cas9 validation by DNA seq

Question

[Lital]

Hi guys!

Our team recently used crispr to delete 45 bases (15 amino acids) from the DNA of a particular protein. To make sure the deletion was done, we ran a 135-base PCR product that contains the sequence we delete + bases from the beginning and end.

This is the first time I work with DNA sequences. Is there a specific protocol I have to work by?

When I do alignment to the unprocessed files, I got that there is a 6-base deletion instead of a 45-base deletion. When I clear sequences and filter, I get a very small number of short sequences.

In addition, we also ran the product of the PCR in gel and there we saw that there is a difference between the WT samples and the DEL samples (the DEL bands were above the WT bands).

Does anyone have an idea?

Thank you!

[ATpoint]

How did you align the sequences? Be sure to use a global aligner such as EMBOSS-Needle which performs end-to-end alignments rather than something like BLAST which is not meant to capture longer deletions and favours local alignments as long as it returns a good alignment score.

Example:

Say the top was the WT sequence and the bottom was the sequence after deletion:

GTAGCGTACTGACGACGTATCTTATCGACGCGCGCGCGCGCGCGTAGCATCTCTTCGACGTACGACATCTACGCTACGACACATCACACTAC
GTAGCGTACTGACGACGTATCTTATCGACGCGCGCGCGCGCGCGTAGCATCTCT                            ATCACACTAC

EMBOSS-Needle gets it correctly:

ref                1 GTAGCGTACTGACGACGTATCTTATCGACGCGCGCGCGCGCGCGTAGCAT     50
                     ||||||||||||||||||||||||||||||||||||||||||||||||||
sanger             1 GTAGCGTACTGACGACGTATCTTATCGACGCGCGCGCGCGCGCGTAGCAT     50

ref               51 CTCTTCGACGTACGACATCTACGCTACGACACATCACACTAC     92
                     ||||                            ||||||||||
sanger            51 CTCT----------------------------ATCACACTAC     64

Whereas BLAST:

Query  1   GTAGCGTACTGACGACGTATCTTATCGAcgcgcgcgcgcgcgcgTAGCATCTCT  54
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  1   GTAGCGTACTGACGACGTATCTTATCGACGCGCGCGCGCGCGCGTAGCATCTCT  54

The alignment score of this alignment is simply higher than of the full-length sequence with all the deletions which create penalty scores. Hence, BLAST is not suitable here.

[Lital]

Thank you for the detailed answer! I used 2 methods STAR and bowtie2 ( I did the alignment twice to make sure). For visualization, I used IGV.

[ATpoint]

Just to confirm, this is Sanger sequencing data you have, right? I would just give the EM a try, the nice thing is that you can really see by-eye how the actual alignment looks base-to-base.

[Lital]

No it's not a Sanger sequencing data, We did it by NGS (Illumina).