The PAM sequence depends upon origin of Cas9 gene. You can use Cas9 of different origin or you can target a gene at 2-3 different regions with different gRNA for getting proper results. Read a recent review with this regard in The Plant Biotechnology journal advance online section.

Thanks. I have actually designed three gRNA and with separate round of transformations.Yet to no avail. I hope to check for the paper though

Just to get a better idea of what we're looking at here:

1. What kind of cells are you working with?

2. What is the nature of the desired mutant? Are you trying to introduce indels, or make a precise change to the nucleotide sequence?

3. What are you using to deliver the CRISPR/Cas9 components? Are you for example injecting Cas9 protein, or are you transfecting a plasmid-based expression system?

1.I work with Filamentous fungi 

2. I intend to cause a double stranded break in the nucleotide sequence of the gene of interest

3. PEG mediated transformation of the WT with the cas 9 expressing plasmid containing the sgRNA.

Thanks