CRISPR: three new developments in the world of gene editing

A new variant for CRISPR

In a new study, researchers from Michigan Medicine at the University of Michigan, US, revealed that using a novel variant to repair DNA can improve both safety and effectiveness of CRISPR-Cas9 for genetic research.

According to the scientists, the safety and efficacy of CRISPR-Cas9 are what continue to hold the gene editing technique back from its full clinical potential.

To develop their variant, the researchers fused a minimal motif consisting of 36 amino acids to a gene-editing nuclease Streptococcus pyogenes Cas9 (spCas9). According to the team, the new meticulous integration Cas9 (miCas9) binds RAD51 through this fusion motif and enriches RAD51 at the target locus.

They explain that the new CRISPR-Cas9 variant therefore improves efficiency when inserting, or knocking in, a gene or DNA fragment to a precise location in the genome. It also reduces the rate of unintended insertions or deletions, known as indels, of base pairs that often happen during gene editing.

“We name it miCas9 to reflect its extraordinary capacity to enable maximum integration, yet with minimal indels, as well as to recognise its development at the University of Michigan,” said senior authors Dr Y Eugene Chen, Jifeng Zhang and Jie Xu. “It provides a ‘one small stone for three birds’ tool in gene editing.”

The researchers say that this fusion motif can also work as a “plug-and-play” module, compatible with other Cas9 variants. Therefore, miCas9 and the minimal fusion motif may find broad applications in gene editing research and therapeutics.

The results of this study were published in Nature Communications.

Using CRISPR-edited CAR T cells to fight against blood cancers

In another study from the University of Pennsylvania, US, researchers have used CRISPR-Cas9 to knock out a protein known to stifle T-cell activation on CAR T cells. According to the team, this enhanced the engineered T-cells’ ability to eliminate blood cancers in pre-clinical studies.

“We have shown, for the first time, that we can successfully use CRISPR-Cas9 to knock out CD5 on CAR T cells and enhance their ability to attack cancer,” said Assistant Professor Marco Ruella, who presented the results at the 62nd American Society of Hematology Annual Meeting & Exposition. “The difference between edited and non-edited CAR T cells was striking in several cancer models.”

The team knocked out the CD5 gene, which encodes for the CD5 protein on the surface of T cells and can inhibit their activation, on CAR T cells using CRISPR-Cas9 and infused them back into mice with T- and B-cell leukaemia or lymphoma.

The team first tested the approach in a T-cell leukaemia model. Anti-CD5 CAR T cells were genetically engineered to seek out CD5 on malignant T cells and attack them. Since CD5 is also expressed on normal T cells, the researchers removed it from CAR T cells, both to avoid the possible killing of other CAR T cells and potentially to unleash CAR T-cell activation that would otherwise be inhibited by the presence of CD5 on these cells.

They found that CD5-deleted anti-CD5 CAR T cells were significantly more potent than CAR T cells without the deletion (wild-type, CD5+) in both in vitro and in vivo experiments, where more than 50 percent of mice were cured at long term.

To test the hypothesis that deletion of CD5 could increase the anti-tumour effect of CAR T cells targeting antigens other than CD5, the results were then validated with CTL019 CAR T cells against CD19+ B-cell leukaemia. The team also found that CD5 knockout led to significantly enhanced CTL019 CAR T-cell anti-tumour efficacy with prolonged complete remissions in the majority of mice.

In a separate analysis, the team reviewed a genomic database of more than 8,000 patients’ tumour biopsies to study their levels of CD5 and found a correlation with outcomes. “Basically, in most cancer types, the less CD5 expressed in T cells, the better the outcome. The level of CD5 in your T cells matters,” Ruella said.

Dr Carl June, one of the study’s authors, added: “Looking at the long term, this could represent a more universal strategy to enhance the anti-tumour effects of CAR T cells. We look forward to building upon these encouraging findings in the next phase of our work.”

Find the abstract of this study here.

Mapping genetic networks with CRISPR

A team from University of California, Berkeley, US, developed a technique using CRISPR to profile cells and rapidly determine all the DNA sequences in the genome that regulate the expression of a specific gene.

“A disease where you might want to use this approach is cancer, where we know certain genes that those cancer cells express and need to express, in order to survive and grow,” said Associate Professor Nicholas Ingolia, whose lab conducted the research. “What this tool would let you do is ask the question: what are the upstream genes – the regulators that are controlling those genes that we know about?”

The researchers say that the new technique simplifies the backtracking along genetic pathways in a cell to identify regulatory genes.

“I sometimes use the analogy that when we walk into a dark room and flip a light switch, we can see what light gets switched on. That light is like a gene and we can tell, when we flip the switch, what genes it turns on. We are already very good at that,” Ingolia added. “What this lets us do is work backward. If we have a light we care about, we want to find out what switches control it. This gives us a way to do that.”

The team say this new technique, dubbed CRISPR interference with barcoded expression reporter sequencing (CiBER-seq), allows the pooling of tens of thousands of CRISPR experiments. The technique does not use fluorescence and employs deep sequencing to directly measure the increased or decreased activity of genes in the pool. Deep sequencing uses high-throughput, long-read next-generation sequencing (NGS) technology to sequence and count all the genes expressed in the pooled samples.

The team’s key innovation was to link each single guide RNA (sgRNA) with a unique, random nucleotide sequence connected to a promoter that will only transcribe it if the gene of interest is also switched on. Each ‘barcode’ reports on the effect of one sgRNA, individually targeting one gene out of a complex pool of thousands of sgRNAs. Deep sequencing revealed the relative abundances of every barcode in a sample and expression of the gene of interest. According to the scientists, for human cells, a researcher might insert more than 200,000 different guide RNAs, targeting each gene multiple times.

In their experiments, the team queried five separate genes in yeast, including genes involved in metabolism, cell division and the cell’s response to stress. The team say that, using their technique, it may be possible to study up to 100 genes simultaneously when CRISPRing the entire genome.

“In one pooled CiBER-seq experiment, in one day, we can find all the upstream regulators for several different target genes, whereas, if you were to use a fluorescence-based technique, each of those targets would take you multiple days of measurement time,” Ingolia said.

The results of this study can be found in Science.