RESUMO
The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Vírus da Leucemia Murina de Moloney , DNA Polimerase Dirigida por RNA , Streptococcus pyogenes , Animais , Humanos , Camundongos , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Vírus da Leucemia Murina de Moloney/genética , DNA Polimerase Dirigida por RNA/genética , Streptococcus pyogenes/genética , Desoxirribonuclease I/genéticaRESUMO
Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.