sgRNA Design & Target Specificity

Specificity of the CRISPR/Cas9 system is determined by the 20-nt guiding sequence of the sgRNA and the presence of a protospacer-adjacent motif (PAM) adjacent to the target sequence in the genome. Some mismatch (3-5 nucleotides) at the PAM-distal end of the sgRNA is tolerated by the system and this can lead to off-target effects. The extent to which this occurs and its significance remains a topic of study and debate.

Kuscu et al. (2014) make their contribution to the debate by using a catalytically inactive Cas9 (dCas9) in conjunction with ChIP-seq. The approach is fairly straightforward – they load dCas9 with sgRNAs and introduce them into cells (in this case human stem cells) and then perform ChIP-seq analysis (crosslink dCas9 to DNA; isolate dCas9-linked DNA; sequence DNA).

The authors tested 12 sgRNAs and found that dCas9 binding specificity varied from 50 off-target sites (6 sgRNAs with this characteristic) to over 1200 binding sites (1 sgRNA). What seemed to be the driver of this off-target binding? The PAM-proximal sequences in the sgRNA!


The authors report that dCas9 generally interacts with each off-target site less frequently than with the on-target site. This is consistent with previous findings that showed that reducing the concentration of sgRNA and Cas9 results in fewer off-target effects. So, keep that in mind – Cas9 concentration makes a difference in controlling off-target effects.

As for sgRNA mismatches and the specificity of dCas9 binding, the authors report that while many off-target targets had near-perfect alignment to the sgRNA sequence proximal to the PAM, some binding sites had as many as 10 consecutive mismatches with PAM-proximal sequences. This is somewhat different from other data using Cas9, suggesting that the modified protein, dCas, might have a somewhat different target specificity.

Despite the off-target binding reported by the authors they suggest that while some bound off-target genomic sites were cleaved by Cas9, the fre¬quency of cleavage at off-target sites was generally substantially lower than at on-target sites.

In sum, off-target binding occurs and careful design of the sgRNA is required. Pay particular attention to PAM-proximal sequences and, to the extent possible, keep the Cas9 concentration as low as practical.

So, this is a topic worth being aware of and our understanding of sgRNA design is evolving. An insect biologist considering using this technology would be well advised to thoroughly explore the latest literature on the topic. This paper will provide you with a foothold into the literature.
Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease
Cem Kuscu, Sevki Arslan, Ritambhara Singh, Jeremy Thorpe & Mazhar Adli
Nature Biotechnology advance online publication Received 24 January; accepted 25 April; published online 18 May 2014; doi:10.1038/nbt.2916




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