Genome editing with programmable base editors in human cells.

Since the revolutionary adaptation of CRISPR-Cas9 for programmable genome editing, the field has expanded rapidly. In 2016, the genome editing toolbox was expanded by the addition of base editors (BEs). These tools install single nucleotide mutations into the genome by directly modifying target DNA bases. While the applications of base editing are broad, they are commonly used to model single-nucleotide variants (SNVs) in mammalian cell lines. In this work, we clearly describe key methods for genome editing using base editors in human cells. Generally, base editors are comprised of an N-terminal deaminase fused to a nickase Cas9 protein (nCas9), with N- and C-terminal nuclear localization signals (NLS). We first discuss their architecture, then we detail considerations for use. This includes accounting for protospacer adjacent motif (PAM) availability near the target nucleotide and sequence motifs favored by the different available deaminase and Cas9 protein combinations. Common available deaminases are also compared, thus enabling readers to select the best BE for their specific needs. After a BE has been selected, we address how to design guide RNAs (gRNAs) for use with base editors and clone gRNA expression constructs. This work also discusses various components of different gRNA expression constructs, and we provide reliable methods for the generation of these constructs, from cloning through transformation and sequencing of the construct to ensure accuracy. After BE selection and gRNA design, these constructs must be delivered into the mammalian cells. As a variety of methods exist to this end, we provide detailed methods for lipid-based transfection, electroporation, and transposition and transduction of components. First, we discuss both forward and reverse transfection methods using cationic lipids. Some cell types are not amenable to transfection with cationic lipids and thus, we provide two protocols for more sensitive cell types: (1) electroporation and (2) genomic encoding of small-molecule-inducible BE construct using piggyBac transposition. The latter method also discusses delivery of gRNAs through lentiviral transduction. Small-molecule inducible BE expression is required for efficient editing in stem cells. To assess editing efficiency, next generation sequencing (NGS) is often used as it provides robust data on genomic sequences, and is able to detect low levels of editing. Amplification and barcoding polymerase chain reaction (PCR) steps are discussed in depth and detailed troubleshooting guides are provided. Should editing efficiency be low for certain targets and cell types, we provide an enrichment strategy utilizing a fluorescent plasmid reporter and fluorescent activated cell sorting (FACS). The reporter design and construct are discussed, as well as sample preparation for FACS. These enriched samples can then be lysed and undergo amplification and barcoding for sequence analysis. If the goal of the experiment is to generate isogenic cell lines using BEs, a protocol is provided specifically for the Namocell, a single-cell sorting instrument that enriches samples similarly to a FACS.