Dear Saif, 

There are some points that you should take in consideration. Is your protein also of bacterial origin?, if not, you should adapt its sequence to Ecoli codon usage. And to be able to be expressed in the chromosome of E.coli, you should also include promoter and stop sequences. Why you said that your should choose an exon site?, this will mean that your protein is of eukaryote origin, isn't it? just remember that bacteria has not intron split system, and transcription and translation are coupled. 

The integration site in E.coli chromosome depends on how big is your protein, and all your system, meaning that if your protein has more than one subunit, all the implied genes should be introduced. 

The best way to select the target is to use a software to design your gRNAs. There are several web based software to design you gRNA. Here is one example. 

https://www.dna20.com/eCommerce/cas9/input

But, you will need to read some more info, as to produce a Knock-in in E.coli y ou will need also to use Lambda-red to get best results. 

Here attached are some papers that i though will be useful for you. There are many more, but for knock-out rather than to knock-in. 

hope this helps you, 

Beatriz Sabater-Munoz

Dear Saif, 

There are some points that you should take in consideration. Is your protein also of bacterial origin?, if not, you should adapt its sequence to Ecoli codon usage. And to be able to be expressed in the chromosome of E.coli, you should also include promoter and stop sequences. Why you said that your should choose an exon site?, this will mean that your protein is of eukaryote origin, isn't it? just remember that bacteria has not intron split system, and transcription and translation are coupled. 

The integration site in E.coli chromosome depends on how big is your protein, and all your system, meaning that if your protein has more than one subunit, all the implied genes should be introduced. 

The best way to select the target is to use a software to design your gRNAs. There are several web based software to design you gRNA. Here is one example. 

https://www.dna20.com/eCommerce/cas9/input

But, you will need to read some more info, as to produce a Knock-in in E.coli y ou will need also to use Lambda-red to get best results. 

Here attached are some papers that i though will be useful for you. There are many more, but for knock-out rather than to knock-in. 

hope this helps you, 

Beatriz Sabater-Munoz

Thank you Beatriz, it was more than helpful.