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John Doench

@johndoench.bsky.social

2.9K followers 760 following 240 posts

Functional genomics @ Broad Institute. Screen all the things!

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John Doench @johndoench.bsky.social · Sep 7

Doomscrolling pause to chat CRISPR libraries! Preprint describes our new, data-driven approach to combine on-target and off-target predictions much more intelligently for *selecting* guides, which we use to develop our newest Cas9 knockout library, Jacquere. Thread: www.biorxiv.org/content/10.1...

Balancing off-target and on-target considerations for optimized Cas9 CRISPR knockout library design

The continued development of high-dimensional CRISPR screen readouts, such as single-cell RNA sequencing and high-content imaging, necessitates compact libraries to enable functional interrogation at ...

www.biorxiv.org

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Reposted by John Doench

Addgene @addgene.bsky.social · 2d

New CRISPR libraries from the @johndoench.bsky.social and Root labs — Jacquere (human) and Julianna (mouse).
A perfect pairing for your next screen. 🍷🧀
https://twp.ai/9PX2kT
https://twp.ai/4irnYP

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Reposted by John Doench

Peter Sagal @petersagal.bsky.social · 10d

I have lived and worked in and around Chicago for 27 years and I have been assaulted less often than Sen Paul has been in his own back yard.

Aaron Rupar @atrupar.com · 10d

Rand Paul: "Chicago is a nightmare. It is literally a war zone."

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Reposted by John Doench

The Tennessee Holler @thetnholler.bsky.social · 10d

79. American citizen.

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Kevin M. Kruse @kevinmkruse.bsky.social · 12d

I'm sorry, what?

Laura Belin @laurarbelin.bsky.social · 12d

Breaking news from Des Moines: ICE has detained Dr. Ian Roberts, the superintendent of Iowa's largest school district.
The Des Moines Public Schools said "We have no confirmed information as to why Dr. Roberts is being detained or the next potential steps."
www.bleedingheartland.com/2025/09/26/i...

ICE detains Des Moines Superintendent Dr. Ian Roberts

Iowa's largest school district had "no confirmed information" on why ICE detained Dr. Ian Roberts, the superintendent since 2023.

www.bleedingheartland.com

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Reposted by John Doench

Jeremy Berg @jeremymberg.bsky.social · 16d

Now with comments... Lots of comments

(A long thread)

1/n

Jeremy Berg @jeremymberg.bsky.social · 16d

Posting without comment...

www.nytimes.com/2025/09/22/o...

Opinion | ‘The Power of Science to Solve Problems Is Almost Limitless’

www.nytimes.com

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Reposted by John Doench

Mark Histed @markhisted.org · 16d

The New York Times piece today about US science is terrible and wrong—in many ways.

I could write a whole article about this, but as one example:

“To close observers, the original crisis began well before any of this…”
No. I’m a close observer of science, and this is incorrect.

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John Doench @johndoench.bsky.social · 18d

this is the exact first thing that popped into my head as well :)

Reposted by John Doench

Barack Obama @barackobama.bsky.social · 20d

After years of complaining about cancel culture, the current administration has taken it to a new and dangerous level by routinely threatening regulatory action against media companies unless they muzzle or fire reporters and commentators it doesn’t like.

Let’s be clear about what happened to Jimmy Kimmel

Trump’s most brazen attack on free speech yet.

www.yahoo.com

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Reposted by John Doench

Barred and Boujee aka Madiba Dennie @audrelawdamercy.bsky.social · 22d

screencap from The Office where Michael Scott says 'I am a victim of a hate crime.' Stanley, exhausted, replies 'That's not what a hate crime is.' Michael then says 'Well, I hated it a lot, okay?'

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Reposted by John Doench

NBC10 Boston @nbcboston.com · 23d

After a co-host suggested that mentally ill homeless people who don't accept services offered to them should be jailed, Brian Kilmeade replied, "Or involuntary lethal injection, or something. Just kill 'em." on.nbcboston.com/JlG99ke

Fox News' Brian Kilmeade apologizes for saying mentally ill homeless people should be executed

Brian Kilmeade suggested to "just kill" mentally ill homeless people who don't accept services offered to them.

on.nbcboston.com

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Reposted by John Doench

Carl Zimmer @carlzimmer.com · 24d

A deep dive into the destruction of US cancer research by @jonathanmahler.bsky.social “It’s an absolutely unmitigated disaster,” a former top official at NIH told him. “It will take decades to recover from this, if we ever do.” Gift link: nyti.ms/48iH3Cr

nyti.ms

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John Doench @johndoench.bsky.social · 27d

Nice work Ben!

