Primary Investigation Into SpCas9 Cleavage Mechanism And Its Application In Biosynthetic Gene Clusters Cloning

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated(Cas) protein, Cas9, is an RNA-guided endonuclease that uses RNA-DNA base pairing to recognize and cleave double-stranded DNA (dsDNA) with a protospacer adjacent motif (PAM). Cas9 can be guided to different target DNA by altering 20 nt (nucleotide) guide sequence of guide RNA. The programmable design and simplicity make CRISPR/Cas9 an important gene editing tool and it has been widely applicated in many organisms.Streptococcus pyogenes Cas9 (SpCas9) is the most widely used Cas9. It strictly requires the presence of NGG PAM sequence in target DNA. In this study, we found firstly that guanosine G1 of 5'-NG1G2-3'in PAM is sufficient but notnecessary for SpCas9-mediated in vitro cleavage at a higher concentration. Importantly, we also revealed for the first time that in vitro annealing of dsDNA with exogenous PAM-presenting oligonucleotides (PAMmers) stimulated SpCas9 cleavage of target dsDNA without PAM, and this might open the way for enhancing its DNA editing abilities.It has been well documented that the HNH and RuvC nucleasedomains of SpCas9 are used for double-stranded DNA cleavage at a position 3 bp upstream of the PAM sequence, yielding a blunt end. Here, we artificially changed relative position of NGG and protospacer, and end-sequenced SpCas9-mediated cleavage products. We found that the cleavage site of complementary strand is completely determined by the location of guide RNA while the location of guide RNA and NGG PAM sequence jointly determine the cleavage site of non-complementary strand.Moreover, it was also observed in our study that NGG location was an important factor that could induce off-target of SpCas9 in vitro. SpCas9 facilitated substantial cleavage of target DNA when NGG overlapped with protospacer by 1 bp or spaced by 1,2 even 3 bp.When NGG was repositioned, the tolerance of SpCas9 to mismatch between target sequence and guide sequence in sgRNA decreased significantly. We also found that DNA configuration can also affect SpCas9 cleavage. SpCas9 showed obvious better cleavage towards supercoiled circular DNA than linear DNA at some off-target sites in vitro. These observations are expected to improve our understanding of SpCas9.On the other hand, SpCas9 shows higher specificity and lower limitation than type II restriction enzymes, it can make SpCas9 show extraordinary talents in in vitro use. Moreover, with the development of DNA sequencing and by bioinformatic analysis, people are full of expectation of abundant biosynthetic gene clusters from nature that encode natural products of great application potential. Frequently, these clusters need to be cloned and heterologous expressed for further study. Here, we combined SpCas9 and Gibson assembly, a highly efficient DNA assembly technology, to clone biosynthetic gene clusters in vitro. Firstly, we successfully cloned an gene cluster that may encode an unknown Tetronate secondary metabolite from deep-sea Shewanella psychrophila WP2 by one-step SpCas9-Gibson assembly. We subquently found the possible product of this gene cluster by heterologous expression in Escherichia coli BL21(DE3). Secondly, we successfully assembled and cloned the complete aminoglycoside Sisomicin gene cluster. The work highlighted the ability of combination of SpCas9 and Gibson assembly for cloning target DNA fragments, especially large DNA fragments with high G+C content and discontinuous DNA fragments.