Protospacer must be a string when initializing cas9

This is just to document an error that @JeffXiePL came across in this notebook:

https://github.com/JeffXiePL/pydna/blob/docs_peilun/docs/notebooks/CRISPR.ipynb

You were trying to instantiate a cas9 object by passing a seqrecord as an argument to the function cas9, that's why you were getting the error. However, the error you committed in the notebook:

TypeError                                 Traceback (most recent call last)
Cell In[12], line 2
      1 # Initializing the Cas9 protein
----> 2 enzyme = cas9(gRNA_sequence)
      4 # Simulating the CRISPR-Cas9 cut
      5 cut_products = target_sequence(enzyme)

TypeError: 'cas9' object is not callable

Is not the error you should be getting. Perhaps you re-assigned cas9 to a variable? Sometimes is good to restart the kernel and re-run the notebook. You should be getting the error below:

TypeError                                 Traceback (most recent call last)
Cell In[30], [line 2](vscode-notebook-cell:?execution_count=30&line=2)
      [1](vscode-notebook-cell:?execution_count=30&line=1) # Initializing the Cas9 protein
----> [2](vscode-notebook-cell:?execution_count=30&line=2) enzyme = cas9(gRNA_sequence)
      [4](vscode-notebook-cell:?execution_count=30&line=4) # Simulating the CRISPR-Cas9 cut
      [5](vscode-notebook-cell:?execution_count=30&line=5) cut_products = target_sequence(enzyme)

File ~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/crispr.py:32, in _cas.__init__(self, protospacer)
     [29](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/crispr.py:29) def __init__(self, protospacer):
     [30](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/crispr.py:30)     self.protospacer = protospacer.upper()
     [31](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/crispr.py:31)     self.compsite = re.compile(
---> [32](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/crispr.py:32)         f"(?=(?P<watson>{self.protospacer}{self.pam}))|(?=(?P<crick>{rc(self.pam)}{rc(self.protospacer)}))",
     [33](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/crispr.py:33)         re.UNICODE,
     [34](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/crispr.py:34)     )

File ~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/utils.py:264, in rc(sequence)
    [259](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/utils.py:259) def rc(sequence: str):
    [260](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/utils.py:260)     """Reverse complement.
    [261](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/utils.py:261) 
    [262](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/utils.py:262)     accepts mixed DNA/RNA
    [263](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/utils.py:263)     """
--> [264](https://file+.vscode-resource.vscode-cdn.net/Users/manu/Documents/OpenSource/summer_internship_2024/pydna/docs/notebooks/~/Documents/OpenSource/summer_internship_2024/pydna/src/pydna/utils.py:264)     return sequence.translate(_complement_table)[::-1]

TypeError: SeqRecord.translate() takes 1 positional argument but 2 were given

This error occurs because cas9 takes a string as an input, not a Dseqrecord. However, note that:

The string should contain the protospacer of the gRNA, not the entire gRNA.
You can extract the protospacer sequence from the entire gRNA with the function protospacer that is in pydna.crispr module.
The gRNA sequence is not only the homologous sequence, but requires some extra sequence that is always the same, I think. I don't remember exactly what the constrains are for cas9, but you should double-check.

A good place to start and see how it works is to see the examples from the tests in tests/test_module_crispr.py, you can take that as a starting example for how the cas9 enzyme works.

Also, you were trying to cut using:

cut_products = target_sequence(enzyme)

Instead, it should be:

cut_products = target_sequence.cut(enzyme)

BjornFJohansson / pydna

Protospacer must be a string when initializing cas9 #257