Hello,

I have tried to use the no-SCAR CRISPR Cas protocol by Reisch and Prather (2017) on E.coli BL21 (DE3) strain and am struggling to get it to work. I have transformed my strain with the cas9 plasmid and the sgRNA plasmid and when I plate on anhydrotetracycline (Atc) plates I get cell death, so my sgRNA is correct and the cas protein is able to cleave the DNA. However when I add my DNA I am only getting wild type sequence back. Could someone please help me, check my DNA sequences, or anything that I have missed as I have been attempting this for several months now.

I have 3 mutations that I need to make, one in the middle of a gene (505) and 2 mutations at the end (1042/1044). I simply need to change them into alanines. My gene is on replichore 1. The sequence for these areas are as follows:

505:

(mutation point is not capitalised)

GTGGCGTTGATCCTGACTCCAGCTCTTTGTGCCACCATGCTGAAACCGATTGCCAAAGGCGATcacGGGGAAGGTAAAAAAGGCTTCTTCGGCTGGTTTAACCGCATGTTCGAGAAGAGCACGCACCAC

1042/1044:

GTTCCGGTATTCTTTGTGGTGGTTCGCCGCCGCTTTAGCCGCAAGAATGAAGATATCGAGcacAGCcatACTGTCGATCATCATTGATACAACGTGTAATCACTAAGGCCGCGTAAGCGGCCTTTTTTATGCATA

For the targeting in 505 I have tried the following site:

GCCAAAGGCGATcacGGGGA AGG (Agg is not included in my sgRNA sequence)

1042:

Gcc ATACTGTCGATCATCATTGA (Gcc not included) Because this is a CCN site I use the reverse complement for the sgRNA which is TCAATGATGATCGACAGTAT (20 bases not including PAM site).

For the 505 site the cell killing is not hugely efficient but it does occur in the Atc plates. I have recently swapped PAM sites and am in the process of continuing this. H1042 sequence leads to highly efficient cell death.

My DNA donor sequences are:

505: C*G*T *TGA TCC TGA CTC CAG CTC TTT GTG CCACCA TGC TGA AAC CGA TTG CTA AGG GGG ACGCCG GCG AAG GTA AAA AAG GCT TCT TCG GCTGGT TTA ACC GCA TGT TCG AGA AGA GC

1042:

T*C*T *TTG TGG TGG TTC GCC GCC GCT TTA GCCGCA AGA ATG AAG ATA TCG Aag ctt cgg caA CTG TCG ATC ATC ATT GAT ACA ACG TGT AATCAC TAA GGC CGC GTA AG

*= phosphorothioate bonds

Both DNA strands have about 50bp homologous arms on either side of the mutation site. The only thing I am confused by is the orientation of the DNA. In the paper they state, “If the target is on replichore 1 (> 3923998 or < 1588774), then the oligo sequence should be complementary sequence to the (+) strand sequence.”

A note from a PI in my lab who has succeeded using CRISPR cas in this gene in the past sent me some that said. “Gene is on the replichore 1 so according to the protocol the primer has to be complementary to the (+) strand. As the gene is on the complementary strand so the oligo sequence will be the same than gene sequence.” So is my DNA in the right orientation as this has confused me?

Sorry for the long question but I cannot figure out why CRISPR cas isn't working. For anyone who has taken the time to try and solve this, thank you sincerely.