Editing stem cell genes will "revolutionize" biomedical research

Applying a dramatically improved method for "editing" genes to human stem cells, University of Wisconsin-Madison professor of neuroscience Su-Chun Zhang has shown a new way to silence genes in stem cells and their progeny at any stage of development.

The advance has advantages in speed and efficiency, says Zhang, and is already being used for basic biological studies.

The invention of gene "knock-outs" earned the 2007 Nobel Prize for its utility in determining what genes do. Oliver Smithies, one of three recipients, performed the winning research at UW-Madison.

The new discovery solves two limitations with existing knock-out techniques. Shutting off a gene too soon can kill a cell or stymie development. And the knock-out techniques did not work well with human embryonic and induced pluripotent stem cells, the do-it-all cells that can transform into any cell in the body.

Now, after 10 years of effort, Zhang, of the Waisman Center, has revealed how to shut off genes in embryonic stem cells after they have been functioning normally. His work is published in the July 2 edition of Cell Stem Cell.

"Timing is the critical advance," says Zhang, who was the first to shape embryonic stem cells into neurons. "Silencing a gene later in development can tell us what it may do in the adult human which we cannot research on. This means you can take out the gene at any given time, in any type of cell."

Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9

Highlights
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Efficient strategy outlined for engineering clonal inducible gene knockout hPSC lines
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Dual-sgRNA targeting is essential for precise biallelic knockin of FRT
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Inducible gene knockout can occur in all cells at any differentiation stages
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Multiple genes can be targeted for inducible knockout

Summary
Precise temporal control of gene expression or deletion is critical for elucidating gene function in biological systems. However, the establishment of human pluripotent stem cell (hPSC) lines with inducible gene knockout (iKO) remains challenging. We explored building iKO hPSC lines by combining CRISPR/Cas9-mediated genome editing with the Flp/FRT and Cre/LoxP system. We found that “dual-sgRNA targeting” is essential for biallelic knockin of FRT sequences to flank the exon. We further developed a strategy to simultaneously insert an activity-controllable recombinase-expressing cassette and remove the drug-resistance gene, thus speeding up the generation of iKO hPSC lines. This two-step strategy was used to establish human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines with iKO of SOX2, PAX6, OTX2, and AGO2, genes that exhibit diverse structural layout and temporal expression patterns. The availability of iKO hPSC lines will substantially transform the way we examine gene function in human cells.

CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology 

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] and transcription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent mutations by introducing doublestranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology 

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] and transcription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent mutations by introducing doublestranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.