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Reposted by John Doench

Ben Kleinstiver @bkleinstiver.bsky.social · 27d

Optimization of a bespoke base editor to treat a severe pediatric vascular disease! 🫀🧬
Our manuscript describes:
1️⃣ Engineering a target-specific BE🧬
2⃣ A *must avoid* bystander edit that occurs with WT SpCas9 BEs! 🙅‍♂️
3⃣ Extension of lifespan after in vivo editing! 🐁✅

www.nature.com/articles/s41...

Treatment of a severe vascular disease using a bespoke CRISPR–Cas9 base editor in mice - Nature Biomedical Engineering

Engineering a mutant-specific customized base editor precisely corrects a mutation while minimizing bystander edits, leading to substantial phenotypic recovery in mouse models of multisystemic smooth ...

www.nature.com

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John Doench @johndoench.bsky.social · Sep 8

Hmm, not sure how to achieve (1) without just not targeting genes in those region of the genome that are prone to low-level chromosomal loss. In other words, seems unlikely that *some* successful guides targeting gene X would lead to this while others would not, right?

John Doench @johndoench.bsky.social · Sep 7

Yup, chromatin can inhibit Cas protein access.

But when a gene is transcribed, or DNA is replicated, chromatin is removed, so Cas proteins have a chance to find their target site.

And, if your gene of interest is never accessible (on) in a cell model, no particular reason to need to knock it out!

John Doench @johndoench.bsky.social · Sep 7

Best for last, the project was led by the incomparable Laura Drepanos, a computationalist in GPP (who, fun fact, will be applying to grad school this fall). I've not seen a more methodical and comprehensive analysis, or read a better first-draft of a manuscript.

I ❤️ this paper.

Happy screening!

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John Doench @johndoench.bsky.social · Sep 7

A more points to wrap things up:
• We also provide the designs for a new mouse library, Julianna
• This new guide selection screen is implemented in CRISPick for Cas9 knockout (off-target for CRISPRi is a another story, stay tuned!)
• Libraries are on their way to @addgene.bsky.social

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John Doench @johndoench.bsky.social · Sep 7

We conclude with a comparison of analysis methods, including a cautionary tale of false positives arising from the use of MAGeCK RRA.

We provide suggestions to "mitigate the reporting of false discoveries that, if left unchecked, would undermine confidence in large-scale perturbational screens."

John Doench @johndoench.bsky.social · Sep 7

We next screened this library and assessed performance via separation of essential and non-essential genes - we see much greater depletion of the former while the distribution of the latter barely budges. So we accomplished our goal: smarter picking of guides increased on-target activity.

John Doench @johndoench.bsky.social · Sep 7

Essentially, by worrying less about off-target activity (because we actually do a pretty good job of predicting it) we're able to design a library with a *much* higher distribution of on-target scores, as assessed by Rule Set 3.

John Doench @johndoench.bsky.social · Sep 7

A lot of details I won't cover here, but essentially we filter out all guides with a CRISPick Aggregate CFD score > 4.8 and then select the 3 guides with the best Rule Set 3 score. This new picking scheme leads to a substantial difference between Jacquere and our prior Brunello+Gattinara libraries

John Doench @johndoench.bsky.social · Sep 7

We then examined three sources of "what are all the genes" and designed Jacquere to target all of them.

John Doench @johndoench.bsky.social · Sep 7

We then used the F1 score to determine the sum of CFD scores that best-distinguished truly-promiscuous from false-positive off-target guides.

We found the best performance if we considered only 1 mismatch in the SDR, at a CFD sum of 4.8, so we define that as our "CRISPick Aggregate CFD" threshold

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John Doench @johndoench.bsky.social · Sep 7

We then asked how well a simple aggregation of CFD scores (as originally proposed in the development of GuideScan, PMID: 28263296) could predict the "promiscuity" of guides, defined as "guides that shouldn't deplete in a viability screen but do anyway" using tiling data of non-essential genes

John Doench @johndoench.bsky.social · Sep 7

Using the Cutting Frequency Determination (CFD) matrix for predicting off-target sites (PMID: 26780180), when we limit to 2 or fewer mismatches in the SDR, the predictions match the measured GuideSeq off-targets sites remarkably well (here, each dot represents a bin of 37 off-target sites)