notebooks/app_workflows/W2_CRISPR-BEST-single.ipynb

{
  "cells": [
    {
      "cell_type": "markdown",
      "metadata": {},
      "source": [
        "## W2-CRISPR-cBEST workflow\n"
      ]
    },
    {
      "cell_type": "markdown",
      "metadata": {},
      "source": [
        "Base-editing with CRISPR-BEST is a DSB-free method that uses a single sgRNA to target a specific genomic location with single-nucleotide–resolution(Figure 3A) which can enable in-vivo protein engineering or as it is often used, for introducing early stop-codons10,12. It can be used to introduce targeted point mutations in the genome of Streptomyces, facilitating studies on gene function and protein engineering. To get started quickly users can download the pCRISPR-cBEST plasmid file, the genome file for S.coelicor (A3) and streptoAIM will generate your plasmids based on the genes you chose to target (Figure 3, S5). "
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 1,
      "metadata": {},
      "outputs": [],
      "source": [
        "import sys\n",
        "import os\n",
        "\n",
        "# Ensure the src directory is in the Python path\n",
        "project_root = os.path.abspath(os.path.join(os.getcwd(), '../../'))\n",
        "if project_root not in sys.path:\n",
        "    sys.path.insert(0, project_root)\n",
        "\n",
        "import os\n",
        "import pandas as pd\n",
        "from pydna.dseqrecord import Dseqrecord\n",
        "from datetime import datetime\n",
        "\n",
        "from streptocad.sequence_loading.sequence_loading import  load_and_process_plasmid, load_and_process_genome_sequences, annotate_dseqrecord, check_and_convert_input, process_specified_gene_sequences_from_record\n",
        "from streptocad.utils import polymerase_dict,ProjectDirectory, extract_metadata_to_dataframe\n",
        "from streptocad.crispr.guideRNAcas3_9_12 import extract_sgRNAs, SgRNAargs\n",
        "from streptocad.cloning.ssDNA_bridging import assemble_plasmids_by_ssDNA_bridging, make_ssDNA_oligos\n",
        "from streptocad.crispr.crispr_best import identify_base_editing_sites, filter_sgrnas_for_base_editing, process_base_editing\n",
        "from streptocad.cloning.plasmid_processing import annotate_plasmid_with_sgrnas\n",
        "from streptocad.primers.primer_generation import find_best_check_primers_from_genome, create_idt_order_dataframe, primers_to_IDT"
      ]
    },
    {
      "cell_type": "markdown",
      "metadata": {},
      "source": [
        "## INPUT"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 2,
      "metadata": {},
      "outputs": [],
      "source": [
        "# Inputs\n",
        "# 1 Add genome of choice (genbank, fasta)\n",
        "path_to_genome = '../../data/genomes/Streptomyces_coelicolor_A3_chromosome.gb'\n",
        "genome = load_and_process_genome_sequences(path_to_genome)[0]\n",
        "\n",
        "# 2 Add plasmid \n",
        "path_to_plasmid = '../../data/plasmids/ pCRISPR-cBEST Sequences.gbk'\n",
        "clean_plasmid = load_and_process_plasmid(path_to_plasmid)\n",
        "\n",
        "# 3 Choose genes to knock out (list)\n",
        "genes_to_KO = ['SCO5087']#,'SCO5088', 'SCO5089', 'SCO5090', 'SCO5091', 'SCO5092', 'SCO5093']\n",
        "#genes_to_KO = ['80000-100000', '4000-7000', '9000-14000','15000-20000']\n",
        "\n",
        "\n",
        "#### Advanced settings ####\n",
        "# 3 Choose polymerase and target melting temperature FOR CHECKING PRIMERS\n",
        "chosen_polymerase = polymerase_dict['Phusion High-Fidelity DNA Polymerase (GC Buffer)']\n",
        "\n",
        "melting_temperature = 65\n",
        "primer_concentration = 0.4 \n",
        "primer_number_increment = 1\n",
        "flanking_region_number = 500\n",
        "\n",
        "# 4 Filtering metrics for sgRNAs\n",
        "gc_upper = 0.99\n",
        "gc_lower = 0.01\n",
        "off_target_seed = 13\n",
        "off_target_upper = 10\n",
        "cas_type='cas9'\n",
        "number_of_sgRNAs_per_group = 5\n",
        "only_stop_codons = True\n",
        "flanking_region = 500\n",
        "\n",
        "# 6 Choose overlapping sequences for our plasmid we can use the following\n",
        "#As per the article **\"CRISPR–Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes\"** we need the following oligoes: \n",
        "#CGGTTGGTAGGATCGACGGC **-N20-** GTTTTAGAGCTAGAAATAGC\n",
        "up_homology = Dseqrecord('cggttggtaggatcgacggc') # V1 homology : CGGTTGGTAGGATCGACGGC\n",
        "dw_homology = Dseqrecord('tatgtcctccgagaccggcc') # V1 homology : GTTTTAGAGCTAGAAATAGC"
      ]
    },
    {
      "cell_type": "markdown",
      "metadata": {},
      "source": [
        "# Computation"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 3,
      "metadata": {},
      "outputs": [
        {
          "name": "stdout",
          "output_type": "stream",
          "text": [
            "pCRISPR-cBEST(#125689)\n"
          ]
        }
      ],
      "source": [
        "print(clean_plasmid.id)\n"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 4,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/plain": [
              "[SeqRecord(seq=Seq('GGCCGTCGACGACGACGCGTCCGCCTGCCTGCTTTTGGCCGGCGGGCCGGGCCG...CCA'), id='pCRISPR-cBEST(#125689)', name='pCRISPR-cBEST', description='C to T base editor for actinomycetes', dbxrefs=[])]"
            ]
          },
          "execution_count": 4,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "from teemi.design.fetch_sequences import read_genbank_files     \n",
        "sequences = read_genbank_files(path_to_plasmid)\n",
        "sequences\n"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 5,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/plain": [
              "25824"
            ]
          },
          "execution_count": 5,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "len(genome.features)\n"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 6,
      "metadata": {},
      "outputs": [
        {
          "name": "stdout",
          "output_type": "stream",
          "text": [
            "False\n"
          ]
        },
        {
          "data": {
            "text/plain": [
              "25824"
            ]
          },
          "execution_count": 6,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "target_dict, genes_to_KO, annotation_input = check_and_convert_input(genes_to_KO)\n",
        "\n",
        "print(annotation_input)\n",
        "if annotation_input == True:\n",
        "    genome = annotate_dseqrecord(genome, target_dict)\n",
        "\n",
        "\n",
        "len(genome.features)"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 7,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/plain": [
              "['SCO5087']"
            ]
          },
          "execution_count": 7,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "genes_to_KO\n"
      ]
    },
    {
      "cell_type": "markdown",
      "metadata": {},
      "source": [
        "We need to check the input genes so we can find sgRNAs. "
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 8,
      "metadata": {},
      "outputs": [
        {
          "name": "stdout",
          "output_type": "stream",
          "text": [
            "Pam was found outside designated locus_tag: SCO5087. To incorporate this extent borders. Skipping to next locus tag.\n",
            "sgRNA generated were outside the designated border in SCO5087. To incorporate this extent borders. Skipping to next locus tag.\n",
            "Pam was found outside designated locus_tag: SCO5087. To incorporate this extent borders. Skipping to next locus tag.\n",
            "sgRNA generated were outside the designated border in SCO5087. To incorporate this extent borders. Skipping to next locus tag.\n",
            "sgRNA generated were outside the designated border in SCO5087. To incorporate this extent borders. Skipping to next locus tag.\n"
          ]
        },
        {
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              "  <thead>\n",
              "    <tr style=\"text-align: right;\">\n",
              "      <th></th>\n",
              "      <th>strain_name</th>\n",
              "      <th>locus_tag</th>\n",
              "      <th>gene_loc</th>\n",
              "      <th>gene_strand</th>\n",
              "      <th>sgrna_strand</th>\n",
              "      <th>sgrna_loc</th>\n",
              "      <th>gc</th>\n",
              "      <th>pam</th>\n",
              "      <th>sgrna</th>\n",
              "      <th>sgrna_seed_sequence</th>\n",
              "      <th>off_target_count</th>\n",
              "    </tr>\n",
              "  </thead>\n",
              "  <tbody>\n",
              "    <tr>\n",
              "      <th>0</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>26</td>\n",
              "      <td>0.80</td>\n",
              "      <td>CGG</td>\n",
              "      <td>TCCACCGGCGCCGCGTCCAG</td>\n",
              "      <td>GCGCCGCGTCCAG</td>\n",
              "      <td>0</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>161</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1189</td>\n",
              "      <td>0.80</td>\n",
              "      <td>TGG</td>\n",
              "      <td>GCTGGGCGCGATCGGCTCGC</td>\n",
              "      <td>GCGATCGGCTCGC</td>\n",
              "      <td>0</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>162</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1181</td>\n",
