1. Sequence submitted - In the context of using gRNA to interfere with gene expression, gRNA should be designed as much as possible in the 2nd or 3rd exon after the start codon ATG. Being too close to the ATG may lead to alternative translation, resulting in the translation of a functional protein with a missing N-terminus. Conversely, if gRNA targets are too close to the stop codon, it might lead to the translation of a protein with a missing C-terminus, which could also be functional. We default to and recommend using the 2nd or 3rd exon after the exon containging ATG.

2. gRNA size - For different Cas proteins, the corresponding gRNA lengths can vary and typically range from 19 to 30 base pairs.

3. Starting nucleotide - The U6 promoter shows a preference for guanine (G) as the starting nucleotide, while the H1 promoter exhibits a preference for adenine (A) as the starting nucleotide.

4. GC content - It is generally recommended to choose sequences with a low GC content. This program defaults to using a GC content range between 35 % and 65 %.

5. Internal repeats - gRNAs lacking internal repeats are strongly preferred. Internal repeats include 6x[G/C], 6x[A/T], repeated Gs/Ts/As/Cs. Sequences containing these internal repeats are discarded.

6. Palindromes - gRNAs lacking palindromes are strongly preferred. Candidates containing palindromes with a length of 8 or more bases are discarded.

7. BLAST - C-terminal of gRNA presenting more than 12 contiguous nucleotides matching with other coding sequences is discarded.

8. Recommend gRNAs - All the gRNAs that remain after rigorous screening criteria theoretically have the potential to effectively target the gene of interest and minimize the possibility of off-target effects.

9. Disclaimer - The effectiveness of gRNA designed using computational methods can enhance target gene silencing possibilities, but experimental validation is necessary to confirm its efficacy in specific biological contexts. Actual results may vary due to various factors, and additional validation is required for conclusive evidence of gRNA performance. For optimal silencing efficacy and to minimize off-target effects, it is recommended to design and use at least two different gRNAs.