I am using the following protocol from a CSH lab manual edited by Doudna on CRISPR, to attempt to retarget sgrna scaffold in a plasmid.
but....I get DNA stuck in the well, not migrating. Suspecting very long, heavy concatenation product? I might try 21 cycles and not 30 as they call for.
protocol:
"2. Order two 60-mer oligonucleotides, one with the sgRNA guide coding sequence (Step 1with the reverse complement of the coding sequence. 3. Dilute each of the two 60-mers to 100 μM in water. 4. Assemble the reaction in a polymerase chain reaction (PCR) tube: Phusion HF buffer (5×) 5 μL dNTP (10 mM total; 2.5 mM each) 0.5 μL Coding sgRNA guide sequence (60-mer; 100 μM) 0.1 μL Reverse complement of sgRNA (60-mer; 100 μM) 0.1 μL pCAS plasmid 40 ng Phusion DNA Polymerase 1 μL H2O to 25 μL
Perform thermocycling with the following profile, (over 300 minute pcr!)
1 cycle 98 ̊C 1 min 30 cycles 98 ̊C 30 sec 58 ̊C 1 min 72 ̊C 10 min 1 cycle 72 ̊C 10 min 4 ̊C Hold 6. Add 1 μL of DpnI, 3 μL of the provided 10× digestion buffer, and 1 μL of water to the reaction and incubate for 6 h at 37 ̊C (or overnight). 7. Transform the DpnI-treated reaction into E. coli competent cells."
I have tried fewer cycles (21), extension time of 5:30, and high GC buffer, dmso, etc.
I also tried lower template concentrations after the DpnI reaction at 50% and 10%, but I got very low pcr yields from those.
Has anyone tried this method? Any tips/pointers?
If it would work, it means not having to obtain a unique sgrna vector for every CRISPR experiment.
CRISPR–Cas9 Genome Engineering in Saccharomyces
cerevisiae Cells
Owen W. Ryan,1 Snigdha Poddar,1 and Jamie H.D. Cate1,2,3,4
1
2Energy Biosciences Institute, University of California, Berkeley, California 94720; Department of Molecular and
3Cell Biology, University of California, Berkeley, California 94720; Department of Chemistry, University of
California, Berkeley, California 94720
This protocol describes a method for CRISPR–Cas9-mediated genome editing that results in scarless
and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results
from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a
uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule
that is used to repair the chromosomal DNA damage by homology-directed repair. For target specif-
icity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA
sequence within the sgRNA cassette. This CRISPR–Cas9 protocol includes methods for (1) cloning the
unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides,
and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically
facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial
polyploid isolates. Therefore, this CRISPR–Cas9-based DNA integration protocol is achievable by
virtually any yeast genetics and molecular biology laboratory.
thank you! - Amy J.
Article Customized one-step preparation of sgRNA transcription templ...
Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing
- Zheng Hu,
- Li Wang,
- Zhaoying Shi,
- Jing Jiang,
- Xiangning Li,
- Yonglong Chen,
- Kai Li &
- Dixian Luo
Cell & Bioscience volume 9, Article number: 87 (2019) Cite this article
I think I may have got this to work! One of my pcr runs on the plasmid has a very faint band in the right place next to the ladder. It was slightly more visible to the eye, hard to photograph with an iphone. There is ladder, an empty well and next to that a faint band.
I had someone else look at it, it isn't just my poor eyesight or wishful thinking! So if all went well that is supercoiled plasmid that has been retargeted by oe pcr ;
the band is sharper to the eye, this is hard to photograph w one person holding a long wave UV and another with iphone!
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