              "      <td>0.75</td>\n",
              "      <td>CGG</td>\n",
              "      <td>GGCCACTCGCTGGGCGCGAT</td>\n",
              "      <td>CGCTGGGCGCGAT</td>\n",
              "      <td>0</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>163</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1172</td>\n",
              "      <td>0.65</td>\n",
              "      <td>GGG</td>\n",
              "      <td>TCGATGGTCGGCCACTCGCT</td>\n",
              "      <td>TCGGCCACTCGCT</td>\n",
              "      <td>0</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>165</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1160</td>\n",
              "      <td>0.50</td>\n",
              "      <td>CGG</td>\n",
              "      <td>AGCTCGATCAAGTCGATGGT</td>\n",
              "      <td>TCAAGTCGATGGT</td>\n",
              "      <td>0</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>...</th>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>231</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>469</td>\n",
              "      <td>0.80</td>\n",
              "      <td>TGG</td>\n",
              "      <td>GGTGGCAGCGGCGACCAGCC</td>\n",
              "      <td>GCGGCGACCAGCC</td>\n",
              "      <td>5</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>42</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>424</td>\n",
              "      <td>0.80</td>\n",
              "      <td>GGG</td>\n",
              "      <td>GGGCGGGGTCGAGGGTGTCC</td>\n",
              "      <td>GTCGAGGGTGTCC</td>\n",
              "      <td>5</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>39</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>394</td>\n",
              "      <td>0.80</td>\n",
              "      <td>CGG</td>\n",
              "      <td>CGCTGGCGGCGAACGCCTCC</td>\n",
              "      <td>GGCGAACGCCTCC</td>\n",
              "      <td>6</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>256</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>307</td>\n",
              "      <td>0.75</td>\n",
              "      <td>AGG</td>\n",
              "      <td>CGACTTCGACCCGGTCGCCG</td>\n",
              "      <td>GACCCGGTCGCCG</td>\n",
              "      <td>7</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>215</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>604</td>\n",
              "      <td>0.90</td>\n",
              "      <td>AGG</td>\n",
              "      <td>CGCCTGGGCGGTCGGCGCCG</td>\n",
              "      <td>GCGGTCGGCGCCG</td>\n",
              "      <td>7</td>\n",
              "    </tr>\n",
              "  </tbody>\n",
              "</table>\n",
              "<p>289 rows × 11 columns</p>\n",
              "</div>"
            ],
            "text/plain": [
              "     strain_name locus_tag  gene_loc  gene_strand  sgrna_strand  sgrna_loc  \\\n",
              "0    NC_003888.3   SCO5087   5529801            1            -1         26   \n",
              "161  NC_003888.3   SCO5087   5529801            1             1       1189   \n",
              "162  NC_003888.3   SCO5087   5529801            1             1       1181   \n",
              "163  NC_003888.3   SCO5087   5529801            1             1       1172   \n",
              "165  NC_003888.3   SCO5087   5529801            1             1       1160   \n",
              "..           ...       ...       ...          ...           ...        ...   \n",
              "231  NC_003888.3   SCO5087   5529801            1             1        469   \n",
              "42   NC_003888.3   SCO5087   5529801            1            -1        424   \n",
              "39   NC_003888.3   SCO5087   5529801            1            -1        394   \n",
              "256  NC_003888.3   SCO5087   5529801            1             1        307   \n",
              "215  NC_003888.3   SCO5087   5529801            1             1        604   \n",
              "\n",
              "       gc  pam                 sgrna sgrna_seed_sequence  off_target_count  \n",
              "0    0.80  CGG  TCCACCGGCGCCGCGTCCAG       GCGCCGCGTCCAG                 0  \n",
              "161  0.80  TGG  GCTGGGCGCGATCGGCTCGC       GCGATCGGCTCGC                 0  \n",
              "162  0.75  CGG  GGCCACTCGCTGGGCGCGAT       CGCTGGGCGCGAT                 0  \n",
              "163  0.65  GGG  TCGATGGTCGGCCACTCGCT       TCGGCCACTCGCT                 0  \n",
              "165  0.50  CGG  AGCTCGATCAAGTCGATGGT       TCAAGTCGATGGT                 0  \n",
              "..    ...  ...                   ...                 ...               ...  \n",
              "231  0.80  TGG  GGTGGCAGCGGCGACCAGCC       GCGGCGACCAGCC                 5  \n",
              "42   0.80  GGG  GGGCGGGGTCGAGGGTGTCC       GTCGAGGGTGTCC                 5  \n",
              "39   0.80  CGG  CGCTGGCGGCGAACGCCTCC       GGCGAACGCCTCC                 6  \n",
              "256  0.75  AGG  CGACTTCGACCCGGTCGCCG       GACCCGGTCGCCG                 7  \n",
              "215  0.90  AGG  CGCCTGGGCGGTCGGCGCCG       GCGGTCGGCGCCG                 7  \n",
              "\n",
              "[289 rows x 11 columns]"
            ]
          },
          "execution_count": 8,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "\n",
        "# Initialize SgRNAargs with desired parameters\n",
        "args = SgRNAargs(genome, \n",
        "                genes_to_KO,\n",
        "                step=['find', 'filter'],\n",
        "                gc_upper = gc_upper,\n",
        "                gc_lower = gc_lower,\n",
        "                off_target_seed = off_target_seed,\n",
        "                off_target_upper = off_target_upper,\n",
        "                cas_type='cas9',\n",
        "                )\n",
        "\n",
        "sgrna_df = extract_sgRNAs(args)\n",
        "sgrna_df"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 9,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/plain": [
              "locus_tag\n",
              "SCO5087    289\n",
              "Name: count, dtype: int64"
            ]
          },
          "execution_count": 9,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "value_counts = sgrna_df['locus_tag'].value_counts()\n",
        "value_counts"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 10,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/plain": [
              "25824"
            ]
          },
          "execution_count": 10,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "len(genome.features)"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 11,
      "metadata": {},
      "outputs": [
        {
          "name": "stdout",
          "output_type": "stream",
          "text": [
            "{'SCO5087': Dseq(-1404)\n",
            "GTGC..ATGA\n",
            "CACG..TACT}\n"
          ]
        },
        {
          "data": {
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              "  <thead>\n",
              "    <tr style=\"text-align: right;\">\n",
              "      <th></th>\n",
              "      <th>strain_name</th>\n",
              "      <th>locus_tag</th>\n",
              "      <th>gene_loc</th>\n",
              "      <th>gene_strand</th>\n",
              "      <th>sgrna_strand</th>\n",
              "      <th>sgrna_loc</th>\n",
              "      <th>gc</th>\n",
              "      <th>pam</th>\n",
              "      <th>sgrna</th>\n",
              "      <th>sgrna_seed_sequence</th>\n",
              "      <th>off_target_count</th>\n",
              "      <th>editing_context</th>\n",
              "      <th>editable_cytosines</th>\n",
              "    </tr>\n",
              "  </thead>\n",
              "  <tbody>\n",
              "    <tr>\n",
              "      <th>0</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>26</td>\n",
              "      <td>0.80</td>\n",
              "      <td>CGG</td>\n",
              "      <td>TCCACCGGCGCCGCGTCCAG</td>\n",
              "      <td>GCGCCGCGTCCAG</td>\n",
              "      <td>0</td>\n",
              "      <td>0,0,0,1</td>\n",
              "      <td>3,5,6,9</td>\n",
              "    </tr>\n",
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              "      <th>161</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
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              "      <td>1189</td>\n",
              "      <td>0.80</td>\n",
              "      <td>TGG</td>\n",
              "      <td>GCTGGGCGCGATCGGCTCGC</td>\n",
              "      <td>GCGATCGGCTCGC</td>\n",
              "      <td>0</td>\n",
              "      <td>1,1</td>\n",
              "      <td>7,9</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>162</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1181</td>\n",
              "      <td>0.75</td>\n",
              "      <td>CGG</td>\n",
              "      <td>GGCCACTCGCTGGGCGCGAT</td>\n",
              "      <td>CGCTGGGCGCGAT</td>\n",
              "      <td>0</td>\n",
              "      <td>1,0,0,0,1</td>\n",
              "      <td>3,4,6,8,10</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>163</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1172</td>\n",
              "      <td>0.65</td>\n",
              "      <td>GGG</td>\n",
              "      <td>TCGATGGTCGGCCACTCGCT</td>\n",
              "      <td>TCGGCCACTCGCT</td>\n",
