Multiplexed Accurate Genome Editing with Short, Trackable, Integrated Cellular barcodes (MAGESTIC) is a platform that builds on the

CRISPR CRISPR (; acronym of clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. Each sequence within an individual prokaryotic CRISPR is d ...

/Cas technique. It further improves CRISPR/Cas by making the gene-editing process more precise. It also increases cell survival during the editing process up to sevenfold. This technology was invented at the Stanford Genome Technology Center in collaboration with the Joint Initiative for Metrology in Biology (JIMB)JIMB website
/ref> which is a coalition of

Stanford University Leland Stanford Junior University, commonly referred to as Stanford University, is a Private university, private research university in Stanford, California, United States. It was founded in 1885 by railroad magnate Leland Stanford (the eighth ...

and the

National Institute of Standards and Technology The National Institute of Standards and Technology (NIST) is an agency of the United States Department of Commerce whose mission is to promote American innovation and industrial competitiveness. NIST's activities are organized into Outline of p ...

Overview

Gene editing is used for a variety of tasks including the modifying of crops, the modifying of bacteria, and the modifying of disease-causing genetic mutations in patients. When only a single edited cell line is required, CRISPR/Cas combined with the endogenous DNA repair efficiency is sufficient to obtain an edited cell line. However, when trying to introduce many edits in multiplex, a higher efficiency of

Homology directed repair Homology-directed repair (HDR) is a mechanism in cells to repair double-strand DNA lesions. The most common form of HDR is homologous recombination. The HDR mechanism can only be used by the cell when there is a homologous piece of DNA presen ...

is required. The MAGESTIC technology has multiple components. One component, the LexA-Fkh1 protein is involved in the process of Donor Recruitment that increases the efficiency of homology directed repair. The second component is a library of CRISPR

Guide RNA Guide RNA (gRNA) or single guide RNA (sgRNA) is a short sequence of RNA that functions as a guide for the Cas9-endonuclease or other Cas-proteins that cut the double-stranded DNA and thereby can be used for gene editing. In bacteria and archaea, ...

s paired with donor DNA which encodes for specified edited to be integrated through homology directed repair. This in turn is linked to a

DNA barcode DNA barcoding is a method of species identification using a short section of DNA from a specific gene or genes. The premise of DNA barcoding is that by comparison with a reference library of such DNA sections (also called "sequences"), an indivi ...

s that allows for specific variants to be tracked in pools, similar to how

Genome-wide CRISPR-Cas9 knockout screens Genome-wide CRISPR-Cas9 knockout screens aim to elucidate the relationship between genotype and phenotype by ablating gene expression on a genome-wide scale and studying the resulting phenotypic alterations. The approach utilises the CRISPR gene ed ...

work, only MAGESTIC is more versatile as it allows for not only loss of function edits, but also DNA Codon changes,

Single-nucleotide polymorphism In genetics and bioinformatics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a ...

Indel Indel (insertion-deletion) is a molecular biology term for an insertion or deletion of bases in the genome of an organism. Indels ≥ 50 bases in length are classified as structural variants. In coding regions of the genome, unless the lengt ...

s, and other types of genetic changes to be introduced and tracked. By improving DNA repair efficiency, using array-synthesized guide–donor oligos for the plasmid-based high-throughput editing, and integrating a genomic barcode to prevent plasmid barcode loss, MAGESTIC leads to more uniform pools with genome integrated stable single copy barcodes and enables robust phenotyping.

Donor Recruitment

Because editing multiple sites in pools can be impacted by a number of factors including ineffective CRISPR

DNA synthesis DNA synthesis is the natural or artificial creation of deoxyribonucleic acid (DNA) molecules. DNA is a macromolecule made up of nucleotide units, which are linked by covalent bonds and hydrogen bonds, in a repeating structure. DNA synthesis occu ...

errors, competition with

Non-homologous end joining Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. It is called "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair ...

and other challenges that occur when building multiplex libraries, MAGESTIC screens required improved DNA repair. This is where the donor recruitment aspect of MAGESTIC comes in. MAGESTIC achieves greater editing efficiency by localizing donor DNA to the site of DNA breaks introduced by a CRISPR cut. A CRISPR machinery cuts at desired locations in the genome, and then MAGESTIC direct the donor DNA to the site of this cut to direct cells to introduce designed edits at the DNA cut sites. This technology is called donor recruitment and relies on a fusion protein that contains one domain recruited to DNA breaks and another domain that binds to the donor DNA. This allows for the production of high quality precision edit pools in yeast, where each cells contains a single edit and a DNA barcode. The donor recruitment aspect of the technology also holds the potential to improve editing efficiency in additional cell types, such as mammalian cells. This may one day prove beneficial to

gene therapies Gene therapy is medical technology that aims to produce a therapeutic effect through the manipulation of gene expression or through altering the biological properties of living cells. The first attempt at modifying human DNA was performed in 1 ...

or other therapeutic editing.

References

{{reflist Gene therapy Medical genetics Gene delivery Applied genetics Biological engineering Biotechnology * 2018 in science