A cloning guide for pRGE vectors

Kabin Xie

Two RNA guided genome editing vectors (pRGE31 and pRGEB31, Figure 1) were developed for gRNA/Cas9 mediated genome editing in rice. Both vectors have same cassette to express gRNA and Cas9. The pRGE31 is smaller than pRGEB31 and is suited to transfect protoplast for transiently expression gRNA and Cas9. Binary vector pRGEB31 is derived from pRGE31 using pCAMBIA1300 backbone, and is used to generate stable transgenic plants by agrobacterium-mediated transformation.

A. Vector information.

Figure 1. Schematic illustration of pRGE31 and pRGEB31 vectors.

B. gRNA design.

1. Choose target sites and gRNA spacer sequence as illustrated in Figure 2. For most model plant species, specific target sites (or gRNA spacer) could be easily obtained from CRISPR-PLANT website (http://www.genome.arizona.edu/crispr).

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Figure 2. RNA guide genome editing using CRISPR-Cas9 system. 2. Design and synthesis oligos for selected spacers (see Figure 3).

Figure 3. Design oligos for pRGE31 or pRGEB31.

C. Cloning

3. Digest the vector as following: pRGE31 or pRGEB31 1 ug 10 X buffer 4 (NEB) 2 ul 10 X BSA 2 ul Bsa I (NEB) 1 ul Add H2O to 20 ul Incubate the digestion at 37oC for 2-4 hours. (optional) Add 0.5 ul of CIP (NEB), and incubate at 37 oC for 30min. Purify digested vector fragment.

2 4. Prepare DNA oligo duplex. Set up phosphorylation and annealing reactions in PCR tubes as following: Forward oligo (100 uM): 1ul Reverse oligo (100 uM): 1ul 10X T4 DNA ligase Buffer: 1ul T4 PNK (NEB) 0.5ul H2O 6.5ul 5. Incubate the tubes in a PCR instrument using following program: 37 oC 60 min 95 oC 10 min Cool down to 25 oC at 0.1 oC/sec Add 190 ul of H2O to dilute the oligo duplex and store in -20 oC. 6. Ligation. Bsa I digested vector: n ul (~50ng) Oligo duplex: 2ul 10X T4 DNA ligase Buffer: 0.5ul T4 ligase (NEB) 1ul Add H2O to 5ul Incubate at 25 oC for 1-3 hours or at 4 oC over night.

7. Transform DH5-alpha using 1-2 ul of ligation product (pRGE3 was selected by

AmpR and pRGEB31 was selected using KanR).

8. Check plasmid by diagnosis digestion. If restrict enzyme site was NOT introduced

in the foreign insertion, check plasmid by Bsa I and Sac I digestion (Bsa I sites will be lost in pRGE vectors with foreign insertion).

9. Confirm the gRNA sequence by Sanger sequencing using M13R(-48) primer.

D. Citation information Xie, K. and Y. Yang (2013). "RNA-guided genome editing in plants using a CRISPR-Cas system." Mol Plant 6(6): 1975-1983. PMID: 23956122

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