              "      <td>0</td>\n",
              "      <td>0</td>\n",
              "      <td>9</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>165</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1160</td>\n",
              "      <td>0.50</td>\n",
              "      <td>CGG</td>\n",
              "      <td>AGCTCGATCAAGTCGATGGT</td>\n",
              "      <td>TCAAGTCGATGGT</td>\n",
              "      <td>0</td>\n",
              "      <td>1,0,0</td>\n",
              "      <td>3,5,9</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>...</th>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>231</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>469</td>\n",
              "      <td>0.80</td>\n",
              "      <td>TGG</td>\n",
              "      <td>GGTGGCAGCGGCGACCAGCC</td>\n",
              "      <td>GCGGCGACCAGCC</td>\n",
              "      <td>5</td>\n",
              "      <td>1,1</td>\n",
              "      <td>6,9</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>42</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>424</td>\n",
              "      <td>0.80</td>\n",
              "      <td>GGG</td>\n",
              "      <td>GGGCGGGGTCGAGGGTGTCC</td>\n",
              "      <td>GTCGAGGGTGTCC</td>\n",
              "      <td>5</td>\n",
              "      <td>1,0</td>\n",
              "      <td>4,10</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>39</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>394</td>\n",
              "      <td>0.80</td>\n",
              "      <td>CGG</td>\n",
              "      <td>CGCTGGCGGCGAACGCCTCC</td>\n",
              "      <td>GGCGAACGCCTCC</td>\n",
              "      <td>6</td>\n",
              "      <td>1,1,1</td>\n",
              "      <td>3,7,10</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>256</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>307</td>\n",
              "      <td>0.75</td>\n",
              "      <td>AGG</td>\n",
              "      <td>CGACTTCGACCCGGTCGCCG</td>\n",
              "      <td>GACCCGGTCGCCG</td>\n",
              "      <td>7</td>\n",
              "      <td>0,0,0</td>\n",
              "      <td>4,7,10</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>215</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>604</td>\n",
              "      <td>0.90</td>\n",
              "      <td>AGG</td>\n",
              "      <td>CGCCTGGGCGGTCGGCGCCG</td>\n",
              "      <td>GCGGTCGGCGCCG</td>\n",
              "      <td>7</td>\n",
              "      <td>1,0,1</td>\n",
              "      <td>3,4,9</td>\n",
              "    </tr>\n",
              "  </tbody>\n",
              "</table>\n",
              "<p>287 rows × 13 columns</p>\n",
              "</div>"
            ],
            "text/plain": [
              "     strain_name locus_tag  gene_loc  gene_strand  sgrna_strand  sgrna_loc  \\\n",
              "0    NC_003888.3   SCO5087   5529801            1            -1         26   \n",
              "161  NC_003888.3   SCO5087   5529801            1             1       1189   \n",
              "162  NC_003888.3   SCO5087   5529801            1             1       1181   \n",
              "163  NC_003888.3   SCO5087   5529801            1             1       1172   \n",
              "165  NC_003888.3   SCO5087   5529801            1             1       1160   \n",
              "..           ...       ...       ...          ...           ...        ...   \n",
              "231  NC_003888.3   SCO5087   5529801            1             1        469   \n",
              "42   NC_003888.3   SCO5087   5529801            1            -1        424   \n",
              "39   NC_003888.3   SCO5087   5529801            1            -1        394   \n",
              "256  NC_003888.3   SCO5087   5529801            1             1        307   \n",
              "215  NC_003888.3   SCO5087   5529801            1             1        604   \n",
              "\n",
              "       gc  pam                 sgrna sgrna_seed_sequence  off_target_count  \\\n",
              "0    0.80  CGG  TCCACCGGCGCCGCGTCCAG       GCGCCGCGTCCAG                 0   \n",
              "161  0.80  TGG  GCTGGGCGCGATCGGCTCGC       GCGATCGGCTCGC                 0   \n",
              "162  0.75  CGG  GGCCACTCGCTGGGCGCGAT       CGCTGGGCGCGAT                 0   \n",
              "163  0.65  GGG  TCGATGGTCGGCCACTCGCT       TCGGCCACTCGCT                 0   \n",
              "165  0.50  CGG  AGCTCGATCAAGTCGATGGT       TCAAGTCGATGGT                 0   \n",
              "..    ...  ...                   ...                 ...               ...   \n",
              "231  0.80  TGG  GGTGGCAGCGGCGACCAGCC       GCGGCGACCAGCC                 5   \n",
              "42   0.80  GGG  GGGCGGGGTCGAGGGTGTCC       GTCGAGGGTGTCC                 5   \n",
              "39   0.80  CGG  CGCTGGCGGCGAACGCCTCC       GGCGAACGCCTCC                 6   \n",
              "256  0.75  AGG  CGACTTCGACCCGGTCGCCG       GACCCGGTCGCCG                 7   \n",
              "215  0.90  AGG  CGCCTGGGCGGTCGGCGCCG       GCGGTCGGCGCCG                 7   \n",
              "\n",
              "    editing_context editable_cytosines  \n",
              "0           0,0,0,1            3,5,6,9  \n",
              "161             1,1                7,9  \n",
              "162       1,0,0,0,1         3,4,6,8,10  \n",
              "163               0                  9  \n",
              "165           1,0,0              3,5,9  \n",
              "..              ...                ...  \n",
              "231             1,1                6,9  \n",
              "42              1,0               4,10  \n",
              "39            1,1,1             3,7,10  \n",
              "256           0,0,0             4,7,10  \n",
              "215           1,0,1              3,4,9  \n",
              "\n",
              "[287 rows x 13 columns]"
            ]
          },
          "execution_count": 11,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "# Load gene sequences from the genome\n",
        "gene_sequences = process_specified_gene_sequences_from_record(genome, genes_to_KO)\n",
        "genes_to_KO_dict = {locus_tag: gene_sequences[locus_tag] for locus_tag in genes_to_KO if locus_tag in gene_sequences}\n",
        "print(genes_to_KO_dict)\n",
        "# Identify and annotate base editing sites\n",
        "sgrna_df_with_editing = identify_base_editing_sites(sgrna_df)\n",
        "\n",
        "# filter out only sgRNAs that result in base-editing\n",
        "filtered_sgrna_df_for_base_editing = filter_sgrnas_for_base_editing(sgrna_df_with_editing)\n",
        "filtered_sgrna_df_for_base_editing"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 12,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/html": [
              "<div>\n",
              "<style scoped>\n",
              "    .dataframe tbody tr th:only-of-type {\n",
              "        vertical-align: middle;\n",
              "    }\n",
              "\n",
              "    .dataframe tbody tr th {\n",
              "        vertical-align: top;\n",
              "    }\n",
              "\n",
              "    .dataframe thead th {\n",
              "        text-align: right;\n",
              "    }\n",
              "</style>\n",
              "<table border=\"1\" class=\"dataframe\">\n",
              "  <thead>\n",
              "    <tr style=\"text-align: right;\">\n",
              "      <th></th>\n",
              "      <th>strain_name</th>\n",
              "      <th>locus_tag</th>\n",
              "      <th>gene_loc</th>\n",
              "      <th>gene_strand</th>\n",
              "      <th>sgrna_strand</th>\n",
              "      <th>sgrna_loc</th>\n",
              "      <th>gc</th>\n",
              "      <th>pam</th>\n",
              "      <th>sgrna</th>\n",
              "      <th>sgrna_seed_sequence</th>\n",
              "      <th>off_target_count</th>\n",
              "      <th>editing_context</th>\n",
              "      <th>editable_cytosines</th>\n",
              "      <th>mutations</th>\n",
              "    </tr>\n",
              "  </thead>\n",
              "  <tbody>\n",
              "    <tr>\n",
              "      <th>0</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>26</td>\n",
              "      <td>0.80</td>\n",
              "      <td>CGG</td>\n",
              "      <td>TCCACCGGCGCCGCGTCCAG</td>\n",
              "      <td>GCGCCGCGTCCAG</td>\n",
              "      <td>0</td>\n",
              "      <td>0,0,0,1</td>\n",
              "      <td>3,5,6,9</td>\n",
              "      <td>V8I</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>161</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1189</td>\n",
              "      <td>0.80</td>\n",
              "      <td>TGG</td>\n",
              "      <td>GCTGGGCGCGATCGGCTCGC</td>\n",
              "      <td>GCGATCGGCTCGC</td>\n",
              "      <td>0</td>\n",
              "      <td>1,1</td>\n",
              "      <td>7,9</td>\n",
              "      <td>A393V</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>162</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1181</td>\n",
              "      <td>0.75</td>\n",
              "      <td>CGG</td>\n",
              "      <td>GGCCACTCGCTGGGCGCGAT</td>\n",
