Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus

Abstract

The Streptococcus thermophilus CRISPR3-Cas (StCas9) system has been shown to mediate DNA cleavage in its original host and in E. coli as well as in vitro. Here, we have reconstituted the StCas9 system in yeast and conducted a systematic optimization of the sgRNA structure, including the minimal length of tracrRNA, loop structure, Match II region, Bulge motif, the minimal length of guide sequence within the crRNA, tolerance of mismatches and target sequence preference. The optimal sgRNA design for the StCas9 system achieved up to 12 and 40 % targeting efficiencies in yeast and human cells, respectively. This study provides important insight into the sequence and structural requirements necessary to develop a targeted and highly efficient eukaryotic gene editing platform using CRISPR-Cas systems.

Assembling the Streptococcus thermophilus clustered regularly interspaced short palindromic repeats (CRISPR) array for multiplex DNA targeting

“…By employing compatible restriction enzyme strategy, we can simply assemble multiple crRNA spacers into the CRISPR array for multiplex targeting. Firstly, we isolated industrial S. thermophilus stains from local yogurt as we did previously [3]. The strains were confirmed by PCR for amplifying the tracrRNA cassette and partial Cas9 fragment ( Figure S1) with primers tracrRNA.F1/R1 and Cas9.F2/R2, respectively (Table S1).…”

“…Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 3 Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated proteins (Cas) derived from bacteria adaptive immune system has been emerged as a powerful and versatile platform for biotechnological, biomedical and transgenic researches. For the type II CRISPR/Cas system, three minimal components, the Cas9 protein, the crRNAs transcribed from the CRISPR locus and the auxiliary trans-activating crRNA (tracrRNA), are sufficient for DNA recognition and targeting [1,2].…”

“…The crRNA::tracrRNA duplex, which can further be fused with a loop to generate a functional single guiding RNA (gRNA or sgRNA), directs the Cas9 protein for the recognition and targeting [2,3]. The single RNA guided CRISPR/Cas9 technology is scalable, affordable and easy to engineer, which makes it the focus of intense development for genome engineering application.…”

“…Considering that a guide sequence of 20 nt within a gRNA, which is derived from the crRNA spacer sequence, is longer enough for guiding the CRISPR/Cas9 nuclease activity [3,8,10], we hypothesized that the crRNA spacer sequence can be reconstituted with a restriction enzyme site (6 bp) and the interested guide sequence (24 bp). By employing compatible restriction enzyme strategy, we can simply assemble multiple crRNA spacers into the CRISPR array for multiplex targeting.…”

“…The two fragments were cloned into the ampicillin resistant (AmpR) pBlueScript II SK cloning vector by SacI/BamHI and BamHI/KpnI sites successively, to generate the parental C0:pCRISPR vector (Figure 1a). Then, we assembled the first DR fragment by directly annealing oligonucleotides (Figure 1b, Table S2) as we usually conducted [3,11], generating the C1:pCRISPR-DR1 sub-cloning vector which would serve as the backbone for further assembling and the negative control for the transformation assays. To mimic the Spacer-DR tandem CRISPR array, 5 different Spacer-DR DNA fragments containing interested crRNA spacer sequence, the adjacent DR and designed restriction enzyme sites [SalI-Interested guide (24 bp)-DR (36 bp)-XhoI-BamHI] were generated by primer extension [12] (Figure 1c, Table S2) and cloned into the XhoI/BamHI sites of the C1:pCRISPR-DR1 sub-cloning vector or the generated pCRISPR serial plasmids.…”