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crispr.md

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liu

  • review current clinical trials and first clincial outcome for base editorsjj

  • alyssa kiona car t triply edited with cd27

  • less of targets

  • homogoneous edits

  • homogoneous edits

  • some PE do better with templates with lots of secondary structure,

tldr; engineer pes to perform better at different types of edits

Regulatory Spacers - Shmakov

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450183/

  • cas express in mini-array with spacer that is complementary to upstream promoter
  • self regulation of expression

Arrays in CRISPR systems are repeat sequences that flank spacers.

A mini-array is an array with only two repeat sequencs (thus one spacer).

These mini arrays encode spacers that are often complementary to the promoter of the mini array itself, inducing a self-regulatory systsem

Clustering and searching for new CRISPR systems - Kannyan (Broad)

cas systems can do complex things * gain and loss of crispr

Polymerase Editors - Erik Sontheimer

Split PE prevents self annealing

Natural RT are bad (M-MLV RT):

  • Fidelity - does not correct in the antisense direction
  • dNTP incoporation rate (naturally limited to dividing cells?)
  • Processivity - length of reverse transcription event

so use DNA Polymerase

592

Lisa Alexander [Metagenomi]

Type V * range from 500 - 1500 aas * 1000s

Claims:

  • long guides - good for eng
  • small guides - good for multiplexing

why?

R Loops - Kevin Aris

negative supercoiling - R loop formation positive supercoiling - R loop dissocation

What are motiffs that cause natural supercoiling

Look up / understand AuRBT

RNA Editing - Korro

A -> I (read as G during translation) ADAR(1-3)

Verve

PKCS9 in non human primates paper

GALNAC - LNP was not to target specific cells but because patients they were treating lack LNP receptors (because of the genetic underpinnings of high LNP)

pkcs9 homozygous fh

what did FDA care about with IND package

  • general off-targets
  • editing of germline cells

Mandana ARbab - HMS

SMN1 ABE8E

explore variety of editing strategies (editors and/or types of mutations that achieve same effect)

AAV 45% transduction efficiency of cell population is the bar

ee pipeline transduction -> cas activity -> desired edit

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10270003/

Intellia

Analysis of offtargets from LNP + editor

Germline editing guidance from FDA (for lentivral therpaies) from 2006

Spatial analysis of editing efficiency amongst ovary

Jing Zeng

in -vivo

intellia

rle of micronuclei in dna damage from cas9

dominique PKU

KJ ABE8E.8 PAH1

look up ONE-seq?

guide engineering

  • RNA / DNA base combinations

Affinia Therapeutics

peptide tropism did not translate well from mouse to monkey

constructing targeted delivery