              "      <td>CGCTGGGCGCGAT</td>\n",
              "      <td>0</td>\n",
              "      <td>1,0,0,0,1</td>\n",
              "      <td>3,4,6,8,10</td>\n",
              "      <td>H389Y, S390L</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>165</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1160</td>\n",
              "      <td>0.50</td>\n",
              "      <td>CGG</td>\n",
              "      <td>AGCTCGATCAAGTCGATGGT</td>\n",
              "      <td>TCAAGTCGATGGT</td>\n",
              "      <td>0</td>\n",
              "      <td>1,0,0</td>\n",
              "      <td>3,5,9</td>\n",
              "      <td>S382L</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>167</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1135</td>\n",
              "      <td>0.75</td>\n",
              "      <td>CGG</td>\n",
              "      <td>CGAACACGCCCGGCGCACTC</td>\n",
              "      <td>GCCCGGCGCACTC</td>\n",
              "      <td>0</td>\n",
              "      <td>0,0,1,0</td>\n",
              "      <td>5,7,9,10</td>\n",
              "      <td>H374Y, A375V</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>...</th>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>105</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>1018</td>\n",
              "      <td>0.75</td>\n",
              "      <td>CGG</td>\n",
              "      <td>GGGACTCGTCGAGGGCGACC</td>\n",
              "      <td>GTCGAGGGCGACC</td>\n",
              "      <td>4</td>\n",
              "      <td>0,0,0</td>\n",
              "      <td>5,7,10</td>\n",
              "      <td>D337N, E338K</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>231</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>469</td>\n",
              "      <td>0.80</td>\n",
              "      <td>TGG</td>\n",
              "      <td>GGTGGCAGCGGCGACCAGCC</td>\n",
              "      <td>GCGGCGACCAGCC</td>\n",
              "      <td>5</td>\n",
              "      <td>1,1</td>\n",
              "      <td>6,9</td>\n",
              "      <td>A152V, A153V</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>42</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>424</td>\n",
              "      <td>0.80</td>\n",
              "      <td>GGG</td>\n",
              "      <td>GGGCGGGGTCGAGGGTGTCC</td>\n",
              "      <td>GTCGAGGGTGTCC</td>\n",
              "      <td>5</td>\n",
              "      <td>1,0</td>\n",
              "      <td>4,10</td>\n",
              "      <td>D139N, A141T</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>39</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>394</td>\n",
              "      <td>0.80</td>\n",
              "      <td>CGG</td>\n",
              "      <td>CGCTGGCGGCGAACGCCTCC</td>\n",
              "      <td>GGCGAACGCCTCC</td>\n",
              "      <td>6</td>\n",
              "      <td>1,1,1</td>\n",
              "      <td>3,7,10</td>\n",
              "      <td>A129T, A130T, S131N</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>215</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>604</td>\n",
              "      <td>0.90</td>\n",
              "      <td>AGG</td>\n",
              "      <td>CGCCTGGGCGGTCGGCGCCG</td>\n",
              "      <td>GCGGTCGGCGCCG</td>\n",
              "      <td>7</td>\n",
              "      <td>1,0,1</td>\n",
              "      <td>3,4,9</td>\n",
              "      <td>A196V, A198V</td>\n",
              "    </tr>\n",
              "  </tbody>\n",
              "</table>\n",
              "<p>260 rows × 14 columns</p>\n",
              "</div>"
            ],
            "text/plain": [
              "     strain_name locus_tag  gene_loc  gene_strand  sgrna_strand  sgrna_loc  \\\n",
              "0    NC_003888.3   SCO5087   5529801            1            -1         26   \n",
              "161  NC_003888.3   SCO5087   5529801            1             1       1189   \n",
              "162  NC_003888.3   SCO5087   5529801            1             1       1181   \n",
              "165  NC_003888.3   SCO5087   5529801            1             1       1160   \n",
              "167  NC_003888.3   SCO5087   5529801            1             1       1135   \n",
              "..           ...       ...       ...          ...           ...        ...   \n",
              "105  NC_003888.3   SCO5087   5529801            1            -1       1018   \n",
              "231  NC_003888.3   SCO5087   5529801            1             1        469   \n",
              "42   NC_003888.3   SCO5087   5529801            1            -1        424   \n",
              "39   NC_003888.3   SCO5087   5529801            1            -1        394   \n",
              "215  NC_003888.3   SCO5087   5529801            1             1        604   \n",
              "\n",
              "       gc  pam                 sgrna sgrna_seed_sequence  off_target_count  \\\n",
              "0    0.80  CGG  TCCACCGGCGCCGCGTCCAG       GCGCCGCGTCCAG                 0   \n",
              "161  0.80  TGG  GCTGGGCGCGATCGGCTCGC       GCGATCGGCTCGC                 0   \n",
              "162  0.75  CGG  GGCCACTCGCTGGGCGCGAT       CGCTGGGCGCGAT                 0   \n",
              "165  0.50  CGG  AGCTCGATCAAGTCGATGGT       TCAAGTCGATGGT                 0   \n",
              "167  0.75  CGG  CGAACACGCCCGGCGCACTC       GCCCGGCGCACTC                 0   \n",
              "..    ...  ...                   ...                 ...               ...   \n",
              "105  0.75  CGG  GGGACTCGTCGAGGGCGACC       GTCGAGGGCGACC                 4   \n",
              "231  0.80  TGG  GGTGGCAGCGGCGACCAGCC       GCGGCGACCAGCC                 5   \n",
              "42   0.80  GGG  GGGCGGGGTCGAGGGTGTCC       GTCGAGGGTGTCC                 5   \n",
              "39   0.80  CGG  CGCTGGCGGCGAACGCCTCC       GGCGAACGCCTCC                 6   \n",
              "215  0.90  AGG  CGCCTGGGCGGTCGGCGCCG       GCGGTCGGCGCCG                 7   \n",
              "\n",
              "    editing_context editable_cytosines            mutations  \n",
              "0           0,0,0,1            3,5,6,9                  V8I  \n",
              "161             1,1                7,9                A393V  \n",
              "162       1,0,0,0,1         3,4,6,8,10         H389Y, S390L  \n",
              "165           1,0,0              3,5,9                S382L  \n",
              "167         0,0,1,0           5,7,9,10         H374Y, A375V  \n",
              "..              ...                ...                  ...  \n",
              "105           0,0,0             5,7,10         D337N, E338K  \n",
              "231             1,1                6,9         A152V, A153V  \n",
              "42              1,0               4,10         D139N, A141T  \n",
              "39            1,1,1             3,7,10  A129T, A130T, S131N  \n",
              "215           1,0,1              3,4,9         A196V, A198V  \n",
              "\n",
              "[260 rows x 14 columns]"
            ]
          },
          "execution_count": 12,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "# Process the DataFrame to apply C-to-T mutations\n",
        "mutated_sgrna_df = process_base_editing(filtered_sgrna_df_for_base_editing, \n",
        "                                        genes_to_KO_dict, \n",
        "                                        only_stop_codons = False, \n",
        "                                        editing_context= False)\n",
        "mutated_sgrna_df"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 13,
      "metadata": {},
      "outputs": [
        {
          "data": {
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              "<div>\n",
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              "        vertical-align: top;\n",
              "    }\n",
              "\n",
              "    .dataframe thead th {\n",
              "        text-align: right;\n",
              "    }\n",
              "</style>\n",
              "<table border=\"1\" class=\"dataframe\">\n",
              "  <thead>\n",
              "    <tr style=\"text-align: right;\">\n",
              "      <th></th>\n",
              "      <th>strain_name</th>\n",
              "      <th>locus_tag</th>\n",
              "      <th>gene_loc</th>\n",
              "      <th>gene_strand</th>\n",
              "      <th>sgrna_strand</th>\n",
              "      <th>sgrna_loc</th>\n",
              "      <th>gc</th>\n",
              "      <th>pam</th>\n",
              "      <th>sgrna</th>\n",
              "      <th>sgrna_seed_sequence</th>\n",
              "      <th>off_target_count</th>\n",
              "      <th>editing_context</th>\n",
              "      <th>editable_cytosines</th>\n",
              "      <th>mutations</th>\n",
              "    </tr>\n",
              "  </thead>\n",
              "  <tbody>\n",
              "    <tr>\n",
              "      <th>180</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>964</td>\n",
              "      <td>0.60</td>\n",
              "      <td>AGG</td>\n",
              "      <td>GTACCACATGACGGGCCTGA</td>\n",
              "      <td>ATGACGGGCCTGA</td>\n",
              "      <td>0</td>\n",
              "      <td>0,0,0</td>\n",
              "      <td>4,5,7</td>\n",
