Connor Rosen Profile picture
Sep 10, 2018 10 tweets 3 min read Read on X
Gene modification in primary human NK cells with Cas9 RNP electroporation - very useful preprint @biorxivpreprints - biorxiv.org/content/early/… Highlights and thoughts… #Immunology #GenomeEditing #CRISPR #PreReview #ASAPBio
The bottom line: Best RNP electroporation conditions resulted in up to 80% deletion, consistently >50%, of 3 tested genes for activated primary human NK cells. Validation of Ncr1 and Cish biology from mouse studies in human NK cells.
Paper starts by demonstrating issues with lentiviral transduction in human NK cells. Electroporation was optimized very thoroughly (>10 buffers and pulse conditions), with ~doubling in efficiency (viability and uptake) to >80% transformed and >80% viable
Cas9-GFP plasmids expressed poorly and no deletion was observed. Following trends from T cell literature, they tried Cas9 RNP electroporation. RNP transformation gave >50% homozygous deletion of CD45, stable by flow - no sequencing validation, but flow data are very clear
Now that manipulation of NK cells was established, they validated Ncr1 (coding for NKp46) role in cytotoxicity against Daudi cells (expressing NKp46 binding proteins). Sorted NKp46 -/- cells were less cytotoxic in both donors, matching mouse data
In the process of validating Ncr1, they also demonstrated deletion in expanded (14 days in vitro) NK cells (proliferating, activated, metabolically active) was substantially higher - up to >80% deletion, and ability to titrate efficiency of deletion by sgRNA concentration
For next validation, knockout of Cish. Knockout was robust, and KO cells had enhanced response to IL-15 stim - more proliferation and more JAK1/STAT5 (both total and phosphorylated - consistent with known role of CIS in degrading JAK)
Knockout in primary human NK cells was efficient (NGS quantification of indels was >60% on average, up to 80% varying a bit by donor/prep) and resulted in increased proliferation with IL-15 and increased cytotoxicity
Knockout in cord blood cells (~40% efficiency) and transfer into humanized mice showed normal NK cell differentiation with loss of Cish, and the derived NK cells had enhanced responses to IL-15 and proliferation ex vivo
Overall, extremely thorough optimization and detailed protocols for human NK cell modification, consistent success across 3 loci, validation of targets from mouse biology in human NK cells - great work and thanks to the authors for putting this comprehensive toolkit together!

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More from @connor_rosen

Sep 25, 2020
Here’s a thread on anti-interferon autoantibodies, viral infections, and human immunology. This is less a covid thread, and more an anti-covid thread, if anything…
Summary: anti-IFN auto-Abs may be reflective of chronic inflammation, and may pre-exist in vulnerable groups at high levels. Presence in severe covid cases may therefore reflect basal immune variation which impacts covid, rather than a special covid-specific phenomenon.
This is prompted based on the new Science paper on autoantibodies against Type I IFNs in severe covid patients (science.sciencemag.org/content/early/…). Basically, ~10% of severe covid patients had high titer anti-IFNa antibodies that were functionally neutralizing.
Read 23 tweets
Jun 2, 2020
The message is very simple. #BlackLivesMatter
I'm grateful that others have put together resources like bit.ly/ANTIRACISMRESO…, which has helped me learn to be better. So You Want To Talk About Race by @IjeomaOluo and How To Be An Antiracist by @DrIbram have been amazing, and I'm looking forward to reading deeper.
I haven't seen a similar central resource for donations, but various recommendations led me to eji.org (Equal Justice initiative, focused on criminal justice reform and education), voterparticipation.org (get people registered to vote)...
Read 5 tweets
Apr 21, 2020
The story about mitochondria being ~10° hotter than the rest of the cell, published @PLOSBiology a couple years ago (journals.plos.org/plosbiology/ar…) got some cool, albeit indirect, support recently @naturemethods#Biophysics #CellBiology
The original paper, which was mainly based on the temperature-dependent fluorescence of a mitochondria-targeting probe, was accompanied by a “Primer” (journals.plos.org/plosbiology/ar…) highlighting potential flaws and implications, a special sort-of-peer-review step by PLOS Biology.
This new awesome resource @naturemethods - nature.com/articles/s4159… - offers some intriguing orthogonal validation. This is a proteome-wide study of protein thermal stability across 13 organisms, conducted using a mass-spec-based approach.
Read 11 tweets
Nov 22, 2019
Super neat story on how cellular quality control impacts the mutational landscape of proteins - beneficial mutations in DHFR during deep mutational scanning are totally altered dependent on cellular QC #Biophysics #Evolution biorxiv.org/content/10.110… Way to go! @KortemmeLab
In an initial DMS experiment on DHFR, there were a large number (25% of all sequences!) of advantageous mutations spread across the whole protein. Reintroduction of QC protein Lon reduced the number of advantageous mutants and lowered average benefit of those mutations
Changes in selection coefficient (the “advantageous-ness”) were most striking at hydrophobic/aromatic residues and buried residues - and these correlate with Tm changes of variants. So Lon seems to be imposing higher standards on DHFR, particularly for destabilizing core mutants
Read 5 tweets
Nov 20, 2019
A couple interesting bits of preliminary data on Langerhans cells and the huLangerin mouse used to study them. Effects of developmental absence of LCs on keratinocytes and T cells, and huLangerin-YFP labels some neurons (check your Cre mice!) #Genetics #Immunology
First - effect of loss of LCs in the huLangerin-DTA mice - biorxiv.org/content/10.110…. Bulk RNA-seq showed changes in keratinocytes and dendritic epidermal T cells, including cell-type specific changes (e.g. loss of IL17 pathway in DETCs).
Unfortunately, underlying data (either gene expression tables or raw RNA-seq data) don’t currently seem available, but hopefully the authors get that up shortly. Will be interesting to look at and prompt some hypotheses about how LCs control homeostasis and development.
Read 5 tweets
Nov 18, 2019
CYTOF analysis of human neutrophils - 7 populations with differing phagocytosis, ROS, and FACS-compatible surface marker phenotypes. Changes in distribution between healthy + melanoma patients. #Immunology #Cancer #Neutrophils biorxiv.org/content/10.110…
Circulating neutrophil precursors, aged neutrophils, and a few populations of mature and immature neutrophils. Melanoma stage correlates with loss of dominant N2 (mature, ROS++, lowly phagocytic), and increase of N5 (immature, non-proliferative, ROS+, lowly phagocytic).
Interesting to note the phagocytosis-SSC staining in Fig4A - big changes in SSC for some populations with zymosan, others not so much - degranulation, different phagosomes…? Similarly, bimodal peaks for ROS production in same populations… Image
Read 4 tweets

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