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Andrew J. Aguirre studied how genomic genomic copy number affects CRISPR-Cas9 screening. Targeting amplified genomic regions can produce a response independent of the targeted gene's usual function, an important distinction when interpreting whether a screening signal demonstrates a gene-specific dependency.^[ref]

Researcher in live-cell genome imaging. Chen and collaborators adapted CRISPR-Cas9 targeting to visualize particular DNA loci in living human cells, connecting sequence recognition with observations of chromosome dynamics.^[ref]

Genome editing deliberately changes the DNA sequence at selected sites in a genome. Changes can include a small insertion or deletion, replacement of a sequence, or introduction of a specific base change. This differs from merely measuring gene expression or transiently changing it without altering the underlying DNA.

In a common CRISPR-Cas9 approach, a guide RNA directs a nuclease to a matching DNA region, and cellular DNA repair determines the resulting sequence change. Repair can disrupt a gene or use a supplied template to introduce a designed change. Le Cong and colleagues demonstrated programmable targeting of endogenous mammalian sites and simultaneous editing at several sites in 2013.^[ref]

A targeted cut is therefore an intermediate operation, not proof of the intended edit. DNA sequencing of the resulting alleles distinguishes the planned change from unintended repair products.

Researcher in genome engineering. Cong and collaborators demonstrated multiplex targeting using CRISPR-Cas9 systems, helping establish a programmable way to alter more than one genomic site in an experiment.^[ref]

Researcher in gene regulation. Gilbert and collaborators developed modular RNA-guided regulation of transcription using CRISPR-Cas9, showing how sequence-directed targeting can control gene activity rather than only cut DNA.^[ref]

Bacterial DNA-recognition and DNA-modifying systems provide ways to mark, map and perturb eukaryotic chromosomes. Their value comes from separating the targeting module from the reporter or effector, so a chromosome becomes experimentally observable without assuming that its sequence alone specifies its behaviour.

For fluorescent operator-repressor chromosome labelling, an integrated array of bacterial operator sequences is bound by a fluorescent repressor fusion. A LacI fusion bound to a lac operator array makes a selected genetic locus visible in a living nucleus. Two differently labelled loci can report separation of sister chromatids, movement relative to the nuclear periphery, or distributions of distances between chromosome regions. Carmen C. Robinett and colleagues demonstrated operator-based imaging in mammalian cells and yeast. The tag reports the marked locus, not every part of the chromosome, and repeated operators or excessive bound repressor can perturb DNA replication and chromatin structure.^[ref]

The bacterial CRISPR-Cas9 recognition mechanism makes targeting programmable. A fluorescent dCas9 fusion and a guide RNA can visualize native repeated sequences; multiple guides can improve detection of a nonrepetitive locus. Baohui Chen and colleagues used this approach to follow human genetic loci. Catalytically inactive targeting avoids making the DNA double-strand break that would itself alter chromosome behaviour. Appropriate controls include guide specificity, signal from an unlabelled locus, and whether tagging changes growth or the measured dynamics.^[ref]

For DamID, bacterial Dam methyltransferase is fused to a nuclear protein. Methylated GATC sites record neighbourhoods visited by the fusion. A fusion to a nuclear-envelope component can identify lamina-associated domains, whereas one to a chromatin-binding protein maps its genomic neighbourhoods. Comparing with untethered Dam helps control accessibility-dependent background. The accumulated methylation signal is a time-integrated proximity record: it need not establish direct binding to every recovered sequence, and resolution depends on GATC distribution and local spreading.^[ref]

Bacterial enzymes also make structural measurements possible. In Hi-C, restriction enzymes generate DNA fragments whose proximity-dependent ligation junctions can be sequenced. In ATAC-seq, a modified bacterial Tn5 transposase inserts sequencing adapters preferentially into accessible DNA. Neither assay directly photographs a fixed chromosome structure: contact frequency and accessibility are different observables. Enzyme sequence preferences, fixation, and cell-to-cell heterogeneity must be controlled. Together, live labelling, proximity recording and sequencing-based assays connect chromosome movement, nuclear neighbourhood and chromatin accessibility, rather than substituting one measurement for all three.