Hi Kunal,

     In most cases ensembl should give transcript information with sequences. Also you should check Vega annotation as it is manually curated hence more accurate. For CRISPRs I would suggest using two gRNAs to cause a deletion. Often removing the first ATG can cause transcription from a later alternate transcription start site and if it is in same frame as original transcript you will get a slightly truncated protein. If you are mutating a transcription factor, you could cause a deletion of the exon encoding the DNA binding (or other function) domain. It is possible that you may not see any phenotype due to compensation by homologs. We frequently mutate both copies of a duplicated gene and often the double mutants show a stronger phenotype.

Using this approach, even if you are not sure of starting ATG, you increase your chances of getting a null mutants.

Sandeep

Hi Kunal, 

In my lab we have found cases where the start codon annotation is incorrect or missing. In cases where you are unsure, I begin by identifying the correct open reading frame (http://www.ncbi.nlm.nih.gov/orffinder/), but you are correct in that the first in-frame ATG is not always the start codon. To find the true start codon, you will want to look for the kozak sequence (which will be just upstream of the start codon) that promotes assembly of the translation machinery. A quick google search showed a couple of programs available that do this automatically (http://atgpr.dbcls.jp), but kozak sequences vary significantly between (and even within) organisms so I would favor manual identification of the strongest kozak you can find based on a literature search. This paper is an excellent reference for zebrafish kozak sequences: PMID: 25248153

Hope this helps!

Jay

To add some point, you may need to consider alternative splicing, where certain form of transcripts can start after the first exon, in exon 4 for example. Check the transcript information in addition to the genome sequence info, that will give you a sense how many transcript variants you're expecting and which form may be responsible for the function you're studying.

if it's a short gene you could also try making two CRISPRs and knock out the whole thing. 

Hi Kunal,

As Pawat suggested it would be worth generating a 2xgRNA to make sure you delete it. Regarding your question about the ATG. There examples in which the first codon of the first exon is not an ATG. For example, in Arabidopsis the first codon of the AGAMOUS gene i is ACG instead of ATG (if I correctly remember it). I hope this information helps.

Just reinforcing, look for Kozak sequence is a good option:

Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger

RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125-48. Review. PubMed PMID:

3313277; PubMed Central PMCID: PMC306349.

https://www.ncbi.nlm.nih.gov/pubmed/3313277

Also check about alternative TSS usage:

http://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-14-331

http://genome.cshlp.org/content/early/2013/10/02/gr.153692.112.full.pdf

Best,

F.

I agree with paired guide RNA approach. It works much more efficient than single guide. 60% paired guide gave me exon deletion and knock out based on 16 genes analyzed, I screened six single cell clones for each and I got homozygous knock outs at protein level. But just in the case of editing (due to inefficient one guide RNA in pair) was quit tricky and efficiency drooped down to less than 20% to get homozygous knock out at protein level. I would suggest select a common exon covering all the isoforms and go for paired guide RNA approach

good luck

According to my experience no even in the absence of isoforms. When we tried to knock out genes, two genes had complete loss of 1st exon still protein was expressed, then scanned downstream Exons and found ATG which might be driving the translation. we did not follow up as our aim was to generate KO cell lines. So I woild suggest to scan the downstream Exons and target the exons with ATG downstream common to all isoforms in case your gene has isoforms

The first ATG in exon 1 of a gene is often, but not always, the start codon in eukaryotic organisms. The start codon is the nucleotide sequence within a messenger RNA (mRNA) that signals the beginning of translation into protein. The most common start codon is ATG, which codes for the amino acid methionine in eukaryotes.

However, there are several reasons why the first ATG in exon 1 might not serve as the start codon:

In summary, while the first ATG in exon 1 is a strong candidate for the start codon, it's not always the case due to various genetic and regulatory factors.

l This protocol list might provide further insights to address this issue.