SPINE: Saturated Programmable INsertion Engineering

Protein domain insertion via programmed oligo libraries

Python script for generating oligo libraries and PCR primers for deep mutational scanning (DMS) of gene of interest. The mutation type for DMS library can be customized based on the personal needs which include single amino acid replacement, deletion and insertion. The oligo library design is meant to be compatible for Golden gate assembly using BsaI.

Python Dependencies

biopython==1.78

numpy==1.26.4

pandas==2.2.1

python-dateutil==2.9.0.post0

pytz==2024.1

six==1.16.0

tzdata==2024.1

Data Formats

Input:

• Targeted genes must be in fasta format and include a minimum of 30 bases surrounding gene.

• Entire plasmid sequence with all the essential genetic context is advised to search for nonspecific amplification. Using linear fragment of the partial plasmid works as long as the gene context of about 100 bp upstream and downstream of the target gene is included.
  

• Multiple sequences can be input for deep mutation scanning in one file.

• Make sure there is no AarI or BsaI restriction enzyme site in the sequence that you included in the fasta, based on what version of SPINE tool you are going to use. For version1, eliminate AarI site For version2, eliminate BsaI site

• Notes: user might get an error report returned if the sequence in the fasta file contains BsmBI. The elimination of BsmBI restriction enzyme site is case specific to Thyer lab's benefit because BsmBI enzyme is part of the lab modular cloning pipeline.

  For the user's benefit, here are three solutions to avoid these problems.

  1. Domesticate the BsmBI site in the sequence by switching it as synonymous codon if the BsmBI site locates within the target gene.

  2. If the BsmBI site is located in the backbone of plasmid, users can choose to fragmentize the plasmid sequence that contain the BsmBI site. Using a linear fragment of the partial plasmid works as long as the gene context of at least 100 bp upstream and downstream of the target gene is included.

  3. For the benefit of running DMS on multiple sequences in the fasta file, adjusting the following line in the code of functional module is recommended. Here I take the file 'SPINE_BsaI.py' as an example.

Eliminate error report of BsmBI and AarI sites detection in template DNA sequence

SPINE_BsaI.py code line 189-190 :

if any([gene.seq.upper().count(cut) for cut in ['GGTCTC', 'GAGACC','CGTCTC', 'GAGACG', 'CACCTGC', 'GCAGGTG']]): 
	raise ValueError('Unwanted Restriction cut sites found. Please input plasmids with these removed.')  

After BsmBI and AarI site removal in line 189-190 :

if any([gene.seq.upper().count(cut) for cut in [ 'GGTCTC', 'GAGACC']): 
	raise ValueError('Unwanted Restriction cut sites found. Please input plasmids with these removed.')  

Delete detection of BsmBI site in generated DMS oligo

SPINE_BsaI.py code line 803-804 :

if (xfrag.upper()[offset:len(tmpseq)+offset].count('CGTCTC') + xfrag.upper()[offset:len(tmpseq)+offset].count('GAGACG')) > 2:
      	raise ValueError('BsmBI site found within insertion fragment')          

Output:

• Final output is fasta format. One file for oligo pools and one file for PCR primers

• Different mutation type function can be run separately. The barcode might be overlapped if the functions are run separately.

• "allmut" function wrapped up everything and generate oligo libraries based on the same fragmentation strategy of the GOI. And it generates all mutation types without the overlap of barcode usage.

• Note: "allmut" and "allmut_noCys" functions generate different length of oligos, because all of the mutation types require to fit into same frame. Based on insertion, deletion and replacement, the length of oligo varies.

• The primers for both of the oligo libraries or plasmid vector within the same fragmentation part are generic.

Notes:

Gene primers should be same for the same sequence, because no matter what the mutation types we introduced here the fragmentation strategy should always be the same based on the size of the GOI

Position arguments

• Gene start is defined as the position / base number of the first base in the '-1 amino acid' of the first amino acid we want to mutagenize.
  e.g. ATGACTGTACCGCAC
  protein sequence : M T V P H
  If the DMS starts from amino acid V, the start position should be base number of A in codon ACT (amino acid T)

• Gene end is defined as base number of the last base in the '+1 codon' of the last codon we want to mutagenize.
  e.g. ATGACTGTACCGCAC
  protein sequence : M T V P H
  If the DMS ends at amino acid P, the end position should be the base number of the last C in codon CAC (amino acid H) (Program will subtract 1 from gene start for python numbering)

To define gene position within given fasta file, add start:# end:# to fasta description. (Otherwise use command line prompts)
'>geneA start:11 end:40'

SPINE versions

There are two versions of SPINE tool generated for different need of DMS library construction based on either using AarI restriction enzyme for assembly or BsaI.