              "      <td>H317Y</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>157</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>1291</td>\n",
              "      <td>0.60</td>\n",
              "      <td>TGG</td>\n",
              "      <td>CGATCTCGACTACGTTCCGC</td>\n",
              "      <td>GACTACGTTCCGC</td>\n",
              "      <td>0</td>\n",
              "      <td>0,0,0</td>\n",
              "      <td>5,7,10</td>\n",
              "      <td>L426F</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>130</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>1270</td>\n",
              "      <td>0.75</td>\n",
              "      <td>AGG</td>\n",
              "      <td>ACTCGGGGTCGCTGGTGCGC</td>\n",
              "      <td>GTCGCTGGTGCGC</td>\n",
              "      <td>0</td>\n",
              "      <td>0,0</td>\n",
              "      <td>4,10</td>\n",
              "      <td>D421N, E423K</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>132</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>1285</td>\n",
              "      <td>0.60</td>\n",
              "      <td>GGG</td>\n",
              "      <td>CGTAGTCGAGATCGCACTCG</td>\n",
              "      <td>GAGATCGCACTCG</td>\n",
              "      <td>0</td>\n",
              "      <td>0</td>\n",
              "      <td>7</td>\n",
              "      <td>D427N</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>133</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>1286</td>\n",
              "      <td>0.55</td>\n",
              "      <td>GGG</td>\n",
              "      <td>ACGTAGTCGAGATCGCACTC</td>\n",
              "      <td>CGAGATCGCACTC</td>\n",
              "      <td>0</td>\n",
              "      <td>0</td>\n",
              "      <td>8</td>\n",
              "      <td>D427N</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>...</th>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "      <td>...</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>108</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>1038</td>\n",
              "      <td>0.70</td>\n",
              "      <td>GGG</td>\n",
              "      <td>GATGTCCGTCGCGTCCGTGC</td>\n",
              "      <td>GTCGCGTCCGTGC</td>\n",
              "      <td>2</td>\n",
              "      <td>0,0,0</td>\n",
              "      <td>6,7,10</td>\n",
              "      <td>D345N</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>254</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>314</td>\n",
              "      <td>0.75</td>\n",
              "      <td>CGG</td>\n",
              "      <td>GACCCGGTCGCCGAGGGCTT</td>\n",
              "      <td>TCGCCGAGGGCTT</td>\n",
              "      <td>2</td>\n",
              "      <td>0,0,0,0</td>\n",
              "      <td>3,4,5,9</td>\n",
              "      <td>P100L</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>255</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>308</td>\n",
              "      <td>0.70</td>\n",
              "      <td>GGG</td>\n",
              "      <td>GACTTCGACCCGGTCGCCGA</td>\n",
              "      <td>ACCCGGTCGCCGA</td>\n",
              "      <td>2</td>\n",
              "      <td>0,0,0,0</td>\n",
              "      <td>3,6,9,10</td>\n",
              "      <td>P100S</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>78</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>745</td>\n",
              "      <td>0.65</td>\n",
              "      <td>GGG</td>\n",
              "      <td>CGACGACGATCGGGGTGATC</td>\n",
              "      <td>GATCGGGGTGATC</td>\n",
              "      <td>2</td>\n",
              "      <td>0,0</td>\n",
              "      <td>4,7</td>\n",
              "      <td>V247I, V248I</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>105</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>1018</td>\n",
              "      <td>0.75</td>\n",
              "      <td>CGG</td>\n",
              "      <td>GGGACTCGTCGAGGGCGACC</td>\n",
              "      <td>GTCGAGGGCGACC</td>\n",
              "      <td>4</td>\n",
              "      <td>0,0,0</td>\n",
              "      <td>5,7,10</td>\n",
              "      <td>D337N, E338K</td>\n",
              "    </tr>\n",
              "  </tbody>\n",
              "</table>\n",
              "<p>73 rows × 14 columns</p>\n",
              "</div>"
            ],
            "text/plain": [
              "     strain_name locus_tag  gene_loc  gene_strand  sgrna_strand  sgrna_loc  \\\n",
              "180  NC_003888.3   SCO5087   5529801            1             1        964   \n",
              "157  NC_003888.3   SCO5087   5529801            1             1       1291   \n",
              "130  NC_003888.3   SCO5087   5529801            1            -1       1270   \n",
              "132  NC_003888.3   SCO5087   5529801            1            -1       1285   \n",
              "133  NC_003888.3   SCO5087   5529801            1            -1       1286   \n",
              "..           ...       ...       ...          ...           ...        ...   \n",
              "108  NC_003888.3   SCO5087   5529801            1            -1       1038   \n",
              "254  NC_003888.3   SCO5087   5529801            1             1        314   \n",
              "255  NC_003888.3   SCO5087   5529801            1             1        308   \n",
              "78   NC_003888.3   SCO5087   5529801            1            -1        745   \n",
              "105  NC_003888.3   SCO5087   5529801            1            -1       1018   \n",
              "\n",
              "       gc  pam                 sgrna sgrna_seed_sequence  off_target_count  \\\n",
              "180  0.60  AGG  GTACCACATGACGGGCCTGA       ATGACGGGCCTGA                 0   \n",
              "157  0.60  TGG  CGATCTCGACTACGTTCCGC       GACTACGTTCCGC                 0   \n",
              "130  0.75  AGG  ACTCGGGGTCGCTGGTGCGC       GTCGCTGGTGCGC                 0   \n",
              "132  0.60  GGG  CGTAGTCGAGATCGCACTCG       GAGATCGCACTCG                 0   \n",
              "133  0.55  GGG  ACGTAGTCGAGATCGCACTC       CGAGATCGCACTC                 0   \n",
              "..    ...  ...                   ...                 ...               ...   \n",
              "108  0.70  GGG  GATGTCCGTCGCGTCCGTGC       GTCGCGTCCGTGC                 2   \n",
              "254  0.75  CGG  GACCCGGTCGCCGAGGGCTT       TCGCCGAGGGCTT                 2   \n",
              "255  0.70  GGG  GACTTCGACCCGGTCGCCGA       ACCCGGTCGCCGA                 2   \n",
              "78   0.65  GGG  CGACGACGATCGGGGTGATC       GATCGGGGTGATC                 2   \n",
              "105  0.75  CGG  GGGACTCGTCGAGGGCGACC       GTCGAGGGCGACC                 4   \n",
              "\n",
              "    editing_context editable_cytosines     mutations  \n",
              "180           0,0,0              4,5,7         H317Y  \n",
              "157           0,0,0             5,7,10         L426F  \n",
              "130             0,0               4,10  D421N, E423K  \n",
              "132               0                  7         D427N  \n",
              "133               0                  8         D427N  \n",
              "..              ...                ...           ...  \n",
              "108           0,0,0             6,7,10         D345N  \n",
              "254         0,0,0,0            3,4,5,9         P100L  \n",
              "255         0,0,0,0           3,6,9,10         P100S  \n",
              "78              0,0                4,7  V247I, V248I  \n",
              "105           0,0,0             5,7,10  D337N, E338K  \n",
              "\n",
              "[73 rows x 14 columns]"
            ]
          },
          "execution_count": 13,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "# Process the DataFrame to apply C-to-T mutations\n",
        "mutated_sgrna_df = process_base_editing(filtered_sgrna_df_for_base_editing, \n",
        "                                        genes_to_KO_dict, \n",
        "                                        only_stop_codons = False, \n",
        "                                        editing_context= True)\n",
        "mutated_sgrna_df"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 14,
      "metadata": {},
      "outputs": [
        {
          "data": {
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              "<table border=\"1\" class=\"dataframe\">\n",
              "  <thead>\n",
              "    <tr style=\"text-align: right;\">\n",
              "      <th></th>\n",
              "      <th>strain_name</th>\n",
              "      <th>locus_tag</th>\n",
              "      <th>gene_loc</th>\n",
              "      <th>gene_strand</th>\n",
              "      <th>sgrna_strand</th>\n",
              "      <th>sgrna_loc</th>\n",
              "      <th>gc</th>\n",
              "      <th>pam</th>\n",
              "      <th>sgrna</th>\n",
              "      <th>sgrna_seed_sequence</th>\n",
              "      <th>off_target_count</th>\n",
              "      <th>editing_context</th>\n",
              "      <th>editable_cytosines</th>\n",
              "      <th>mutations</th>\n",
              "    </tr>\n",
              "  </thead>\n",
              "  <tbody>\n",
              "    <tr>\n",
              "      <th>258</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>283</td>\n",
              "      <td>0.75</td>\n",
              "      <td>AGG</td>\n",
