Version Compatibility

Reference examples tested with: BioPython 1.83+

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show <package> then help(module.function) to check signatures

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Prime Editing Design

"Design a prime editing guide for my point mutation" → Generate pegRNA sequences (spacer, scaffold, RT template, PBS) for precise genomic modifications without double-strand breaks, optimizing PBS length and RT template for editing efficiency.

• Python: PrimeDesign algorithms with Bio.Seq for sequence handling

pegRNA Structure

pegRNA components:
1. Spacer (20nt) - guides Cas9 to target site
2. Scaffold - Cas9 binding sequence
3. RT template - encodes the desired edit
4. PBS (primer binding site) - anneals to nicked strand

        Spacer (20nt)      Scaffold     RT template    PBS
    5'─[NNNNNNNNNNNNNNNNNNNN]─[scaffold]─[edit]─────[PBS]─3'

Design pegRNA for Point Mutation

from Bio.Seq import Seq

def design_pegrna_substitution(target_seq, edit_pos, new_base, pbs_length=13, rt_length=15):
    '''Design pegRNA for a point mutation

    Args:
        target_seq: ~100bp sequence centered on edit site
        edit_pos: Position of nucleotide to change (0-indexed in target_seq)
        new_base: New nucleotide (A, C, G, or T)
        pbs_length: Primer binding site length (13-17nt optimal)
                   Shorter = less stable, Longer = more secondary structure
        rt_length: RT template length including edit (10-20nt for substitutions)

    Returns:
        dict with pegRNA components
    '''
    target_seq = target_seq.upper()

    # Find nick site (3bp upstream of PAM, which is 3bp after edit for +strand)
    # For substitution, nick should be close to edit site
    nick_pos = edit_pos + 3  # Adjust based on PAM location

    # Spacer: 20nt upstream of PAM
    spacer_start = nick_pos - 17  # Nick is 3bp upstream of PAM
    spacer = target_seq[spacer_start:spacer_start + 20]

    # PBS: Reverse complement of sequence just upstream of nick
    pbs_region = target_seq[nick_pos - pbs_length:nick_pos]
    pbs = str(Seq(pbs_region).reverse_complement())

    # RT template: Contains the edit
    # Sequence from nick site, with edit incorporated
    rt_region = list(target_seq[nick_pos:nick_pos + rt_length])

    # Incorporate the edit
    edit_offset = edit_pos - nick_pos
    if 0 <= edit_offset < len(rt_region):
        rt_region[edit_offset] = new_base

    rt_template = str(Seq(''.join(rt_region)).reverse_complement())

    return {
        'spacer': spacer,
        'pbs': pbs,
        'rt_template': rt_template,
        'pbs_length': pbs_length,
        'rt_length': rt_length,
        'edit_type': 'substitution'
    }