Version1 AarI:

• Version1 generates DMS oligo fragment with AarI type II restriction enzyme for further cloning.

In version1, main file 'main_spine.py' is paired with functional module 'SPINE_v7.py'

• SPINE_v7 contains all of the single amino acid mutation function and also the integrated version for bulk deep mutation scanning

  DMS: single aa replacement

  S_INS/S_DEL: single aa insertion / deletion

  allmut: combination of all single aa mutation types

  allmut_noCys: exclude introduction of Cysteine from the all mutation types

• SPINE_v7 also allows for multiple input of GOI sequences in one input file

• SPINE_v7 carries counter to generate the diversity of final oligo libraries

• 'Fasta_converter.ipynb' file locates in the diretory of 'tests' folder. It requires Juypter notebook to run the code. Fasta_converter.ipynb is used to convert fasta file to csv file. It contains a function to deduplicate the oligos by identifying BsaI as token and extract oligo sequence translating it into amino acid. The final deduplicating will be run based on the repeat of amino acid sequence. The duplicate is caused by insertion and deletion. (e.g. amino acid: NDK. Inserting D at the second and third position are the same -- NDDK. For deletion, if there are continuous same amino acids, such as NDDK, deletion of the second or the third amino acid generates same result )

  This deduplicating function is integrated into the functional module of SPINE version2 (main_spine_BsaI.py), so no more separate deduplicating is needed using version 2. When using version 1, deduplicating checkpoint is required. (Coder's comments: if necessary, I will fix it later.)

• 'Fa_to_csv_oligoP.ipynb' file locates in the diretory of 'tests' folder.It requires Juypter notebook to run the code. The directory of oligo.fa or primer.fa needs to be changed based on user's directory or file name. This function helps to convert fasta file to csv file which makes the ordering of sequence easier.

Running test for version1

'oligoLen' can be customized based on the ordering requirement for oligo length. e.g. 230 bp or 150 bp

Domain Insertion Scanning: Note: Domain Insertion scanning is directly from the original script. I did not make any changes to that.

python3.8 main_spine.py -wDir tests -geneFile test.fa -oligoLen 230 -mutationType DIS

Deep Mutational Scanning:

python3.8 main_spine.py -wDir tests -geneFile test.fa -oligoLen 230 -mutationType allmut -usage ecoli

python3.8 main_spine.py -wDir tests -geneFile test.fa -oligoLen 230 -mutationType DMS -usage ecoli

Version2 BsaI:

• Version2 generates DMS oligo fragment with BsaI type II restriction enzyme for further cloning.

In version2, main file 'main_spine_BsaI.py' is paired with functional module 'SPINE_BsaI.py'

• SPINE_BsaI is modified to generate DMS library with type II site BsaI. It has been troubleshooted to fix the problem of introducing extra BsaI site into the oligo pool after the oligo generation from DMS.
• Other features of version2 is similar as version1.

Running test for version2

'oligoLen' can be customized based on the ordering requirement for oligo length. e.g. 230 bp or 150 bp

Deep Mutational Scanning:

python3.8 main_spine_BsaI.py -wDir tests -geneFile test.fa -oligoLen 150 -mutationType allmut -usage ecoli | tee output.txt 

Usage

optional arguments:
-h, --help                 show this help message and exit
-wDir WDIR                 Working directory for fasta files and output folder
-geneFile GENEFILE         Input all gene sequences including backbone in a fasta
                           format. Place all in one fasta file. Name description
                           can include start and end points (>gene1 start:1
                           end:2)
-handle HANDLE             Genetic handle for domain insertion.  This is important
                           for defining the linker. Currently uses BsaI (4 base
                           overhang), but this can be swapped for SapI (3 base
                           overhang).
-matchSequences            Find similar sequences between genes to avoid printing
                           the same oligos multiple times. Default: No matching
-oligoLen OLIGOLEN         Synthesized oligo length
-fragmentLen FRAGMENTLEN   Maximum length of gene fragment
-overlap OVERLAP           Enter number of bases to extend each fragment for
                           overlap. This could help with insertion coverage close to
                           fragment boundary. Overlap does not add additional
                           insertions and thus no additional oligos.
-mutationType              Run deep insertion scan "DIS", deep mutation scan for single amino acid replacement "DMS", single amino acid deletion "S_DEL", 
			  single amino acid insertion "S_INS" or combine all single amino acid mutation together(replacement, deletion, insertion) "allmut".
			  Or exclude any introduction of 'Cys' into the mutation library "allmut_noCys" 
-usage USAGE               Default is "human". Or select "ecoli"