Hi Isabela,

Yes, it is possible to delete large segments of DNA using CRISPR/Cas9. According to an editorial (link below), the efficiency of large segment deletion will be low (10 - 30%) and this will create different population of cells (genomic mosaicism). To improve the efficacy, they recommend using 4 different guide RNAs (sgRNAs) and possibly small-molecule inhibitors that suppress DNA repair.

Article Large-scale genomic deletions mediated by CRISPR/Cas9 system

In principle, one sgRNA creates a double strand break (DSB). The efficiency (probability, p) of creating a single DSB can be assumed as 80 - 90% (p=0.8 - 0.9). To delete a large piece of genomic segment, atleast two sgRNA will be needed to cut at either ends of the genomic region. Hence, the theoretical efficiency of deletion will be the product of combined efficiency of individual sgRNAs, (i.e., 0.8x0.8 = 64% to 0.9x0.9 = 81%). Once the DSBs are created, the cell will try to repair the DNA damage (non-homologous end-joining (NHEJ), Homologous recombination, Ligases), which will further reduce the final outcome.

Probably, if the CRISPR/Cas9 transfection is accompanied by a DNA-exonuclease (e.g., Lambda or T5 Exonuclease, highly efficient enzymes, https://www.neb.com/tools-and-resources/selection-charts/properties-of-exonucleases-and-endonucleases), this might start degrading the blunt ends created by the CRISPR/Cas9 and may increase the deletion efficiency. This suggestion is purely a speculation.

In the following video, Prof. Dana Carroll discusses how the deletion events can be predicted using micro-homology and tailoring the sgRNA (Video: 14:00 to 25:00 minutes, article reference: DOI: 10.1101/gr.171322.113; http://genome.cshlp.org/content/24/6/1012)

Video: https://www.youtube.com/watch?v=upzSmpnxNig

Best wishes.

Hi Isabela,

Yes, it is possible to delete large segments of DNA using CRISPR/Cas9. According to an editorial (link below), the efficiency of large segment deletion will be low (10 - 30%) and this will create different population of cells (genomic mosaicism). To improve the efficacy, they recommend using 4 different guide RNAs (sgRNAs) and possibly small-molecule inhibitors that suppress DNA repair.

Article Large-scale genomic deletions mediated by CRISPR/Cas9 system

In principle, one sgRNA creates a double strand break (DSB). The efficiency (probability, p) of creating a single DSB can be assumed as 80 - 90% (p=0.8 - 0.9). To delete a large piece of genomic segment, atleast two sgRNA will be needed to cut at either ends of the genomic region. Hence, the theoretical efficiency of deletion will be the product of combined efficiency of individual sgRNAs, (i.e., 0.8x0.8 = 64% to 0.9x0.9 = 81%). Once the DSBs are created, the cell will try to repair the DNA damage (non-homologous end-joining (NHEJ), Homologous recombination, Ligases), which will further reduce the final outcome.

Probably, if the CRISPR/Cas9 transfection is accompanied by a DNA-exonuclease (e.g., Lambda or T5 Exonuclease, highly efficient enzymes, https://www.neb.com/tools-and-resources/selection-charts/properties-of-exonucleases-and-endonucleases), this might start degrading the blunt ends created by the CRISPR/Cas9 and may increase the deletion efficiency. This suggestion is purely a speculation.

In the following video, Prof. Dana Carroll discusses how the deletion events can be predicted using micro-homology and tailoring the sgRNA (Video: 14:00 to 25:00 minutes, article reference: DOI: 10.1101/gr.171322.113; http://genome.cshlp.org/content/24/6/1012)

Video: https://www.youtube.com/watch?v=upzSmpnxNig

Best wishes.

Thanks a lot for all the information, Shubaash!

Shubaash Anthiya R G Helped me too. Thanks a lot.

Oi Isabela Ichihara de Barros ! Que legal a sua pergunta! Você conseguiu fazer a deleção? Eu queria uma deleção de 400kb nas iPSC nos meus pacientes. Eles têm uma duplicação e eu queria retirá-la para resgatar o fenótipo. Gostaria de saber se você teve êxito no seu experimento. Abraços e sucesso na sua pesquisa!!