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Yale Team Combines DIA-MS With CRISPR to Study Down Syndrome Protein


NEW YORK (GenomeWeb) – A team led by researchers at Yale University has used data independent acquisition mass spectrometry (DIA-MS) combined with CRISPR gene deletion to study the function of a protein linked to Down Syndrome and tumor immunity.

Described in a study published this month in Proteomics, the effort looked at the proteomic effects of knockout of the non‐histone chromosomal protein HMG‐14 (HMGN1), which is known to play a role in regulating chromatin structure.

Using two-hour DIA mass spec runs on a Thermo Fisher Scientific Orbitrap Lumos instrument, the researchers were able to reproducibly quantify roughly 6,200 proteins in HeLa cells with CRISPR-deleted HMGN1, confirming deletion of the target gene and identifying 147 proteins with expression levels that changed significantly following knockout of this gene.

According to Yansheng Liu, assistant professor of pharmacology at Yale and senior author on the study, the paper offers a proof-of-concept that DIA mass spec can be a rapid and effective method for confirming the outcome of CRISPR experiments while also assessing the downstream effects of the targeted genetic alterations.

Liu said the study also observed significant differences at the proteomic level between different HeLa cell clones used in the experiment, which he suggested builds upon a paper published in Nature Biotechnology in which he and co-authors including Swiss Federal Institute of Technology, Zurich researcher Ruedi Aebersold, in whose lab Liu was a post-doc, found that HeLa cell populations presumed to be homogenous varied significantly across laboratories at the genomic, transcriptomic, proteomic, and phenotypic level.

Liu and his colleagues also sought to demonstrate that off-target effects of CRISPR experiments could also be detected at the proteomic level using DIA mass spec, but he said that in this case their findings were inconclusive.

CRISPR-Cas9 gene editing allows researchers to alter DNA in a highly targeted manner, allowing for the deletion of particular sequences and/or the introduction of new genetic material. Follow-up experiments are typically required to assess the efficacy of these alterations and to identify any off-target edits.

This follow up is often done using PCR or western blotting to determine whether the target sequence was successfully altered and if the target protein was effectively knocked out. Next-generation sequencing has also been used to assess the efficacy of CRISPR experiments as well as to look for off-target effects.

The development and advancement of DIA mass spec suggested to Liu that this work might be possible to do on the proteomic level, he said.

"I looked in the literature to see if there were assessments of the outcome of CRISPR experiments done by proteomics, and I didn't actually find very many," he said, noting that while he came across several shotgun mass spec studies, they didn't typically look in a rigorously quantitative way at variation across clone and dish replicates.

DIA provides a relatively rapid and simple approach to reproducibly quantifying large numbers of proteins across large numbers of samples. This, Liu said, means that researchers can both confirm the effectiveness of a CRISPR experiments and look at the impact of the gene edit on the protein level at the same time.

In the Proteomics study, the researchers found consistent knock-out of HMGN1 across the three different HeLa cell clones (A, B, and C) that they analyzed. However, they found that clone C different significantly from A and B at the proteomics level, with measurements of the three clone C replicates clustering separately from the three A and B replicates, which clustered as one.

Liu suggested this divergence occurred during the growth period required to go from the single CRISPR-edited cells to cell populations large enough for mass spec analysis and added that it was evidence of the heterogeneity he and his co-authors observed in the Nature Biotechnology paper.

"These [HeLa] cell lines are known to be genomically unstable, and I think proteomics actually provides [researchers] a very good opportunity to identify these clone effects in CRISPR experiments," he said.

The approach was less successful in identifying off-target CRISPR edits, in part, he suggested, because many of these off-target alterations occur in intronic regions as opposed to in exons, and so may not be as directly reflected at the protein level.

The researchers detected "one or two," proteins with fold changes potentially due to off-target editing, but "the changes were very low and the p-values were not significant," Liu said. "I think if you are really interested in the off-target effects, proteomics is not yet comparable to sequencing."

The study's focus on HMGN1 stemmed from Liu's interest in questions around gene dosage imbalance, a condition in which a cell or organism has more copies than normal of a particular gene and which is presumed to play a key role in conditions like Down syndrome and other aneuploidies.

In 2017, Liu and co-authors including Aebersold published a study in Nature Communications profiling the proteome of fibroblast cells taken from people with Down syndrome to assess the effects of gene dosage at the protein level.

In that study, HMGN1 "really jumped out as a protein that was highly relevant to Down syndrome," he said, adding that additional work looking at overexpression at the transcript level in Down syndrome also identified HMGN1 as linked to the condition.

Liu said that he is now working with researchers using CRISPR to replicate the effects of certain genetics diseases that he hopes to study at the proteomic level. Additionally, he is working with collaborators to obtain additional aneuploidy samples that he plans to analyze at the proteomic level to continue his investigation of gene dosage imbalances.