In some cases, no design results are returned using the default parameters. Below are potential causes and suggestions for modifying the default settings to obtain designs:

• Your gene may have non-overlapping transcripts. The default CRISPR Design Tool settings require targeting of all transcripts. In Advanced Options, specific transcripts may be Excluded or Allowed, rather than Required, to loosen the design criteria.
• You might be using protein-coding gene settings with a noncoding gene target. Go back to the Design Purpose and select: Long non-coding RNA locus.
• Your gene does not contain a NGG PAM. In Advanced Options, try including NAG PAM or uncheck “Require cuts to be in CDS”.
• All potential target sites have GC percentages outside the default parameters. In Advanced Options, expand the GC Percentage settings.
• Your gene does not contain target sites with more than two mismatches to PAM-adjacent sites in the selected genome. Deselect the Specificity Check. Keep in mind that the designs without Specificity Check are very likely to off-target.

In some cases, no crRNA design results are returned using the default parameters. Below are examples of why the default settings may return no crRNA designs, and suggestions for obtaining designs:

• Your DNA sequence does not contain a NGG PAM. In advanced options, try including NAG PAM.
• Your DNA sequence has target sites with GC percentages outside the default parameters. In Advanced Options, expand the default GC Percentage settings.
• Your DNA sequence only contains target sites that have possible off-targets to the selected genome. Expand the provided DNA sequence or deselect the Specificity Check.

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