immune checkpoint inhibitor (ICI) enterocolitis was explored. Patients who began ICIs between July 2010 and September 2020 (n = 10,907) were included. The exposure group included patients with evidence of appendectomy prior to ICIs in operative notes (n = 380).

The evolution of pituitary cysts in growth hormone-treated children.

Objectives: We have previously shown that pituitary cysts may affect growth hormone secretion. This study sought to determine cyst evolution during growth hormone treatment in children.

Methods: Forty-nine patients with short stature, a pituitary cyst, and at least two brain MRI scans were included.

Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination.