How far we can understand the extent to which our genes directly affect cell, tissue, and organ function depends on our ability to precisely modify the genetic code and analyze the consequences of those modifications. CRISPR, for clustered regularly interspaced short palindromic repeats, technology has revolutionized our ability to carry out gene editing, but the system isn’t foolproof. It’s akin to using scissors to cut out words from text on a piece of paper, but without the precision to cut out individual letters or understand how these modifications to the text might affect meaning.
Scientists at the Joint Institute of Metrology and Biology (JIMB)—a collaboration between Stanford University and the National Institute of Standards and Technology (NIST)—have now developed a CRISPR-Cas9–based platform that can make precise, single-nucleotide changes in target DNA at the genome-wide level. The new technology has been dubbed MAGESTIC because it is a “multiplexed, accurate genome-editing” tool that uses “short, trackable, integrated cellular” barcodes. Tests using the MAGESTIC platform to carry out high-throughput, precise gene editing and evaluation in the yeast model Saccharomyces cerevisiae demonstrated fivefold increases in editing efficiency and as well as sevenfold increases in cell survival.
“MAGESTIC is like an advancement in the 'control F' [find text] operation of a word-processing program, with the replace-text command allowing a desired change,” comments JIMB researcher Kevin Roy, Ph.D., who is one of the platform’s developers, and lead author of the team’s published paper in Nature Biotechnology. “This lets us really poke at the cell in a very precise way and see how the change affects cell function. Then we can compare the actual effects of each variant with the computationally predicted effects, and ultimately improve models for predicting how genetic variants impact health and disease.”
The JIMB researchers report on the development of MAGESTIC and initial tests with the system in a paper entitled “Multiplexed Precision Genome Editing with Trackable Genomic Barcodes in Yeast.”
CRISPR screens have been widely used to modify genes by engineering in small insertions/deletions (indels), and introducing premature termination codons (PTCs) in open reading frames (ORFs), but, as the authors point out, “few methods have been developed to introduce specific amino acid and nucleotide variants at the gnome scale.” And while high-throughput genome-editing technologies have been reported in prokaryotes and yeast, these platforms also haven’t been used to study genetic variation. “Dissecting complex genotype-phenotype relationships has remained a central obstacle in quantitative genetics despite major technological advances in sequencing and genome editing,” the team writes.
The MAGESTIC CRISPR-Cas9 platform developed by the JIMB team has been designed to address current hurdles in gene-editing technology, and “overcomes major shortcomings in currently employed approaches,” the authors write. MAGESTIC enables researchers to make single-nucleotide variations genome wide and quantitatively assess the effects of each genetic modification. And, unlike previous platforms, which edit each variant in different experiments, MAGESTIC is carried out in a single tube, making single base-pair changes in individual cells among millions of otherwise identical cells.
One of the major differentiators of the MAGESTIC platform is that it actively recruits the donor DNA to the double-stranded breaks caused by Cas9 scissors using array-synthesized guide RNA/donor DNA (guide-donor) oligonucleotides. This enables a more than fivefold increase in precision editing efficiency, the authors state. Another of the major features that sets MAGESTIC apart from other multiplexed CRISPR editing approaches is that it uses genome-integrated barcodes that are tagged onto the end of the guide-donor sequence. Using traditional approaches, the barcodes are embedded in the plasmids, which are then inherited by daughter cells and multiply. However, the plasmid barcode system is not completely accurate, and so can give false measures of cell number.
Instead, the MAGESTIC platform integrates the barcodes directly into the yeast cell chromosomes, which is a far more stable, easily tracked system, the researchers claim. “…we introduce stable, genome-integrated barcodes instead of plasmid barcodes, thereby enabling marker-free variant tracking and one-to-one correspondence of barcode counts to stain abundance.”
The barcodes effectively also function to help distinguish cells that carry the same guide-donor pair, but which result from different editing events, so ”providing internal replicates and serving as single-cell tracers,” the team notes. Overall, genomic integration of the barcode offers several advantages, they point out. “(1) Phenotyping is not confined to environments requiring marker selection, (2) each cell harbors only a single barcode rather than a variable copy number plasmid, and (3) thousands of individual strains can be readily isolated and identified en masse from a mutant pool using recombinase-directed indexing.”
The authors claim that MAGESTIC will springboard our understanding of the genotype–environment–phenotype relationship and fill current gaps in our ability to compare single point edits, one by one, so that we can more accurately unpick the effects of mutation and variation on disease. “Overall, MAGESTIC enables tens of thousands of specific genetic variants across the genome to be created in a manner that is compatible with robust phenotyping across hundreds of conditions,” they write.
“We are reaching a state where we have not only achieved the ability to sequence the order of base pairs in genomes but we can also make changes to them,” comments JIMB's Lars Steinmetz, Ph.D., professor of genetics at Stanford University, group leader at the European Molecular Biology Laboratory (EMBL), and senior author on the team’s paper. “We still need a better understanding of the consequences of our edits. With MAGESTIC it's like being able to make small edits to individual letters in a book, and being able to see what effect it has on the meaning of the text. Our donor recruitment method also allows the new piece of information to be placed at exactly the right page where the cut occurred.”