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Genome Editing

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 already 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 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.

Protocols

This homology-directed insertion-based CRISPR gene-editing protocol enables knockout of all alleles of a target gene in the polyploid Drosophila S2R+ cell line, using either two sequential rounds of homology-directed insertion or a single round with a donor vector containing four different sgRNAs.

Protocol | | Nature Protocols

The authors describe DISCOVER-seq, a method to detect off-targets of CRISPR–Cas genome editing based on ChIP-seq analysis of MRE11 recruitment to DSBs, and subsequent bioinformatics analysis of sequencing data using the BLENDER pipeline.

Protocol | | Nature Protocols

Research

The heterochromatic knob (hk4S) on Arabidopsis chromosome 4 prevents the recombination between accessions with and without hk4S. Here, via egg-cell specific expression of the Cas9 nuclease, the authors demonstrate targeted reversal of the 1.1 Mb long hk4S-inversion in Col-0 and restore the crossovers with Ler-1.

Article | Open Access | | Nature Communications

Garcia-Marques et al. present CLADES, an innovative approach to study neuronal lineages based on CRISPR. Inspired by synthetic biology, CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a predetermined order.

Technical Report | | Nature Neuroscience

Developing cyanobacteria as CO2-neutral cell factories relies on the knowledge of the regulation mechanisms for growth and metabolism. Here, the authors develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803 and screens genes potentially affecting growth and L-lactate tolerance and production.

Article | Open Access | | Nature Communications

A single therapeutic base edit of the BCL11A enhancer in human HSPCs can ameliorate cellular defects associated with sickle cell disease and β-thalassemia in vitro and efficiently induce fetal hemoglobin expression upon engraftment in mice in vivo.

Letter | | Nature Medicine

Existing examples of targeted gene insertion in plants either rely on a selectable marker gene or result in short DNA inserts. Here, the authors use an optimized CRISPR-Cas9 method to insert a 5.2 kb carotenoid biosynthesis cassette into genomic safe harbors in rice, and obtain marker-free lines with high carotenoid content.

Article | Open Access | | Nature Communications

Selectable markers are widely used in cell engineering but there is only a limited variety to choose from. Here the authors split markers using inteins, allowing up to six transgene integration events to be selected for with one marker.

Article | Open Access | | Nature Communications

One third of verified gene knock outs with CRISPR still show residual protein expression owing to translation reinitiation or exon skipping. Several proteins are still functional. The authors call for a systematic analysis of protein levels after genome editing.

Analysis | | Nature Methods

Previous gene editing in haematopoietic stem cells (HSCs) has focussed on a heterogeneous CD34+ population. Here, the authors demonstrate high efficiency CRISPR/Cas9-based editing of purified long-term HSCs using non-homologous end joining and homology-directed repair, by directing isoform-specific expression of GATA1.

Article | Open Access | | Nature Communications

CRISPR activation (CRISPRa) can target select genes and, rather than being used to delete them, can be used to activate their expression. Chen and colleagues use a CRISPRa-based approach to drive the expression of multiple endogenous genes in tumors and presentation of the antigens encoded, thus enhancing antitumor immune responses.

Article | | Nature Immunology

Dystrophin-deficient mice are used to test corrective strategies for Duchenne muscular dystrophy, but evaluation of dystrophin expression requires collection of tissue samples from specific muscles and time points. Here, the authors generate mice in which dystrophin expression is coupled to luciferase, and show that bioluminescence allows non-invasive monitoring of dystrophin expression following genome editing.

Article | Open Access | | Nature Communications

The role of CTCF-bound insulator elements in enhancer-gene interactions and transcriptional regulation remains poorly understood. Here, the authors investigate multiple epigenome editing strategies for perturbing individual CTCF-bound insulators, and evaluate their effects on genome topology and transcription.

Article | Open Access | | Nature Communications

Mucopolysaccharidosis type I (MPSI) is a lysosomal storage disease caused by insufficient iduronidase (IDUA) activity. Here, the authors use an ex vivo genome editing approach to overexpress IDUA in human hematopoietic stem and progenitor cells and show it can phenotypically correct MSPI in mouse model.

