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CRISPR Cas9 gene editing can cause deletions

The CRISPR-Cas9 system is now thought to be the method for gene editing therapy in humans with minimal side-effects. However, significant off-target effects were until recently believed to be just insertions and deletions (indels) of less than 20 base pairs. Therefore, the CRISPR-Cas9 system is assumed to be very specific making it a useful tool for gene editing in cells. However, this approach may not be as straightforward as it sounds.

Figure 1: Crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM (Anders et al. 2014).  


Ihry et al. recently reported that p53 inhibits the CRISPR-Cas9 system in human pluripotent stem cells (hPSCs). The research group found that double-strand breaks induced by Cas9 are toxic and kill most hPSCs.

Next, Kosicki et al. recently showed that DNA breaks introduced by single-guide RNA-Cas9 frequently resolved into deletions extending over many kilobases. This research group used long-read sequencing and long-range PCR genotyping for the study of DNA break repair mechanisms. Additionally, the researcher observed lesions distal to the cut site and crossover events as well. As a result, Kosicki et al. reason, the observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.

A third study (Haapaniemi et al. 2018) reported that genome editing by CRISPR-Cas9 induced a p53-mediated DNA damage response. The cell cycle was arrested in immortalized human retinal pigment epithelial cells. However, inhibition of p53 prevented the damage response and increased the rate of homologous recombination from a donor template.

As a result, more research is needed since correcting somatic mutations in human cells using CRISPR-Cas9 may cause more harm than doing good. Employing bridged nucleic acids (BNAs) for more selective gene editing may also improve the selectivity of the CRISPR-Cas9 system.

Reference

Carolin Anders, Ole Niewoehner, Alessia Duerst, and Martin Jinek; Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014 Sep 25; 513(7519): 569–573.

Haapaniemi, Emma, Botla, Sandeep, Persson, Jenna, Schmierer, Bernhard, Taipale, Jussi; 2018. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nature Medicine 24, 927-930 (2018).

Ihry, Robert J., Worringer, Kathleen A., Salick, Max R., Frias, Elizabeth, Ho, Daniel, Theriault, Kraig, Kommineni, Sravya, Chen, Julie, Sondey, Marie, Ye, Chaoyang, Randhawa, Ranjit, Kulkarni, Tripti, Yang, Zinger, McAllister, Gregory, Russ, Carsten, Reece-Hoyes, John, Forrester, William, Hoffman, Gregory R., Dolmetsch, Ricardo, Kaykas, Ajamete;  p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nature Medicine 24, 939-946 (2018).

Kosicki, Michael, Tomberg, Kärt, Bradley, Allan; Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nature Biotechnology 2018/07/16/online, 36, 765-771 (2018).

http://dx.doi.org/10.1038/nbt.4192. https://www.nature.com/articles/nbt.4192#supplementary-information

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