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Recent advances in genome editing technologies have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Programmable nucleases, particularly the CRISPR/Cas system, are revolutionizing our ability to interrogate the function of the genome and can potentially be used clinically to correct or introduce genetic mutations to treat diseases that are refractory to traditional therapies. This collection of recent articles from the Nature Research journals provides an overview of current progress in developing targeted genome editing technologies. A selection of protocols for using and adapting these tools in your own lab is also included.
This protocol describes prime editing (PE) and twinPE experiments as well as the design and optimization of pegRNAs. The authors provide guidelines for selecting the proper PE system for a given application and how to perform PE in mammalian cells.
This protocol for base editing in cultured mammalian cells provides guidelines for choosing target sites, appropriate base editor variants and delivery strategies, as well as detailing the computational analysis of base-editing outcomes using CRISPResso2.
GUIDE-seq (genome-wide unbiased identification of double-stranded breaks enabled by sequencing) is a sensitive, unbiased, genome-wide method for defining the specificity of genome-editing nucleases in living cells.
Bacterial genome-wide gene fitness is assessed by CRISPRi-seq. The procedure includes a pipeline for single-guide RNA library design, workflows for pooled CRISPRi library construction, growth assays, sequencing and read analysis fitness quantification.
The authors present a protocol for using the CRISPR–Cas9 genome editing system to knock out a gene of interest in human intestinal tissue–derived enteroids by lentiviral transduction and single-cell cloning.
This protocol uses PlantPegDesigner to design and optimize prime editing guide RNA and engineered plant prime editor vectors for efficient prime editing in monocot plants.
The authors provide a versatile gene therapy approach that is mutation- and gene size–independent, using dCas9-VPR–based transcriptional activation of functionally equivalent genes. They show how to apply this for gene therapy for inherited retinal dystrophies in mice as an example.
The authors provide protocols for rapid and scalable genome engineering in somatic cells of the liver and pancreas through both viral and nonviral delivery of CRISPR components into living mice.
This chromosome-engineering protocol generates heritable chromosomal rearrangements in A. thaliana; by combining SaCas9 with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements can be made and homozygous lines can be established.