              "      <td>CCGTTCACAGGTCGCGGCGG</td>\n",
              "      <td>CAGGTCGCGGCGG</td>\n",
              "      <td>0</td>\n",
              "      <td>0,0</td>\n",
              "      <td>6,8</td>\n",
              "      <td>S90L, Q91*</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>54</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>522</td>\n",
              "      <td>0.70</td>\n",
              "      <td>CGG</td>\n",
              "      <td>GTCGACCTCCCAGTCACGGC</td>\n",
              "      <td>TCCCAGTCACGGC</td>\n",
              "      <td>0</td>\n",
              "      <td>0,0,0,0,0</td>\n",
              "      <td>3,6,7,9,10</td>\n",
              "      <td>W171*, E172K, V173I, D174N</td>\n",
              "    </tr>\n",
              "  </tbody>\n",
              "</table>\n",
              "</div>"
            ],
            "text/plain": [
              "     strain_name locus_tag  gene_loc  gene_strand  sgrna_strand  sgrna_loc  \\\n",
              "258  NC_003888.3   SCO5087   5529801            1             1        283   \n",
              "54   NC_003888.3   SCO5087   5529801            1            -1        522   \n",
              "\n",
              "       gc  pam                 sgrna sgrna_seed_sequence  off_target_count  \\\n",
              "258  0.75  AGG  CCGTTCACAGGTCGCGGCGG       CAGGTCGCGGCGG                 0   \n",
              "54   0.70  CGG  GTCGACCTCCCAGTCACGGC       TCCCAGTCACGGC                 0   \n",
              "\n",
              "    editing_context editable_cytosines                   mutations  \n",
              "258             0,0                6,8                  S90L, Q91*  \n",
              "54        0,0,0,0,0         3,6,7,9,10  W171*, E172K, V173I, D174N  "
            ]
          },
          "execution_count": 14,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "# Process the DataFrame to apply C-to-T mutations\n",
        "mutated_sgrna_df = process_base_editing(filtered_sgrna_df_for_base_editing, \n",
        "                                        genes_to_KO_dict, \n",
        "                                        only_stop_codons = True, \n",
        "                                        editing_context= True)\n",
        "mutated_sgrna_df"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 15,
      "metadata": {},
      "outputs": [
        {
          "data": {
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              "<table border=\"1\" class=\"dataframe\">\n",
              "  <thead>\n",
              "    <tr style=\"text-align: right;\">\n",
              "      <th></th>\n",
              "      <th>strain_name</th>\n",
              "      <th>locus_tag</th>\n",
              "      <th>gene_loc</th>\n",
              "      <th>gene_strand</th>\n",
              "      <th>sgrna_strand</th>\n",
              "      <th>sgrna_loc</th>\n",
              "      <th>gc</th>\n",
              "      <th>pam</th>\n",
              "      <th>sgrna</th>\n",
              "      <th>sgrna_seed_sequence</th>\n",
              "      <th>off_target_count</th>\n",
              "      <th>editing_context</th>\n",
              "      <th>editable_cytosines</th>\n",
              "      <th>mutations</th>\n",
              "    </tr>\n",
              "  </thead>\n",
              "  <tbody>\n",
              "    <tr>\n",
              "      <th>258</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>1</td>\n",
              "      <td>283</td>\n",
              "      <td>0.75</td>\n",
              "      <td>AGG</td>\n",
              "      <td>CCGTTCACAGGTCGCGGCGG</td>\n",
              "      <td>CAGGTCGCGGCGG</td>\n",
              "      <td>0</td>\n",
              "      <td>0,0</td>\n",
              "      <td>6,8</td>\n",
              "      <td>S90L, Q91*</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>54</th>\n",
              "      <td>NC_003888.3</td>\n",
              "      <td>SCO5087</td>\n",
              "      <td>5529801</td>\n",
              "      <td>1</td>\n",
              "      <td>-1</td>\n",
              "      <td>522</td>\n",
              "      <td>0.70</td>\n",
              "      <td>CGG</td>\n",
              "      <td>GTCGACCTCCCAGTCACGGC</td>\n",
              "      <td>TCCCAGTCACGGC</td>\n",
              "      <td>0</td>\n",
              "      <td>0,0,0,0,0</td>\n",
              "      <td>3,6,7,9,10</td>\n",
              "      <td>W171*, E172K, V173I, D174N</td>\n",
              "    </tr>\n",
              "  </tbody>\n",
              "</table>\n",
              "</div>"
            ],
            "text/plain": [
              "     strain_name locus_tag  gene_loc  gene_strand  sgrna_strand  sgrna_loc  \\\n",
              "258  NC_003888.3   SCO5087   5529801            1             1        283   \n",
              "54   NC_003888.3   SCO5087   5529801            1            -1        522   \n",
              "\n",
              "       gc  pam                 sgrna sgrna_seed_sequence  off_target_count  \\\n",
              "258  0.75  AGG  CCGTTCACAGGTCGCGGCGG       CAGGTCGCGGCGG                 0   \n",
              "54   0.70  CGG  GTCGACCTCCCAGTCACGGC       TCCCAGTCACGGC                 0   \n",
              "\n",
              "    editing_context editable_cytosines                   mutations  \n",
              "258             0,0                6,8                  S90L, Q91*  \n",
              "54        0,0,0,0,0         3,6,7,9,10  W171*, E172K, V173I, D174N  "
            ]
          },
          "execution_count": 15,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "# Filter the DataFrame to retain only up to 5 sgRNA sequences per locus_tag\n",
        "filtered_df = mutated_sgrna_df.groupby('locus_tag').head(number_of_sgRNAs_per_group)\n",
        "filtered_df"
      ]
    },
    {
      "cell_type": "markdown",
      "metadata": {},
      "source": [
        "## Output"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 16,
      "metadata": {},
      "outputs": [],
      "source": [
        "# MAke oligoes\n",
        "list_of_ssDNAs = make_ssDNA_oligos(filtered_df, upstream_ovh = up_homology,\n",
        "                      downstream_ovh=dw_homology)\n",
        "\n",
        "\n",
        "# cut plasmid\n",
        "from Bio.Restriction import NcoI, NheI\n",
        "linearized_plasmid = sorted(clean_plasmid.cut(NcoI,NheI ), key=lambda x: len(x), reverse=True)[0]\n",
        "\n",
        "# assemble plasmid\n",
        "sgRNA_vectors = assemble_plasmids_by_ssDNA_bridging(list_of_ssDNAs,linearized_plasmid)"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 17,
      "metadata": {},
      "outputs": [],
      "source": [
        "# Constructing a meaningful name, ID, and description for the assembled plasmid using user input\n",
        "targeting_info = []\n",
        "for index, row in filtered_df.iterrows():\n",
        "    formatted_str = f\"pCRISPR-BEST_{row['locus_tag']}_p{row['sgrna_loc']}\"\n",
        "    targeting_info.append(formatted_str)\n",
        "\n",
        "for i in range(len(sgRNA_vectors)):\n",
        "    sgRNA_vectors[i].name = f'{targeting_info[i]}_#{i+1}'\n",
        "    sgRNA_vectors[i].id = sgRNA_vectors[i].name  # Using the same value for ID as for name for simplicity\n",
        "    sgRNA_vectors[i].description = f'Assembled plasmid targeting {\", \".join(genes_to_KO)} for base-editing, assembled using StreptoCAD.'\n",
        "\n",
        "# annotate plasmids\n",
        "for plasmid in sgRNA_vectors: \n",
        "    annotate_plasmid_with_sgrnas(plasmid, filtered_df)\n",
        "\n",
        "# PRINT\n",
        "print_plasmids = False\n",
        "if print_plasmids: \n",
        "    for vector in sgRNA_vectors: \n",
        "        vector.write(f\"../../data/plasmids/sgRNA_plasmids_pCRISPR_cBEST/{vector.id}.gb\")"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 18,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/html": [
              "<div>\n",
              "<style scoped>\n",
              "    .dataframe tbody tr th:only-of-type {\n",
              "        vertical-align: middle;\n",
              "    }\n",
              "\n",
              "    .dataframe tbody tr th {\n",
              "        vertical-align: top;\n",
              "    }\n",
              "\n",
              "    .dataframe thead th {\n",
              "        text-align: right;\n",
              "    }\n",
              "</style>\n",
              "<table border=\"1\" class=\"dataframe\">\n",
              "  <thead>\n",
              "    <tr style=\"text-align: right;\">\n",
              "      <th></th>\n",
              "      <th>plasmid_name</th>\n",
              "      <th>date</th>\n",
              "      <th>original_plasmid</th>\n",
              "      <th>integration</th>\n",
              "      <th>size</th>\n",
              "    </tr>\n",
              "  </thead>\n",
              "  <tbody>\n",
              "    <tr>\n",
              "      <th>0</th>\n",
              "      <td>pCRISPR-BEST_SCO5087_p283_#1</td>\n",
              "      <td>2024-09-03</td>\n",
              "      <td>pCRISPR-cBEST(#125689)</td>\n",
              "      <td>sgRNA_SCO5087(283)</td>\n",
              "      <td>11817</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>1</th>\n",
              "      <td>pCRISPR-BEST_SCO5087_p522_#2</td>\n",
