Paper of the month April 2018

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

In many bacteria and archaea, CRISPR/Cas9 functions as the acquired immunity system against viruses and plasmids by targeting and cutting the exogenous  DNA. Scientists exploit the DNA recognition and cutting property of CRISPR/Cas9 as “scissor” for genome editing. The Cas9 enzyme is led to the specific genome locus by a single-guide RNA (sgRNA), which works like a guiding dog for blind people, and cut that site. Theoretically, sgRNA sequence can be designed to generate a cut at any site desired. However, the Protospacer Adjacent Motif (PAM)`availability has to be taken into account. The presence of the PAM nearby the target facilitates recognition of the Cas9 protein which then cleaves the target DNA. CRISPR/Cas9 of different bacteria or archaea requires various PAM sequences. The most used Cas9 variant is spCas9, which derived from Streptococcus pyogenes, and possesses the least restrictive PAM DNA sequence known as NGG. Theoretically, about 1/16 of the genome contain the sequence of NGG. But spCas9 cannot process the sequences without NGG nearby. In the recent paper of David R. Liu’s group, they found the method to evolve a new version of Cas9 able to recognise other PAMS which are different from the canonical one.

In this paper, the authors tackled the challenges by taking advantage of the phage-assisted continuous evolution (PACE) system. Phages are bacterial virus that can infect and replicate in bacteria. In this assay, they use the phages  lacking the infectious gene (gene III), so that they need  exogenous gene III to be infectious and spread. The exogenous gene III expression is carried out by the plasmid containing gene III in E.coli, but its expression is under the control of the bacterial RNA polymerase. The RNA polymerase in that E.coli is not sufficient due to the lack of the ω subunit. The authors package the phage with the DNA that express SpCas9 fused to the ω subunit of the RNA polymerase, so that when the spCas9 binds to the designated PAM sequences situated upstream of the gene III will lead to the hybridization of the functional RNA polymerase and downstream expression of gene III and `the phage infectivity will be restored The principle of this amazing assay is simple: only if the phage evolves the desired protein, the latter will promote the expression of the Gene III by the host cells and the phage will replicate and spread. With this approach the authors can mimic the evolution process in a bioreactor and they could obtain different Cas9 variants (called x.Cas9). Striking is the finding for example of the xCas9(3.7) able to recognise up to 5 different PAMs! This xCas9, along with the other variants, showed a cleavage activity comparable or even higher than the spCas9. This cleavage activity was also evaluated in human cell lines (HEK293, U2OS) and the results left no doubt about the xCas9 efficiency. Moreover, they were showed to be employable also for targeted gene-activation, by fusing the xCas9 to a transcriptional activator like VPR – a protein increasing gene expression.

Although the enhanced PAMs recognition spectrum may correlate with augmented off-target activity - cleaving undesired genomic locations both being NGG and non-NGG sequences - and so loss of specificity by the Cas9, the data provided shows that the xCas9 variants retain their specificity and they are even more accurate than the spCas9 itself!

We highly recommend reading this paper, where the evidences provided on the one hand, more insights about which are the amino acidic residues that rule the PAM recognition within the Cas9 and on the other hand, the authors offered an enhanced tool able to dramatically broaden the targetable genes within the genome.