Parallels Between CRISPR-Cas9 and RNAi

Dr. Arun Kuman

Arun Kumar K. P, Ph.D., Head, Laboratory of Molecular Genetics, Centre for DNA Fingerprinting & Diagnostics, Hyderabad, India MORE ABOUT THE AUTHOR

CRISPR-Cas9 based genome engineering is gaining ground from its first publication in 2012 (Jinek et al, 2012). The technique is being tested in different model and non-model organisms. The number of publications is increasing exponentially for the last couple of years.

Recently, Barrangou et al (2015) have published an interesting review on this new technology by drawing parallels between CRISPR-Cas9 based genome editing and RNAi based gene silencing.

guide RNA relative to target sequence

guide RNA relative to target sequence

As CRISPR-Cas9 system is emerging as a versatile tool in genome editing it is necessary to know the complete landscape of this system. The authors in this review tried to give a perspective of CRISPR-Cas9 along with RNAi. To explain the whole phenomenon they chose several comparable aspects between CRISPR-Cas9 and RNAi with respect to efficiency, off target effects, genome screening tools, in vivo studies and molecular consequences. As off targets are a major problem in genome editing technologies the authors covered certain developments such as introducing mutations in sgRNAs, delivery of multiple sgRNAs for a single target etc. In addition to it various screening strategies, which are used in RNAi have also been explained.

CRISPR-Cas9 system does not interfere with the endogenous machinery of cell as it is edited at the level of DNA within the nucleus; sometimes it is a major problem with siRNAs or shRNAs, which may lead to cell death (Doudna et al, 2014).

Parallel timelines for the development and use of RNAi and CRISPR/Cas9
Image from Barrangou et al (2015)

For phase III entry therapies RNAi cannot be applied solely due to reversible nature. But it can be overcome with CRISPRa and other dCas9-based approaches. This system can be more advantageous since there is no requirement for continuous delivery of shRNAs or siRNAs and the effect of CRISPR-Cas9 can be maintained throughout generations; whereas it is transient in case of siRNA based knock down.

It is reported that Cas9 undergoes conformational changes when it is bound to sgRNA and further upon to dsDNA when it is bound to target sequence (Ban et al 2015). Since, Cas9 bound to off target sequence will be in inactive state, it does not induce double strand breaks in the off target sequence (Doudna et al, 2014). So focusing on the modifications in sgRNA or Cas9 protein can be effective in minimizing the off target effects.

RNAi

RNAi

This review is particularly interesting as it compares the CRISPR-Cas9 with RNAi with respect to effectiveness, specificity and off-target effects that are significant factors for applying genome engineering across wide variety of species.

It also talks about the technologies that can be adapted from RNAi for the improvement of CRISPR-Cas9 for faster evolution.

It has also pointed out the difficulties and challenges in delivery of complexes in comparison with RNAi, such as ease of use, overcoming methodologies and advantages with the continuous effect of CRISPR-Cas9 in contrast to transient effects with RNAi.

The authors ended the review with a hope to develop a methodology, which can overcome difficulties faced during RNAi development. This review will be very helpful for researchers working on genome editing as it gives unique and comprehensive view of CRISPR-Cas9 system in comparison with RNAi.

Barrangou R, Birmingham A, Wiemann S, Beijersbergen RL, Hornung V and Smith AB (2015) Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference. Nucleic Acids Research (first published online March 23) doi:10.1093/nar/gkv226.

References:

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA and Charpentier E. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337:816-821. DOI: 10.1126/science.1225829.

Doudna JA and Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9. Science 346: 1077.  DOI: 10.1126/science.1258096.

Ban T, Zhu J, Melcher K and Xu E (2015) Structural mechanisms of RNA recognition: sequence-specificand non-specific RNA-binding proteins and the Cas9-RNA-DNAcomplex. Cell. Mol. Life Sci. 72: 1045–1058. DOI: 10.1007/s00018-014-1779-9.

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