Symbolic recording of signalling and cis-regulatory element activity to DNA.

Measurements of gene expression or signal transduction activity are conventionally performed using methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm in which such biological activities are stably recorded to the genome. Enhancer-driven genomic recording of transcriptional activity in multiplex (ENGRAM) is based on the signal-dependent production of prime editing guide RNAs that mediate the insertion of signal-specific barcodes (symbols) into a genomically encoded recording unit.

Precise genomic deletions using paired prime editing.

Current methods to delete genomic sequences are based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 and pairs of single-guide RNAs (sgRNAs), but can be inefficient and imprecise, with errors including small indels as well as unintended large deletions and more complex rearrangements. In the present study, we describe a prime editing-based method, PRIME-Del, which induces a deletion using a pair of prime editing sgRNAs (pegRNAs) that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair. PRIME-Del achieves markedly higher precision than CRISPR-Cas9 and sgRNA pairs in programming deletions up to 10 kb, with 1-30% editing efficiency.

A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens.

Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq).

Accurate classification of BRCA1 variants with saturation genome editing.

Variants of uncertain significance fundamentally limit the clinical utility of genetic information. The challenge they pose is epitomized by BRCA1, a tumour suppressor gene in which germline loss-of-function variants predispose women to breast and ovarian cancer. Although BRCA1 has been sequenced in millions of women, the risk associated with most newly observed variants cannot be definitively assigned.