Journal: Plant Methods

Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

doi: 10.1186/s13007-017-0236-9

Figure Lengend Snippet: Construction and schematic of plasmid. a pEn-Chimera-ccdB. A cassette consisting of chloramphenicol resistance gene ( CmR ) and the ccdB gene was PCR-amplified and inserted between the AtU6-26(P) promoter and the sgRNA of pEn-Chimera using the In-Fusion ® HD cloning strategy as described in “ ” section. Plasmid pEn-Chimera-ccdB is used as template in PCR for fusing the 20-nucleotide protospacer sequence to the AtU6-26 promoter and sgRNA. Using the ccdB gene virtually eliminated any background colonies, which could arise due to incomplete digestion of pEn-Chimera using the restriction enzymes-based cloning method. b pDe-Cas9-D10A-2 gRNA: Schematic illustration of pDe-Cas9-D10A after two gRNA constructs, gRNA1 and gRNA2, are directly cloned in this vector using the In-Fusion ® HD cloning system. c pUC57GW: this is an in-house constructed Gateway ® -compatible Entry vector, which, in contrast to commonly used Gateway ® Entry/DONR vectors, contains the ccdB and Chloranphenicol ( CmR ) resistance genes. This unique design allows efficient cloning of gRNAs constructs in this vector using the In-Fusion ® HD cloning system without any background colonies. Please see “ ” and “ ” section for details

Article Snippet: A number of online tools are available for designing gRNA [ ], and we chose to use a free-of-cost gRNA design tool available online (DNA 2.0 Inc, https://www.dna20.com/ ).

Techniques: Plasmid Preparation, Amplification, Cloning, Sequencing, Construct, Clone Assay

Journal: Plant Methods

Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

doi: 10.1186/s13007-017-0236-9

Figure Lengend Snippet: Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

Article Snippet: A number of online tools are available for designing gRNA [ ], and we chose to use a free-of-cost gRNA design tool available online (DNA 2.0 Inc, https://www.dna20.com/ ).

Techniques: Cloning, CRISPR, Amplification, Plasmid Preparation, Sequencing, Expressing

Journal: Plant Methods

Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

doi: 10.1186/s13007-017-0236-9

Figure Lengend Snippet: Overview of In-Fusion ® -based cloning of two gRNA targets for paired nickases (Cas9-D10A). a Illustration of cloning strategy. Schematics of final gRNA cassette is shown in the top panel. Using a set of four universal primers (p1F, p2F, g1R and g2R) and four target-specific primers (g1F and p1R for protospacer target 1, and g2F and p2R for protospacer target 2), four fragments, A, B, C and D are PCR amplified using pEn-Chimera-ccdB plasmid in Round 1 PCR. In Round 2 PCR, using primers p1F and g1R, fragments A and B are fused resulting in fragment AB, and using primers p2F and g2R, fragments C and D are fused resulting in fragment CD. In Step 3, fragments AB and CD are cloned into pDe-Cas9-D10A or pUC57GW using the In-Fusion ® HD cloning system. b A representative gel picture showing PCR fragments of YFP upper panel, SlMLO1, NbPDS and mCherry lower panel. Expected sizes of each fragment are shown on the left. c Protospacer sequences of the targeted genes ( YFP upper panel, NbPDS middle panel, and mCherry lower panel) are highlighted in purple background and the PAM sequences NGG in red background

Article Snippet: A number of online tools are available for designing gRNA [ ], and we chose to use a free-of-cost gRNA design tool available online (DNA 2.0 Inc, https://www.dna20.com/ ).

Techniques: Cloning, Amplification, Plasmid Preparation, Clone Assay

Journal: Plant Methods

Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

doi: 10.1186/s13007-017-0236-9

Figure Lengend Snippet: Mutation detection using Agrobacterium -mediated transient expression in N. benthamiana. a Schematic illustrations showing the locations of gRNAs, PAM positions, and primers in the genes , NbPDS , PIP2 - - mCherry and YFP targeted for genome editing. b Resolvase assay for detecting mutation in NbPDS and mCherry targeted by the gRNA constructs pDe-Cas9-D10A-gNbPDS and pDe-Cas9-D10A-gmCherry. High fidelity PCR was performed with primers NbPDS-F and NbPDS-R for the NbPDS target, and mCherry-F and mCherry-R for the mCherry target (panel A and Table ) using template DNA isolated from leaf spots on N. benthamiana infiltrated with Agrobacterium tumefaciens carrying the indicated CRISPR–Cas9 constructs; N. benthamiana was stably transformed with the PIP2 - - mCherry gene, which served as a transgene target for the gmCherry gRNA. Amplicons were subjected to Resolvase assays (Guide-it™ Mutation Detection Kit, Cat#631443, Clontech) and reactions were run on a 1.5% agarose gels. Digested fragments, which result from gRNA-induced mutations, are indicated by *, and their sizes are ~ 570 and ~ 150 bp for NbPDS, and ~ 277 and ~ 150 bp for mCherry. Undigested fragments are 727 bp for NbPDS and 427 bp for mCherry. Lower panel shows a representative N. benthamiana leaf infiltrated in three independent sites with the indicated CRISPR–Cas9 constructs. c Surveyor (CEL II)—nuclease assay for detecting mutation in a transiently transformed YFP gene carried on pGWB415-35S::HA-YFP plasmid. PCR was performed with forward primer (35SpromF) and reverse primer (EYFPStopXhoR) (panel A and Table ) using template DNA from Wild Type N. benthamiana co-transformed with pGWB415-35S::HA-YFP along with either pDe-Cas9-D10A-gYFP or pDe-Cas9-D10A-gNbPDS (negative control). Surveyor (CEL II)—nuclease assay was performed with amplicons and reactions were run on 1.5% agarose gels (mismatch-specific Surveyor nuclease, Surveyor ® Mutation Detection Kit; Cat#706025, IDTdna.com). gRNA-induced mutations are revealed by the digested ~ 700 and ~ 400 bp fragments, marked by *; the undigested fragment is 1067 bp, which is shown in both lanes. Lower panel shows a representative N. benthamiana leaf infiltrated in three independent sites with the indicated CRISPR–Cas9 constructs

Article Snippet: A number of online tools are available for designing gRNA [ ], and we chose to use a free-of-cost gRNA design tool available online (DNA 2.0 Inc, https://www.dna20.com/ ).

Techniques: Mutagenesis, Expressing, Construct, Isolation, CRISPR, Stable Transfection, Transformation Assay, Nuclease Assay, Plasmid Preparation, Negative Control