CRISPRGuru @CRISPRGuru@qoto.org

The CRISPR-KRISPR method can also be useful for characterizing knock-in loci in other model organisms, including cell lines!

Random insertions are identified by strategically placing the guide RNAs (gRNAs) used in the CRISPR-KRISPR assay: one gRNA in one of the homology arms and the second binding to an overlapping region between the insert and the other arm. See article for more details.

In the process of simplifying the Circle-Seq method, we realized that our method offers much more: it can also find out if the pieces of donor DNAs (used for inserting a gene at the CRISPR cut site) are inserted correctly and if they are inserted elsewhere (random insertions).

We did not find off target cleavages in genetically engineered mouse (GEM) models. It is already well established that off-target cleavages are not common in GEM mice. It is nicely documented in this paper. https://www.nature.com/articles/nmeth.3408

We named our method as CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching PRotocol)

The method uses very less DNA and it can do much more than just finding out off target cleavages in the genome.

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CRISPRGuru @CRISPRGuru
🧵The CRISPR-KRISPR method was a collaborative work from
@Masato_Ohtsuka
lab. His talented team of bioinformaticians came up with a strategy to simplify Circle-Seq, a popular approach for detecting off target editing events
nature.com
CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets
https://www.nature.com/articles/nmeth.4278

Prof: Your CRISPR editing experiment worked?
Student: CRISPR won’t EDIT, it only CUTS✂️
P: Who edits then?
S: Cell does. But it is unpredictable
P: How can you find out?
S: Using CRISPR-KRISPR
P: What?
S: Yeah, CRISPR-KRISPR, a new method. See paper https://genomebiology.biomedcentral.com/articles/10.1186/s13059-022-02779-8