Scarless genome editing: progress towards understanding genotype–phenotype relationships

Abstract

The ability to predict phenotype from genotype has been an elusive goal for the biological sciences for several decades. Progress decoding genotype–phenotype relationships has been hampered by the challenge of introducing precise genetic changes to specific genomic locations. Here we provide a comparative review of the major techniques that have been historically used to make genetic changes in cells as well as the development of the CRISPR technology which enabled the ability to make marker-free disruptions in endogenous genomic locations. We also discuss how the achievement of truly scarless genome editing has required further adjustments of the original CRISPR method. We conclude by examining recently developed genome editing methods which are not reliant on the induction of a DNA double strand break and discuss the future of both genome engineering and the study of genotype–phenotype relationships.

Glossary

Directed Evolution

A process in which a specific DNA element (often an open reading frame or enzymatic binding site on a protein) is mutagenized to create a large library. This library is then put through a specific in vitro screening process, generally for multiple rounds, to identify mutations which give rise to a desired effect on the assayed activity or function

DNA Binding Motif

A region of a protein which is capable of binding to a specific DNA sequence

Donor Template

An (often) short piece of DNA containing desired DNA edits flanked by regions homologous to the region to be edited. Cells may use it as a repair template during homologous -recombination based DNA-repair mechanism

Double Strand Break

A type of DNA damage in which both strands have been severed in close proximity to each other. This results in the creation of two distinct strands from the original

Endogenous DNA

The native DNA of a cell

Genome Editing

The process of intentionally altering (adding, removing, or changing) the genome of a cell

Genome Scarring

The introduction of unwanted or unintended edits to cellular DNA during genome editing

Genotype

The DNA composition of a genomic region or of the entire genome

Homologous recombination based DNA repair

A type of DNA damage repair mechanism in which the cell uses a homologous region to the one which has been cut as a template to repair the cut strand. During natural repair, this template is generally the sister chromosome of the one being cut, but during genome editing this template is generally synthetic DNA introduced to the cell for this purpose

Mutagenesis

The process of inducing random mutations to a piece of DNA or to an entire genome. Historically this has been done using radiation or mutagenic chemicals. A mutagenized stretch of DNA will contain a number of random mutations of all types

Nuclease

A protein which is capable of inducing a DNA double strand break

Phenotype

A behavior of a cell which is observable to a researcher

Plasmid-based expression system

A means of gene expression in which the gene of interest is placed into a non-integrating plasmid and transformed into a cell