pinellolab / crispritz Goto Github PK

Hi there,

I am trying to index genome using PAM NNGRRT with 21bp guide RNA, however, the index-genome always crashed. Any idea of what is going on?

Thanks.

George

FYI, there is still a bit of python 2 specific code in CRISPRitz, such as:

Hi @lucapinello ,

May I ask how the annotation file - hg38Annotation.zip was derived?

Thanks a lot.

Hi. Thank you for creating this tool. I have been able to execute and utilize all the other tools successfully, except for generate-report for which I receive the error below. Is this a true syntax warning or is there something wrong with my annotation summary file?

/opt/conda/opt/crispritz/Python_Scripts/Plot/radar_chart.py:74: SyntaxWarning: "is" with a literal. Did you mean "=="?
if '-pop' is sys.argv[:]:
Traceback (most recent call last):
File "/opt/conda/opt/crispritz/Python_Scripts/Plot/radar_chart.py", line 51, in
inAnnotation_ref = open(annotation_ref, 'r').readlines()
FileNotFoundError: [Errno 2] No such file or directory: 'no'

Sorry if this is just a me issue. I am very new to coding and CRISPR tools. Thank you.

Hi There,

Can you tell me how you generated the annotation file -- hg38Annotation.zip?

Thanks a lot.

Hey,

Do you support mismatches and bulges in the PAM? I tried to run the tool and it seems that it does not handle this. Even when I trying to index the genome with NNN PAM (meaning that when I will perform a search for off-targets so that it would take into account all possible PAMs in the DNA sequences), and performing the search for off-targets with NGG PAM, the tool forced the PAM of my guide to be NNN in order to avoid mismatches in the PAM.

Another question:
Does your tool support bulges in both DNA and RNA simultaneously?

Thank you

Hi There,

Thanks for making this valuable and innovative program available.
I am interested in incorporating it into my current pipeline.
However, I wonder if it is possible to add one more column into the results from the search module to indicate the original guide sequence?

Thanks a lot.

Best Regards,
Huanle

Hi @lucapinello ,

thank you and your colleagues for developing crispritz.
I have a question regarding the output radar chart of crispritz.
I can not understand the radar chart?
In the example image, what is the MAX VALUE?
What are the numbers in the parentheses? Are they the number of guides or features?
What are those decimal numbers? Are they proportions accounting for the total number of guides in the same category (defined using the number of mismatches) or the area size of the inner circles?

Thanks a lot and I look forward to your help.

Hi,

I find this software potentially useful and I would add this to my future pipeline. Is it difficult/possible to add an early stopping option which would limit number of reported off-targets per guide? The logic here is that there is no value in a guide that already has more than N off-targets.

Ideally this would report off-targets with low mismatch/gap first. Is it possible?

Best,
Kornel

Hi Crispritz developers,

Thanks for developing and bringing crisprtiz to the community.
I have a layman's question regarding inferring cutting sites from the predicted (off-) targets.
Basically, when a gRNA match to the same target region but with bulges introduced to different positions in the target DNA, will the cutting sites vary? aka, will the position of the bulge alter the 3nt-proximal-PAM-cutting-site rule.

The attached image contains one such example.
Thanks a lot in advance for your help!

Based on the position field -- how would one figure out the start and end positions from the search output. Is it dependent on strand ie start = pos and end = pos+length of guide+pam for + strand and start = pos-length of guide-pam and end = pos for + strand? Thanks!

Hi there,

I am wondering how the -score works only for NGG or I need to tweak it for some other PAM?

Thanks.

George

I'm just getting into gene editing, and I'm interested in the tools you're developing. I ran into a problem when running the' index-genome 'function.

terminate called after throwing an instance of 'std::out_of_range' what(): basic_string::substr: __pos (which is 1) > this->size() (which is 0)

I checked the source suspected a problem here. https://github.com/pinellolab/CRISPRitz/blob/master/sourceCode/CRISPR-Cas-Tree/aho-Corasick.cpp

if ((i - (pamlimit - 1)) <= (l - (pamlen + max_bulges)))  //save the pam position only if possible for a guide to attach that position(avoid out of bound)
{
    indices_private.push_back(-(i - (pamlimit - 1)));
}

But I'm not so sure, so ask.

When attempting to run a crispritz search on a non-human primate genome, using the following command:
'python ~/miniconda3/pkgs/crispritz-2.6.6-py39h243b37b_0/bin/crispritz.py search Mnem_1.0/ 20bp-NGG-SpCas9.txt gla-NM-gRNA-1.txt gla.Mnem1 -mm 4 -t -scores Mnem_1.0/'

I get the following error::

'Search START USED THREADS 8 ANALYZING CHROMOSOME NW_012020308.1 Macaca nemestrina isolate M95218 unplaced genomic scaffold, Mnem_1.0 Scaffold999, whole genome shotgun sequence (Total progress: 100.0%) terminate called after throwing an instance of 'std::length_error' what(): vector::_M_default_append Search END Search runtime: 24.96618342399597 seconds Traceback (most recent call last): File "/home/va/miniconda3/pkgs/crispritz-2.6.6-py39h243b37b_0/opt/crispritz/Python_Scripts/Scores/scores.py", line 211, in save_targ_scor.write(next(result).strip() + '\tCFD\n') # Add header StopIteration `

Genome is unindexed since we were not planning on doing a bulge search, at least not initially. The gRNA and Cas9 text files are attached.

Software versions:
WSL2 on Windows 11 with Ubuntu 20.04
Python 3.9

gla-NM-gRNA-1.txt
20bp-NGG-SpCas9.txt