              "      <td>2024-09-03</td>\n",
              "      <td>pCRISPR-cBEST(#125689)</td>\n",
              "      <td>sgRNA_SCO5087(522)</td>\n",
              "      <td>11817</td>\n",
              "    </tr>\n",
              "  </tbody>\n",
              "</table>\n",
              "</div>"
            ],
            "text/plain": [
              "                   plasmid_name        date        original_plasmid  \\\n",
              "0  pCRISPR-BEST_SCO5087_p283_#1  2024-09-03  pCRISPR-cBEST(#125689)   \n",
              "1  pCRISPR-BEST_SCO5087_p522_#2  2024-09-03  pCRISPR-cBEST(#125689)   \n",
              "\n",
              "          integration   size  \n",
              "0  sgRNA_SCO5087(283)  11817  \n",
              "1  sgRNA_SCO5087(522)  11817  "
            ]
          },
          "execution_count": 18,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "integration_names = filtered_df.apply(lambda row: f\"sgRNA_{row['locus_tag']}({row['sgrna_loc']})\", axis=1).tolist()\n",
        "plasmid_metadata_df = extract_metadata_to_dataframe(sgRNA_vectors,\n",
        "                                                    clean_plasmid,\n",
        "                                                    integration_names)\n",
        "\n",
        "plasmid_metadata_df"
      ]
    },
    {
      "cell_type": "markdown",
      "metadata": {},
      "source": [
        "# IDT primers"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 19,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/html": [
              "<div>\n",
              "<style scoped>\n",
              "    .dataframe tbody tr th:only-of-type {\n",
              "        vertical-align: middle;\n",
              "    }\n",
              "\n",
              "    .dataframe tbody tr th {\n",
              "        vertical-align: top;\n",
              "    }\n",
              "\n",
              "    .dataframe thead th {\n",
              "        text-align: right;\n",
              "    }\n",
              "</style>\n",
              "<table border=\"1\" class=\"dataframe\">\n",
              "  <thead>\n",
              "    <tr style=\"text-align: right;\">\n",
              "      <th></th>\n",
              "      <th>Name</th>\n",
              "      <th>Sequence</th>\n",
              "      <th>Concentration</th>\n",
              "      <th>Purification</th>\n",
              "    </tr>\n",
              "  </thead>\n",
              "  <tbody>\n",
              "    <tr>\n",
              "      <th>0</th>\n",
              "      <td>SCO5087_loc_283</td>\n",
              "      <td>cggttggtaggatcgacggcCCGTTCACAGGTCGCGGCGGtatgtc...</td>\n",
              "      <td>25nm</td>\n",
              "      <td>STD</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>1</th>\n",
              "      <td>SCO5087_loc_522</td>\n",
              "      <td>cggttggtaggatcgacggcGTCGACCTCCCAGTCACGGCtatgtc...</td>\n",
              "      <td>25nm</td>\n",
              "      <td>STD</td>\n",
              "    </tr>\n",
              "  </tbody>\n",
              "</table>\n",
              "</div>"
            ],
            "text/plain": [
              "              Name                                           Sequence  \\\n",
              "0  SCO5087_loc_283  cggttggtaggatcgacggcCCGTTCACAGGTCGCGGCGGtatgtc...   \n",
              "1  SCO5087_loc_522  cggttggtaggatcgacggcGTCGACCTCCCAGTCACGGCtatgtc...   \n",
              "\n",
              "  Concentration Purification  \n",
              "0          25nm          STD  \n",
              "1          25nm          STD  "
            ]
          },
          "execution_count": 19,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "idt_df1 = primers_to_IDT(list_of_ssDNAs)\n",
        "idt_df1"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 20,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/plain": [
              "['SCO5087']"
            ]
          },
          "execution_count": 20,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "genes_to_KO"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 21,
      "metadata": {},
      "outputs": [],
      "source": [
        "# Getting checking primers\n",
        "checking_primers_df = find_best_check_primers_from_genome(genome, \n",
        "                                       genes_to_KO, \n",
        "                                       flanking_region=flanking_region,\n",
        "                                       target_tm = melting_temperature, \n",
        "                                        primer_concentration = primer_concentration, \n",
        "                                        polymerase = chosen_polymerase)\n",
        "checking_primers_df.to_clipboard()"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 22,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/html": [
              "<div>\n",
              "<style scoped>\n",
              "    .dataframe tbody tr th:only-of-type {\n",
              "        vertical-align: middle;\n",
              "    }\n",
              "\n",
              "    .dataframe tbody tr th {\n",
              "        vertical-align: top;\n",
              "    }\n",
              "\n",
              "    .dataframe thead th {\n",
              "        text-align: right;\n",
              "    }\n",
              "</style>\n",
              "<table border=\"1\" class=\"dataframe\">\n",
              "  <thead>\n",
              "    <tr style=\"text-align: right;\">\n",
              "      <th></th>\n",
              "      <th>locus tag</th>\n",
              "      <th>f_primer_name</th>\n",
              "      <th>r_primer_name</th>\n",
              "      <th>f_primer_sequences(5-3)</th>\n",
              "      <th>r_primer_sequences(5-3)</th>\n",
              "      <th>f_tm</th>\n",
              "      <th>r_tm</th>\n",
              "      <th>ta</th>\n",
              "      <th>flanking_region</th>\n",
              "      <th>annealing_temperature</th>\n",
              "      <th>...</th>\n",
              "      <th>homodimer_reverse_tm</th>\n",
              "      <th>homodimer_reverse_deltaG (kcal/mol)</th>\n",
              "      <th>heterodimer_tm</th>\n",
              "      <th>heterodimer_deltaG (kcal/mol)</th>\n",
              "      <th>hairpin_forward_structure_found</th>\n",
              "      <th>hairpin_forward_tm</th>\n",
              "      <th>hairpin_forward_deltaG (kcal/mol)</th>\n",
              "      <th>hairpin_reverse_structure_found</th>\n",
              "      <th>hairpin_reverse_tm</th>\n",
              "      <th>hairpin_reverse_deltaG (kcal/mol)</th>\n",
              "    </tr>\n",
              "  </thead>\n",
              "  <tbody>\n",
              "    <tr>\n",
              "      <th>0</th>\n",
              "      <td>SCO5087</td>\n",
              "      <td>SCO5087_fwd_checking_primer</td>\n",
              "      <td>SCO5087_rev_checking_primer</td>\n",
              "      <td>GACGATTCGGCCCGTG</td>\n",
              "      <td>CAGGGCGTCCAGGC</td>\n",
              "      <td>59</td>\n",
              "      <td>58</td>\n",
              "      <td>61</td>\n",
              "      <td>500</td>\n",
              "      <td>61</td>\n",
              "      <td>...</td>\n",
              "      <td>9.706246</td>\n",
              "      <td>-3.415427</td>\n",
              "      <td>1.918567</td>\n",
              "      <td>-2.396641</td>\n",
              "      <td>False</td>\n",
              "      <td>0.0</td>\n",
              "      <td>0.0</td>\n",
              "      <td>False</td>\n",
              "      <td>0.0</td>\n",
              "      <td>0.0</td>\n",
              "    </tr>\n",
              "  </tbody>\n",
              "</table>\n",
              "<p>1 rows × 23 columns</p>\n",
              "</div>"
            ],
            "text/plain": [
              "  locus tag                f_primer_name                r_primer_name  \\\n",
              "0   SCO5087  SCO5087_fwd_checking_primer  SCO5087_rev_checking_primer   \n",
              "\n",
              "  f_primer_sequences(5-3) r_primer_sequences(5-3) f_tm r_tm  ta  \\\n",
              "0        GACGATTCGGCCCGTG          CAGGGCGTCCAGGC   59   58  61   \n",
              "\n",
              "  flanking_region  annealing_temperature  ... homodimer_reverse_tm  \\\n",
              "0             500                     61  ...             9.706246   \n",
              "\n",
              "   homodimer_reverse_deltaG (kcal/mol)  heterodimer_tm  \\\n",
              "0                            -3.415427        1.918567   \n",
              "\n",
              "   heterodimer_deltaG (kcal/mol)  hairpin_forward_structure_found  \\\n",
              "0                      -2.396641                            False   \n",
              "\n",
              "   hairpin_forward_tm  hairpin_forward_deltaG (kcal/mol)  \\\n",
              "0                 0.0                                0.0   \n",
              "\n",
              "   hairpin_reverse_structure_found  hairpin_reverse_tm  \\\n",
              "0                            False                 0.0   \n",
              "\n",
              "   hairpin_reverse_deltaG (kcal/mol)  \n",
              "0                                0.0  \n",
              "\n",
              "[1 rows x 23 columns]"
            ]
          },
