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In a CRISPR-Cas9 experiment, the protein cuts the site matching the cRNA part of the gRNA. My question is: How many cuts are possible if multiples sites matching the cRNA are found in the cell?

Especially, considering DNA is made of multiple chromosomes, if more than one chromosome have a site matching the cRNA, will they all be cut? Does it depend of the quantity of Cas9 protein brought in during the experiment (meaning is some Cas9 protein "consumpted" at each cut)?

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    $\begingroup$ The main limitation on guide RNA design is the availability of a PAM site (NGG for Cas9) proximal to the target sequence. Addgene has a great article on gRNA design that may address some of your questions. $\endgroup$
    – acvill
    Dec 23, 2022 at 17:46
  • $\begingroup$ @acvill Thank you I'll check the link. But I'm not sure that it adresses the question: the question is not about designing what sequence the gRNA should have, but what are the possibilities of sequence (everything - magic versus a pool of possibilities) $\endgroup$ Dec 23, 2022 at 18:11
  • $\begingroup$ It seems like your question is less about gRNAs and more about oligonucleotide synthesis. Insomuch that gRNAs are just oligonucleotides, any gRNA sequence can be created by solid-phage chemical synthesis, either as a DNA construct to clone into an expression vector or as a ready-to-load RNA for use with purified Cas9. $\endgroup$
    – acvill
    Dec 23, 2022 at 18:26
  • $\begingroup$ @acvill thank you the link you gave between oligonucleotide synthesis and gRNA does give me elements to search for. So every gRNA could be synthetized? $\endgroup$ Dec 23, 2022 at 20:27

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On a single molecule level, Cas9 from Streptococcus pyogenes cuts but does not move to other targets for a few days (Raper et al. 2018). In contrast, Cas9 from Staphylococcus aureus cuts multiple targets, though also it takes hours for a single round (Yourik et al. 2019). However, inside a cell, even S. pyogenes Cas9 is rapidly dislodged either by RNA polymerase (Clarke et al. 2018) or FACT histone chaperone (Wang et al. 2020). So even if only one Cas9-sgRNA complex enters a cell, it will be able to cut multiple targets. There are several methods to measure the efficiency of Cas9 cleavage of multiple target and off-target sites that differ in accuracy and sensitivity. The PCR amplification-free method, RGEN-seq, is the latest innovation in this field (Kuzin et al. 2021).

References

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  • $\begingroup$ Thanks but I have a misunderstanding: are'nt "However, inside a cell, even S. pyogenes Cas9 is rapidly dislodged either by RNA polymeras" and "So even if only one Cas9-sgRNA complex enters a cell, it will be able to cut multiple targets" contradictory? I might have misunderstood, could you please clarify? $\endgroup$ Feb 18, 2023 at 21:15

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