enhancing crispr/cas9 targeting in cancer patients
Image by TheDigitalArtist via Pixabay licensed under CC0

Nowadays, CRISPR/Cas9 technology is the gold standard for knocking out alleles in any biological system through its capacity for inducing double strand breaks (DSB). In most cases, these DSBs provoke a cellular repair response triggering a kind of mutations called indels (insertions/deletions) at the cleavage point. When the cleavage point breaks a coding sequence, these mutations could generate a nonfunctional allele (null allele) by breaking the reading frame at the coding sequence (frameshift or off-frame mutations).

However, due to the variable size of the indel mutation, generating a null allele with high frequency using a single guide (sgRNA) is not always possible. In fact, it is a random issue, and in a minor proportion, several in-frame mutations preserving the reading frame (e.g. +3/-3 bp or 3 multiples) are always generated.

A possible solution would be to use two or more sgRNAs at the same time to knock out the target gene at several key areas to guarantee the null result. Nevertheless, a high proportion of off-targets would be increased with each new sgRNA added, and a high number of DSBs would be created. In this sense, it has been demonstrated that more DSBs would induce a stronger p53-mediated DNA damage response and, even worse, the possibilities of creating more complex chromosome rearrangements.

All these undesirable effects could limit the therapeutic efficiency of gene therapy strategies based on disrupting oncogene expression using CRISPR/Cas9 technology, and, therefore, it must be considered carefully. Thus, in this cancer cell context, both an efficient Cas9-sgRNA cell delivery and, importantly, a high capacity for generating null mutations are essential. In this way, most knockout studies show sgRNAs targeting different positions within the chosen exon, most of them central position avoiding exon boundaries. In most of these cases, the sgRNA design follows only off-target criteria. However, for those cases in which cellular selection is not possible and only one sgRNA can be used, we hypothesize it is possible to enhance the null effect with a sgRNA that targets splice site consensus sequences or close to them.

In this work, we have compared the null effect induced by CRISPR guides directed to the exon boundary, close to the splice donor (SD) site versus guides targeting a central position in the key exon. Following this approach, we have demonstrated that a new possibility for generating a null allele is added: the probability of destroying the SD site and, therefore, the splicing pathway.

To test this hypothesis, we compared the null efficiency of both kinds of guides to abolish the expression of 2 different genes and 1 oncogene, in-vitro and in-vivo, in human cells and in mouse cells.

enhancing crispr/cas9 targeting in cancer patients

An edited mouse with CRISPR for knocking-out Tyr gene. Image courtesy Manuel Sánchez-Martín

We have compared the null efficiency of both guides and, in all cases, a higher number of null alleles was always obtained by the splice donor guides. We also confirmed these results at the protein level. We also targeted the oncogene BCR/ABLp210 in a human cell line K562 to abrogate their survival effect, and again we detected a high number of apoptotic cells using the SD guide. Importantly, this result confirms the putative therapeutic potential of CRISPR tools in human cancer.

In order to test whether these results can be reproduced in vivo, we microinjected mouse zygotes with both kinds of guides directed to TYR gen, and similar results in null effect were obtained. When the manipulated embryos were implanted, a higher number of albino or mosaic mice were generated with the SD guide.

We concluded that Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency and could be relevant in future clinical assays in human cancer.

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