مقالات پذیرفته شده در سومین کنگره بین المللی زیست پزشکی
Construction of an all-in-one CRISPR/Cas9 vector for CRIS-PITCh system
Construction of an all-in-one CRISPR/Cas9 vector for CRIS-PITCh system
Samaneh Ghanbari,1,*Sana tabatabaei,2Fatemeh Davami,3Pezhman Fard-Esfahani,4Masoumeh Azizi,5
1. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 2. Dept. of Biotechnology, College of Science, University of Tehran, Tehran, Iran 3. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 4. Department of BiochemistryPasteur Institute of IranTehranIran 5. Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Ira
Introduction: CRISPR/Cas9 as a new genome engineering technology enables gene knock-in by utilizing DNA DSB repair pathways. Although homologous recombination (HR) is a well-established repair pathway which is harnessed for gene knock-in, construction of donor vector for HR- mediated knock-in is complicated since it requires long homology arms. Moreover, HR frequencies are variable, so it cannot be applied to every cell type. For the first time in 2014, Nakade et al. developed an alternative method for gene knock-in termed the PITCh (Precise Integration into Target Chromosome) system which utilizes microhomology-mediated end-joining (MMEJ) pathway. This system does not rely on HR, so long homology arms are not needed. However, linearization of the donor vector is required for MMEJ-mediated knock-in so this system rely on two different gRNAs, one universal gRNA named PITCh gRNA which should be cloned into CRIS-PITCh vector and its target site should be added into both 5′ and 3′ of microhomology arms in donor vector to be in vivo linearized and the other gRNA for targeting the genomic site. The protocol published for construction of all-in-one CRISPR-Cas9 vector for CRIS-PITCh is a two-step golden gate assembly of genome targeting gRNA and universal PITCh gRNA, consequently cloning of PITCh gRNA is required every time that new genome targeting gRNA wants to be cloned. Besides the plasmid used in this protocol (px330A-1) does not include any reporter gene, which makes it impossible to assess transfection efficiency or fluorescent enrichment which is pivotal in knock-in projects. In this research, we construct an all in one CRIS-PITCh vector into which PITCh gRNA cassette has been cloned therefore only one-step cloning of genome targeting gRNA is needed. Furthermore, the reporter gene in the vector will enable subsequent assessment of transfection efficiency as well as fluorescent enrichment.
Methods: Plasmid construction
Oligonucleotides for PITCh-gRNA were synthesized (Metabion, Germany), phosphorylated and annealed. Annealed gRNA was inserted into bbsI digested pU6-(BbsI) _CBh-Cas9-T2A-mCherry (addgene, plasmid 64324) and transformed into chemically competent E. coli TOP10F', the cells were plated on LB agar containing 100 µg/ml ampicillin and 15 µg/ml tetracycline. Ampicillin resistant colonies were screened by colony PCR for desired insert using u6 forward primer and PITCh reverse primer. Plasmid was extracted from a positive clone, digested with AflIII and EcoRI and analyzed by gel electrophoresis. Then the entire cassette of PITCh-gRNA including U6 promoter- PITCh gRNA- scaffold was PCR amplified using primers containing XbaI and KpnI sites, the PCR product was cloned into XbaI and KpnI digested wild- type pU6-(BbsI) _CBh-Cas9-T2A-mCherry. Cloning was verified with colony PCR, and Sanger sequencing (Macrogen, Korea).
Results: To verify PITCh-gRNA cloning into bbsI site, transformants were screened by colony PCR using u6 forward and PITCh reverse primers leading to a 273 bp amplicon. The vector named PITCh vector was then digested with AflIII and EcoRI. Fragments were verified by gel electrophoresis. For cloning of U6-PITCh gRNA-scaffold cassette into XbaI and KpnI sites, the cassette was PCR amplified and 441 bp amplicon was confirmed by gel electrophoresis. Colony PCR using ORI-f forward and PITCh reverse primer results in 1018 bp amplicon in four clones. Finally, plasmid was sent for sequencing, the blast result shows the perfect match of in silico cloned U6-gRNA-scaffold using snapgene with sequencing result.
Conclusion: In summary, we have constructed an improved version of all-in-one CRISPR/Cas9 vector for CRIS-PITCh system. This vector has two advantages in comparison to the old one. First, PITCh gRNA expression cassette is already cloned so it eliminates two-step golden gate assembly of two gRNAs. Second, the vector contains red fluorescent protein, mCherry which co-express with Cas9, therefore, make it possible to assess transfection efficiency or fluorescent enrichment of CRIS-PITCh vector following transfection. It has been shown in the previous study that fluorescent enrichment resulting in a threefold increase in the number of knocked-in cells, application of this approach is therefore worth considering for the PITCh system.