High Throughput CRISPR Approach Adopted for Profiling Genomic Variants

High Throughput CRISPR Approach Adopted for Profiling Genomic Variants

The CRISPR gene editing tool allows scientists to make precise cuts to double-stranded DNA to alter defective genes and add new genetic material. The technique has been shown to result in little to no off-target alterations and gene function can be quickly and easily changed. However, analysing the effects of each gene edit is a time-consuming process since the analysis requires each gene edit to be assessed one at a time. It can therefore take several weeks to determine the outcomes of each genetic alteration.

However, a team of researchers at the University of California, Los Angeles have tweaked CRISPR technology to speed up the process, adopting a high throughput CRISPR approach to monitor the outcome of thousands or even tens of thousands of gene edits in the time it has previously taken to monitor just a few.

The increased speed means it will now be far easier for researchers to identify genetic changes that will be most beneficial and will disrupt the function of genes that are contributing to the cause of disease.

CRISPR gene editing uses guide RNA to identify a specific location on the genome and a protein called Cas9 to make the cut to the DNA. By doing so, the gene can be disabled. It is also possible to insert a new piece of DNA into the cut site.

For the technique to work, the correct guide RNA is required along with the correct section of DNA to insert. While making gene edits one at a time is possible, making multiple edits using correctly matched pairs of DNA segments and guide RNAs and delivering those pairs to many thousands of cells at the same time has been a challenge.

The UCLA researchers have devised a method that allows multiple edits to be performed and monitored. The researchers developed a method of physically connecting the guide RNA to their corresponding DNA patches and delivering them to cells.

The researchers explained in their paper, ”We developed a CRISPR-library-based approach for highly efficient and precise genome-wide variant engineering. We used our method to examine the functional consequences of premature-termination codons (PTCs) at different locations within all annotated essential genes in yeast.”

Yeast was used rather than human cells as the effects of the edits are easier to observe. The researchers grew millions of yeast cells, added a set of guide RNA and DNA patches, and were able to study the effects of around 10,000 mutations separately. After four days the researchers were able to see which cells survived and which did not, and thus determine which edits were damaging and which were harmless.

The researchers identified genes that had large dispensable sections that could be altered without changing gene function and some genes that were thought to be essential for survival turned out to be dispensable.

“We can now edit the genome in thousands of different ways, while observing positive or negative effects on cells,” said lead author Leonid Kruglyak. “Our ultimate goal is to help scientists zero in on the genetic culprit for a disease, leading doctors to a firm diagnosis and allowing patients to obtain the most effective treatment.”

The research is detailed in the paper – Highly parallel genome variant engineering with CRISPR–Cas9 – recently published in Nature Genetics.

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