CRISPR-Cas9
CRISPR/Cas9 series: a compelling journey from a primitive bacterial defence system to an advanced human gene-editing technology
19/01/2022

CRISPR/Cas9 series: a compelling journey from a primitive bacterial defence system to an advanced human gene-editing technology

The side effects of CRISPR / Cas9 and the current strategies to mitigate them

Summary:

  • The CRISPR/Cas9 system often not only cuts at the intended site of the genome, but also in other unwanted places, which are called off-targets;
  • Off-target events are mainly due to sequence homology between sgRNA and genomic DNA other than the target one, as well as to the degree of purity and stability of sgRNA/Cas9 complex inside the cells;
  • Researchers have implemented numerous strategies to minimise off-target effects and increase the safety of the CRISPR/Cas9 system.

The CRISPR/Cas9 system functions in bacteria and archaea as an RNA-based, adaptive immune system. It is able to direct the Cas9 protein to the complementary DNA of an invading virus, and thus cut and destroy it. As we have learned in the previous articles (CRISPR1, CRISPR2), CRISPR/Cas9 is a bacterial-derived gene editing tool that exploits DNA repair systems of cells, providing an opportunity to make gene therapy in humans. To enable its gene-targeting capacity, CRISPR system can be replicated using the following three minimal components:

  1. the DNA-cutting Cas9 protein;
  2. a sequence-specific CRISPR RNA (crRNA);
  3. an auxiliary trans-activating RNA (tracrRNA).

The crRNA and tracrRNA duplexes can also be fused to generate a chimeric single-guide RNA (sgRNA) [1]. The first 20 nucleotides of the sgRNA are complementary to the target DNA sequence, followed by a sequence called the Protospacer Adjacent Motif (PAM) [2]. Simply by changing the sgRNA sequence, the researchers can make Cas9 reach the gene of interest and cut exactly into the target DNA sequence adjacent to the PAM [3].

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Despite the CRISPR/Cas9 system demonstrated to be an unparalleled genome-editing technology, there are still several concerns to its large-scale therapeutic and clinical applications.

CRISPR/Cas9 is an extremely powerful system, but also very inaccurate [4]. The sgRNA induces Cas9 to cut at other sites other than the intended on-target one, then causing the so-called “off-target” mutations with very high frequency (>50%) [5-6]. An off-target event is a side-effect of Cas9-mediated genome-editing, resulting in DNA alterations that can lead to unpredictable adverse biological outcomes. This off-target cleavage activity is the consequence of little differences (or mismatches) between the sgRNA and the target DNA sequence. Cas9 protein fails to notice them, cutting the wrong sequence as if it were the one actually targeted.

When the 20 nucleotides of sgRNA are designed by researchers to be complementary to the DNA sequence of the gene of interest, it means that sgRNA and targeted DNA sequences perfectly match the one with the other. Nevertheless, other DNA sequences in the host cell may be partially similar to the motif targeted by sgRNA/Cas9. Similar but not identical sequences to the one of interest are recognized and cleaved by Cas9 as well, and the number of off-target sites strongly depends on how many of these homologous sequences exist within the genome.

Off-target effects might be cell-type-specific and highly dependent on the integrity of the DNA repair pathways of a particular cell type [7]. Moreover, the degree of purity of Cas9 protein and sgRNA has been found to affect the specificity of cleavage activity. It has been reported that the time Cas9 spends inside the cells is directly proportional to the increase in off-target events [8], so it would be advantageous that sgRNA/Cas9 complex cleaves target DNA almost immediately after delivery and is degraded rapidly in cells.

To be able to bring the CRISPR/Cas9 technology into the healthcare system, the need to effectively reduce off-target events has become a priority. Actually, off-target effects in gene editing represent a not negligible risk for patients safety, as they can cause DNA changes that could evolve in cancer or other undesired outcomes. In light of these considerations, several approaches have been proposed to minimise off-target effects.

A first approach is based on altering the sgRNA sequence. Specifically, the truncation of sgRNA or the addition of two specific nucleotides called guanines at the end of sgRNA improves target specificity, decreasing undesired mutations at some off-target sites under 1%. However, modifications of sgRNA sequence to enhance Cas9 specificity without compromising on-target efficiency have not provided reliable results [9]. A second promising strategy for minimising off-target effects is to control the concentration and formulation of sgRNA/Cas9 complex, in order to finely control its stability and behaviour inside the cells [10]. Other approaches focus on the modification of Cas9 itself. In particular, to further improve DNA cleavage specificity, fusions of inactive Cas9 with the FokI nuclease domain have been generated. Modified Cas9 is able to edit target DNA with significantly higher specificity than its wild-type counterpart, and to reduce enormously off-target events [11].

Many other next generation tools have been emerging in recent years to make the CRISPR/Cas9 system suitable for clinical applications. Advances in bioinformatics and genetic engineering are providing tools to improve sgRNA and Cas9 in terms of efficacy and safety [12].

While researchers are racing to mitigate off-target effects, clinicians may be forced to choose between administering the gene therapy and incurring the risk of off-target mutations or giving up this therapy and miss the opportunity to treat the patient. Currently, over 20 clinical trials have been approved for the use of the CRISPR/Cas9 technology in patients [13]. The implications of cost/benefit ratio of the increasing use of CRISPR/Cas9 are then leading to demand for strong regulatory institutions and medical ethics boards to prevent any abuse or moral transgressions, as in the case of the (in)famous chinese CRISPR babies which will be the next topic of this series.

References:

  1. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–821. doi: 10.1126/science.1225829.
  2. Anders C, Niewoehner O, Duerst A, Jinek M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014;513(7519):569-73. doi: 10.1038/nature13579.
  3. Ceasar SA, Rajan V, Prykhozhij SV, Berman JN, Ignacimuthu S. Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9. Biochim Biophys Acta. 2016;1863(9):2333-44. doi: 10.1016/j.bbamcr.2016.06.009. 
  4. Lin Y, Cradick TJ, Brown MT, et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 2014;42(11):7473-7485. doi:10.1093/nar/gku402
  5. Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH. Off-target Effects in CRISPR/Cas9-mediated Genome Engineering. Mol Ther Nucleic Acids. 2015 Nov 17;4(11):e264. doi: 10.1038/mtna.2015.37. PMID: 26575098; PMCID: PMC4877446.
  6. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013;31(9):822-6. doi: 10.1038/nbt.2623.
  7. Lin S, Staahl BT, Alla RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife. 2014;3:e04766. doi:10.7554/eLife.04766.
  8. Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 2015;208:44-53. doi: 10.1016/j.jbiotec.2015.04.024. 
  9. Han HA, Pang JKS, Soh BS. Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing. J Mol Med (Berl). 2020;98(5):615-632. doi:10.1007/s00109-020-01893-z.
  10. Alkan F, Wenzel A, Anthon C, Havgaard JH, Gorodkin J. CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters. Genome Biol. 2018;19(1):177. doi: 10.1186/s13059-018-1534-x.
  11. Saifaldeen M, Al-Ansari DE, Ramotar D, Aouida M. CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering. Cells. 2020;9(11):2518. doi: 10.3390/cells9112518.
  12. Naeem M, Majeed S, Hoque MZ, Ahmad I. Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing. Cells. 2020;9(7):1608. Published 2020 Jul 2. doi:10.3390/cells9071608.
  13. Uddin F, Rudin CM, Sen T. CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future. Front Oncol. 2020;10:1387. doi: 10.3389/fonc.2020.01387.
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