What are the considerations for target design in CRISPR?

1. The length of sgRNA is about 18~20 nucleotides;

2. For the base composition of sgRNA sequence, sgRNA with NGG in 3 'end is optional; NGG is commonly referred to as the PAM sequence, and the PAM sequence varies with the strain from which Cas9 is derived, so when designing gRNA targets, PAM at the 3 'end needs to be consistent with the strain. Note that the sequence of sgRNA cannot contain the sequence of PAM.

3. For the base composition of sgRNA sequence, 4 or more T should be avoided in the seed sequence (13 bases before PAM), while GC% content is generally about 40%~60%.

4. The matching number of seed sequences in sgRNA and off-target sites should be as low as possible;

5. If the U6 or T7 promoter is used to drive the expression of sgRNA, the 5' -end base of sgRNA should be considered as G or GG to improve its transcription efficiency;

6. If Cas9 is to destroy the target gene, the gRNA target should be close to the 5 'end of the gene, that is, the n-end of the protein coding, so as to increase the possibility of gene destruction;

7. The presence of SNPs should be detected in the genomic sequence corresponding to the sgRNA targeting binding site;

8. For the analysis of the off-target effect of the whole gene, the maximum number of mismatched bases allowed at the off-target site should be considered, and a minimum of 5 bases is recommended. Focus on the number of base mismatches between the seed sequence and the non-seed sequence, and whether the off-target site is located in the coding region of the gene, etc., and whether there is an off-target site with base insertion or deletion;