Assembling the Streptococcus thermophilus clustered regularly interspaced short palindromic repeats (CRISPR) array for multiplex DNA targetingby Lijun Guo, Kun Xu, Zhiyuan Liu, Cunfang Zhang, Ying Xin, Zhiying Zhang

Analytical Biochemistry


Molecular Biology / Biochemistry / Biophysics / Cell Biology


Accepted Manuscript

Notes & Tips

Assembling the Streptococcus thermophilus CRISPR array for multiplex DNA targeting

Lijun Guo, Kun Xu, Zhiyuan Liu, Cunfang Zhang, Ying Xin, Zhiying Zhang

PII: S0003-2697(15)00095-0


Reference: YABIO 11998

To appear in: Analytical Biochemistry

Received Date: 2 January 2015

Revised Date: 25 February 2015

Accepted Date: 26 February 2015

Please cite this article as: L. Guo, K. Xu, Z. Liu, C. Zhang, Y. Xin, Z. Zhang, Assembling the Streptococcus thermophilus CRISPR array for multiplex DNA targeting, Analytical Biochemistry (2015), doi: 10.1016/j.ab.2015.02.028

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Assembling the Streptococcus thermophilus CRISPR array for multiplex DNA targeting

Lijun Guo#, 1, Kun Xu#, 1, Zhiyuan Liu1, Cunfang Zhang1, 2, Ying Xin1, Zhiying Zhang*, 1 1 College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi, 712100, China 2 Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology,

Chinese Academy of Sciences, Xining, Qinghai, 810008, China *To whom correspondence should be addressed. Tel: +86-029-87092102; Fax: +86-029-87092164; Email: # Co-first authors

Subject category: DNA Recombinant Techniques and Nucleic Acids

Short title: Assembling CRISPR array for multiplex targeting 2


In addition to the advantages of scalable, affordable and easy to engineer, CRISPR/Cas technology possesses the superiority for multiplex targeting, which remains laborious and inconvenient to be achieved by cloning multiple gRNA expressing cassettes. Here, we report a simple CRISPR array assembling method which will facilitate the multiplex targeting usage. Firstly, the Streptococcus thermophilus

CRISPR3/Cas locus was cloned. Secondly, different CRISPR arrays were assembled with different crRNA spacers. Transformation assays using different Escherichia coli strains demonstrated efficient plasmid

DNA targeting, and we achieved targeting efficiency up to 95% with an assembled CRISPR array with three crRNA spacers.

Key words:Cas9; CRISPR array; CRISPR/Cas; multiplex targeting; Streptococcus thermophilus 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. Thus, a dozen of CRISPR/Cas systems derived from different bacterial strains have been developed and emerged using similar strategies.

Furthermore, compared with the designed zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas technology possesses the superiority for multiplex targeting.

However, we found that the currently popular multiplex usages mostly preferred to involve several different RNA Pol III promoters in a single vector with each promoter driving one gRNA expression [4-6].

An alternative method used a single RNA Pol III promoter (tRNA) for multiple gRNA co-expression. The gRNA co-expression product was designed to be processed internally by HDV ribozyme [7]. Unfortunately, it is very laborious and inconvenient for these methods to clone and assemble the multiple Pol III-gRNA or

HDV-gRNA cassettes [4-7]. Then, we asked whether we could simply mimic the bacterial CRISPR array for multiplex targeting, and the answer was yes as previously reported with a synthetized CRISPR array containing three direct repeats (DRs) and two crRNA spacers [8, 9]. But when we designed a CRISPR array harboring four DRs and three crRNA spacers, the synthesis was not suggested by the company (Genscript, Nanjing, China) due to the difficulty and high mutagenesis. Hence, there remains a need for 4 approaches to artificially assemble the CRISPR array, which will facilitate the multiplex targeting usage for CRISPR/Cas technology. Here, we report the cloning of the Streptococcus thermophilus (S. thermophilus) CRISPR3/Cas locus, the detailed design for the assembling of CRISPR arrays (Figure 1) and the functional assays using different Escherichia coli (E. coli) strains (Figure 2).

The native S. thermophilus CRISPR3 array consists of short conserved direct repeats (DRs; 36 bp) interspaced by unique crRNA spacers of the same size (30 bp), which involves complicated spacer assembling and precrRNA maturing processes. 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. 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). Secondly, colony PCR reactions were performed with primers tracrRNA.F1/crLeader.R1 and Term.F3/R3 (Table S1) for a ~6.7 kb fragment (consisting of the tracrRNA cassette, the locus of four Cas genes and the CRISPR leader sequence) and the 103 bp CRISPR terminator. 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