hey,
I am designing gRNA target site to check the endogenous expression of protein in zebrafish. For this I am using chop-chop site. But most of the sites are in exonic region. I couldn't find best target site with restriction site in it. So, one site is just in intron 1 which i could find with efficiency more than 50%.
I am doing this for the first time. I appreciate guidance and any other consideration in this process.
Thank you
Nawaz Jia ---- Nawaz, The link below is from the US National Library of Medicine, a part of the NIH. Since I'm not certain I fully understand best "guidance" in this area -- I'm sending a link to a 2022 article published in RNA Biology, titled, "Splice site proximity influences alternative exon definition”. — In the abstract they say, “However, when alternative splice sites are embedded within large flanking introns (exon definition), the 5′ and 3′ splice sites closest to each other across the exon are preferentially selected. Thus, alternative splicing decisions are influenced by the intron and exon definition modes of splice site recognition. The results demonstrate that the spliceosome pairs splice sites that are closest in proximity within the unit of initial splice site selection.”
Hopes this helps………
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9225289/
Yes, it can be. intron 1 can be used as a target site for CRISPR-Cas9 mediated knock-in, but there are several important considerations to keep in mind when designing your experiment:
1. Guide RNA Design: The guide RNA (gRNA) must be designed to target a sequence within intron 1. The target sequence should be unique to avoid off-target effects and must be adjacent to a protospacer adjacent motif (PAM) sequence (usually NGG for Cas9 from Streptococcus pyogenes).
2. Homology Directed Repair (HDR): For a knock-in experiment, the desired DNA sequence to be inserted must be flanked by homology arms (sequences matching the genomic DNA on either side of the cut site). These homology arms are crucial for HDR, which is the process by which the cell repairs the DNA double-strand break using the provided DNA template, incorporating the new sequence into the genome.
3. Intron Functionality: While introns are non-coding regions, they can contain regulatory elements such as enhancers, silencers, or splicing signals. Disruption of these elements by CRISPR-Cas9 or the inserted sequence could affect gene expression or splicing. Therefore, it’s important to consider the potential impact on the gene's function.
4. Efficiency and Screening: The efficiency of CRISPR-Cas9 mediated knock-in can be variable. It may be necessary to screen a large number of clones to identify those that have successfully incorporated the desired sequence at the correct location.
5. Validation: After generating clones, thorough validation is required to confirm the correct insertion of the sequence. This includes sequencing the target region to verify the precise insertion and to check for any unintended mutations or off-target effects. hope it helps you