Can this be accomplished using Cas9 and two sgRNAs targeting genomic sites over 100 kb away from each other? I want to do this in a plant that is transformable, but for which there are no reports of using CRISPR. If it is feasible, what range of editing frequency can I expect to achieve? Any literature sources on the use of gene editing for long deletions in plants generally?
I think that people have already reported doing that in plants. In animal, researchers can even delete an arm of chromosome or a whole chromosome with CRISPR system. The editing efficiency can be vary depending on many factors, such as the choose of sgRNA, the promoter you use to drive Cas9 (some papers have reported it), the delivery form of Cas9/sgRNA (by DNA or by ribonucleoproteins (RNP)?), the delivery method (by agrobacterium, by bombardment, by PEG-mediated, or by lipoinfection?).
I am sorry that the case I mentioned in my previous answer is in animal cells, not plant cells. The authors have successfully removed up to 100 kb of targeted DNA sequences in human embryonic stem cells. But, they used a new CRISPR system called CRISPR-Cas3 (Dolan et al. 2019).
The paper:
Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas
Mol Cell. 2019 Jun 6;74(5):936-950.e5. doi: 10.1016/j.molcel.2019.03.014. Epub 2019 Apr 8.
In your question, you also asked "Any literature sources on the use of gene editing for long deletions in plants generally?". I am wondering whether you saw the paper by Durr et al. below:
Durr et al. (2018) successfully used CRISPR-Cas9 to create heritable chromosomal deletions to study gene function in plants. A genomic region flanking the ISU1 locus (~12 kb), containing a large cluster of defensin-like genes and non-coding regulatory sequences, was selected for deletion.
Hi Chris,
You can find some information from this reference. They could produce 115-245 kb deletions.
"Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice" Zhou et al., 2014. Nucleic Acids Res. 2014;42(17):10903-14. doi: 10.1093/nar/gku806. Epub 2014 Sep 8.
Good luck!
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