Article | Open Access | | Nature Communications

Single-particle tracking PALM (sptPALM) provides quantitative information in vivo if the protein of interest remains in a single diffusional state during track acquisition. Here the authors develop a custom-built sptPALM microscope and a Monte-Carlo based diffusion distribution analysis to study dynamic DNA-dCas9 interactions in live bacteria.

Article | Open Access | | Nature Communications

Cystic fibrosis is caused by mutations in the CFTR chloride channel. Here, the authors develop a gene therapy approach using the programmable nuclease AsCas12a to correct a splicing mutation in CFTR, and show efficient repair of the mutation and recovery of CFTR function in patient-derived organoids and airway epithelial cells.

Article | Open Access | | Nature Communications

AsCpf1 is an alternative nuclease to Cas9 for CRISPR mediated genome engineering. Here the authors demonstrate functional genomic screens with AsCpf1 that minimize library size with no loss in gene targeting efficiency.

Article | Open Access | | Nature Communications

Here, the authors show that sequential treatment with long-acting slow-effective release ART and AAV9- based delivery of CRISPR-Cas9 results in undetectable levels of virus and integrated DNA in a subset of humanized HIV-1 infected mice. This proof-of-concept study suggests that HIV-1 elimination is possible.

Article | Open Access | | Nature Communications

Reviews

cis-Regulation therapy (CRT) — modifying the activity of gene-regulatory elements — is emerging as a potential approach to treat genetic diseases. Here, Matharu and Ahituv assess emerging CRT technologies and present proof-of-concept studies in cell and animal models. Key factors to be considered for the translation of CRT into the clinic are discussed.

Review Article | | Nature Reviews Drug Discovery

The newest CRISPR–Cas genome editing technologies enable precise and simplified formation of crops with increased yield, quality, disease resistance and herbicide resistance, as well as accelerated domestication. Recent breakthroughs in CRISPR–Cas plant biotechnologies improve reagent delivery, gene regulation, multiplexed gene editing and directed evolution.

Review Article | | Nature Reviews Molecular Cell Biology

CRISPR–Cas systems have revolutionized genome editing, and the CRISPR–Cas toolkit has been expanding to include single-base editing enzymes, targeting RNA and fusing inactive Cas proteins to effectors that regulate various nuclear processes. Consequently, CRISPR–Cas systems are being tested for gene and cell therapies.

Review Article | | Nature Reviews Molecular Cell Biology

Genome editing through direct editing of bases holds promise for achieving precise genomic changes at single-nucleotide resolution while minimizing the occurrence of potentially mutagenic double-strand DNA breaks. In this Review, Rees and Liu provide a comprehensive account of the state of the art of base editing of DNA and RNA, including the progressive improvements to methodologies, understanding and avoiding unintended edits, cellular and organismal delivery of editing reagents and diverse applications in research and therapeutic settings.

Review Article | | Nature Reviews Genetics

News and views

CRISPR–Cas gene editors are now both moving into the clinic and being embraced as a means to find and validate drug targets. But for Jennifer Doudna, who helped pioneer this promise with her work at UC Berkeley, the full potential of these tools will only be unleashed when they can be used at scale. To this end, Doudna and colleagues partnered last year with GlaxoSmithKline to launch the Laboratory for Genomic Research (LGR), a US$67 million effort aimed at industrializing the CRISPR–Cas workflow for the detailed exploration of human genetics. One year on, she spoke with Asher Mullard about her hopes for CRISPR–Cas editors as drug discovery tools, the types of projects the LGR is working on and the challenges they face.

An Audience With | | Nature Reviews Drug Discovery

The simultaneous removal of endogenous T-cell receptor α-chains and β-chains, and the orthotopic placement of an exogenous receptor in human T cells via CRISPR gene-editing, prevents the mispairing between endogenous and transgenic receptors while preserving the cells’ function.

News & Views | | Nature Biomedical Engineering

A new genome editing strategy called prime editing uses a catalytically impaired Cas9 fused to an engineered reverse transcriptase to write desired genetic sequence information directly into a target locus.

Research Highlight | | Nature Reviews Genetics