          "execution_count": 22,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "checking_primers_df"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 23,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/html": [
              "<div>\n",
              "<style scoped>\n",
              "    .dataframe tbody tr th:only-of-type {\n",
              "        vertical-align: middle;\n",
              "    }\n",
              "\n",
              "    .dataframe tbody tr th {\n",
              "        vertical-align: top;\n",
              "    }\n",
              "\n",
              "    .dataframe thead th {\n",
              "        text-align: right;\n",
              "    }\n",
              "</style>\n",
              "<table border=\"1\" class=\"dataframe\">\n",
              "  <thead>\n",
              "    <tr style=\"text-align: right;\">\n",
              "      <th></th>\n",
              "      <th>Name</th>\n",
              "      <th>Sequence</th>\n",
              "      <th>Concentration</th>\n",
              "      <th>Purification</th>\n",
              "    </tr>\n",
              "  </thead>\n",
              "  <tbody>\n",
              "    <tr>\n",
              "      <th>0</th>\n",
              "      <td>SCO5087_fwd_checking_primer</td>\n",
              "      <td>GACGATTCGGCCCGTG</td>\n",
              "      <td>25nm</td>\n",
              "      <td>STD</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>1</th>\n",
              "      <td>SCO5087_rev_checking_primer</td>\n",
              "      <td>CAGGGCGTCCAGGC</td>\n",
              "      <td>25nm</td>\n",
              "      <td>STD</td>\n",
              "    </tr>\n",
              "  </tbody>\n",
              "</table>\n",
              "</div>"
            ],
            "text/plain": [
              "                          Name          Sequence Concentration Purification\n",
              "0  SCO5087_fwd_checking_primer  GACGATTCGGCCCGTG          25nm          STD\n",
              "1  SCO5087_rev_checking_primer    CAGGGCGTCCAGGC          25nm          STD"
            ]
          },
          "execution_count": 23,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "idt_df2 = create_idt_order_dataframe(checking_primers_df)\n",
        "idt_df2"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 24,
      "metadata": {},
      "outputs": [
        {
          "data": {
            "text/html": [
              "<div>\n",
              "<style scoped>\n",
              "    .dataframe tbody tr th:only-of-type {\n",
              "        vertical-align: middle;\n",
              "    }\n",
              "\n",
              "    .dataframe tbody tr th {\n",
              "        vertical-align: top;\n",
              "    }\n",
              "\n",
              "    .dataframe thead th {\n",
              "        text-align: right;\n",
              "    }\n",
              "</style>\n",
              "<table border=\"1\" class=\"dataframe\">\n",
              "  <thead>\n",
              "    <tr style=\"text-align: right;\">\n",
              "      <th></th>\n",
              "      <th>Name</th>\n",
              "      <th>Sequence</th>\n",
              "      <th>Concentration</th>\n",
              "      <th>Purification</th>\n",
              "    </tr>\n",
              "  </thead>\n",
              "  <tbody>\n",
              "    <tr>\n",
              "      <th>0</th>\n",
              "      <td>SCO5087_loc_283</td>\n",
              "      <td>cggttggtaggatcgacggcCCGTTCACAGGTCGCGGCGGtatgtc...</td>\n",
              "      <td>25nm</td>\n",
              "      <td>STD</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>1</th>\n",
              "      <td>SCO5087_loc_522</td>\n",
              "      <td>cggttggtaggatcgacggcGTCGACCTCCCAGTCACGGCtatgtc...</td>\n",
              "      <td>25nm</td>\n",
              "      <td>STD</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>0</th>\n",
              "      <td>SCO5087_fwd_checking_primer</td>\n",
              "      <td>GACGATTCGGCCCGTG</td>\n",
              "      <td>25nm</td>\n",
              "      <td>STD</td>\n",
              "    </tr>\n",
              "    <tr>\n",
              "      <th>1</th>\n",
              "      <td>SCO5087_rev_checking_primer</td>\n",
              "      <td>CAGGGCGTCCAGGC</td>\n",
              "      <td>25nm</td>\n",
              "      <td>STD</td>\n",
              "    </tr>\n",
              "  </tbody>\n",
              "</table>\n",
              "</div>"
            ],
            "text/plain": [
              "                          Name  \\\n",
              "0              SCO5087_loc_283   \n",
              "1              SCO5087_loc_522   \n",
              "0  SCO5087_fwd_checking_primer   \n",
              "1  SCO5087_rev_checking_primer   \n",
              "\n",
              "                                            Sequence Concentration  \\\n",
              "0  cggttggtaggatcgacggcCCGTTCACAGGTCGCGGCGGtatgtc...          25nm   \n",
              "1  cggttggtaggatcgacggcGTCGACCTCCCAGTCACGGCtatgtc...          25nm   \n",
              "0                                   GACGATTCGGCCCGTG          25nm   \n",
              "1                                     CAGGGCGTCCAGGC          25nm   \n",
              "\n",
              "  Purification  \n",
              "0          STD  \n",
              "1          STD  \n",
              "0          STD  \n",
              "1          STD  "
            ]
          },
          "execution_count": 24,
          "metadata": {},
          "output_type": "execute_result"
        }
      ],
      "source": [
        "full_idt = pd.concat([idt_df1, idt_df2])\n",
        "full_idt"
      ]
    },
    {
      "cell_type": "markdown",
      "metadata": {},
      "source": [
        "## Folder with all the generated I/O"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": 25,
      "metadata": {},
      "outputs": [],
      "source": [
        "input_files = [\n",
        "    {\"name\": \"input_genome.gb\", \"content\": genome},\n",
        "    {\"name\": \"input_plasmid.gb\", \"content\": clean_plasmid}\n",
        "]\n",
        "\n",
        "output_files = [\n",
        "    {\"name\": \"cBEST_w_sgRNAs.gb\", \"content\": sgRNA_vectors}, # LIST OF Dseqrecords\n",
        "    {\"name\": \"primer_df.csv\", \"content\": checking_primers_df},\n",
        "    {\"name\": \"full_idt.csv\", \"content\": full_idt},\n",
        "    {\"name\": \"mutated_sgrna_df.csv\", \"content\": mutated_sgrna_df},\n",
        "    {\"name\": \"filtered_df.csv\", \"content\": filtered_df},\n",
        "    {\"name\": \"plasmid_metadata_df.csv\", \"content\": plasmid_metadata_df},\n",
        "\n",
        "]\n",
        "\n",
        "input_values = {\n",
        "    \"genes_to_knockout\": genes_to_KO,\n",
        "    \"polymerase_settings\": {\n",
        "        \"chosen_polymerase\": chosen_polymerase,\n",
        "        \"melting_temperature\": melting_temperature,\n",
        "        \"primer_concentration\": primer_concentration,\n",
        "        \"primer_number_increment\": primer_number_increment,\n",
        "        \"flanking_region_number\": flanking_region_number\n",
        "    },\n",
        "    \"filtering_metrics\": {\n",
        "        \"gc_upper\": gc_upper,\n",
        "        \"gc_lower\": gc_lower,\n",
        "        \"off_target_seed\": off_target_seed,\n",
        "        \"off_target_upper\": off_target_upper,\n",
        "        \"cas_type\": cas_type,\n",
        "        \"number_of_sgRNAs_per_group\": number_of_sgRNAs_per_group\n",
        "    },\n",
        "    \"overlapping_sequences\": {\n",
        "        \"up_homology\": str(up_homology),\n",
        "        \"dw_homology\": str(dw_homology)\n",
        "    }\n",
        "}\n",
        "\n",
        "\n",
        "# Paths to Markdown files\n",
        "markdown_file_paths = [\n",
        "    \"../../protocols/conjugation_protcol.md\",\n",
        "    \"../../protocols/single_target_crispr_plasmid_protcol.md\"\n",
        "\n",
        "]\n",
        "\n",
        "# Data and time\n",
        "timestamp = datetime.utcnow().isoformat()\n",
        "\n",
        "# Create project directory structure\n",
        "project_directory = ProjectDirectory(\n",
        "    project_name=f\"CRISPR_cBEST_workflow_{timestamp}\",\n",
        "    input_files=input_files,\n",
        "    output_files=output_files,\n",
        "    input_values=input_values,\n",
        "    markdown_file_paths=markdown_file_paths\n",
        ")\n",
        "\n",
        "\n",
        "# DO You want to save the folder? \n",
        "save_zip_folder = False \n",
        "\n",
        "if save_zip_folder: \n",
        "    # Generate the project directory structure and get the zip content\n",
        "    zip_content = project_directory.create_directory_structure(create_directories=False)\n",
        "\n",
        "    # Save the zip file to disk (optional)\n",
        "    with open(\"project_structure.zip\", \"wb\") as f:\n",
        "        f.write(zip_content)"
      ]
    },
    {
      "cell_type": "code",
      "execution_count": null,
      "metadata": {},
      "outputs": [],
      "source": []
    }
  ],
  "metadata": {
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      "display_name": "constrain",
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