<div style="font-family:Hoefler Text; font-size: 14px; color: "black" >After running GuideMaker, the designed gRNA output can be downloaded and with minor adjustments, the targets can be ordered as oligos for cloning. Pooled oligonucleotides can be purchased from several vendors, including those listed below. Pool sizes vary from 100 to over 200,000 oligonucleotides. Vendor specifications for the number of oligos, oligo length, and cost per bp vary widely. For bacterial genome-scale experiments, as of 2021, Genscript offers pool sizes of 12,472 and 91,766 with up to 79 bp per oligo for list prices of $1600 and $4,000, respectively.
Some example vendors are:
</div> <div style="font-family:Hoefler Text; font-size: 14px; color: "black" >Most pools require amplification before cloning to convert the ssDNA to dsDNA and increase the concentration for efficient cloning. Accordingly, adding a constant region at the 3' end for primer binding is recommended. Sub-pools can also be amplified by adding unique constant regions to some oligos, enabling the large-scale synthesis to be split amongst organisms or specific targets in a single organism. Because Golden Gate cloning utilizes restriction enzymes, filtering gRNA designs with the cognate restriction enzyme recognition sites is necessary, a feature found in GuideMaker. A general protocol for cloning pooled gRNA from synthesized oligonucleotides from IDT is linked below, though similar workflows can be used for pools from other vendors.
<div style="font-family:Hoefler Text; font-size: 14px; color: "black" >After the experiment, the cells are collected and DNA is isolated. The target sequence is then amplified and adaptors for high-throughput sequencing added. Several data analysis pipelines have been developed to identify target sequences over-represented or under-represented in the pool. The manuscript by Wang et al. (2019) provides a protocol for using a high-quality tool with these capabilities. </div>
<div style="font-family:Hoefler Text; font-size: 14px; color: "black" >Wang, B., Wang, M., Zhang, W. et al. Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute. Nat Protoc 14, 756–780 (2019). https://doi.org/10.1038/s41596-018-0113-7 </div>