Seven technologies to watch in 2023

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG capable of targeting an expanded set of NGN PAMs, and further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN>NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

Increases in (14)C concentrations in tree rings could be attributed to cosmic-ray events, as have increases in (10)Be and nitrate in ice cores. The record of the past 3,000 years in the IntCal09 data set, which is a time series at 5-year intervals describing the (14)C content of trees over a period of approximately 10,000 years, shows three periods during which (14)C increased at a rate greater than 3‰ over 10 years. Two of these periods have been measured at high time resolution, but neither showed increases on a timescale of about 1 year (refs 11 and 12). Here we report (14)C measurements in annual rings of Japanese cedar trees from ad 750 to ad 820 (the remaining period), with 1- and 2-year resolution. We find a rapid increase of about 12‰ in the (14)C content from ad 774 to 775, which is about 20 times larger than the change attributed to ordinary solar modulation. When averaged over 10 years, the data are consistent with the decadal IntCal (14)C data from North American and European trees. We argue that neither a solar flare nor a local supernova is likely to have been responsible.

A proteomics tool capable of identifying single proteins would be important for cell biology research and applications. Here, we demonstrate a nanopore-based single-molecule peptide reader sensitive to single–amino acid substitutions within individual peptides. A DNA-peptide conjugate was pulled through the biological nanopore MspA by the DNA helicase Hel308. Reading the ion current signal through the nanopore enabled discrimination of single–amino acid substitutions in single reads. Molecular dynamics simulations showed these signals to result from size exclusion and pore binding. We also demonstrate the capability to “rewind” peptide reads, obtaining numerous independent reads of the same molecule, yielding an error rate of <10−6 in single amino acid variant identification. These proof-of-concept experiments constitute a promising basis for the development of a single-molecule protein fingerprinting and analysis technology.