bio-genome-engineering-grna-design

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.

Guide RNA Design

"Design guide RNAs for my CRISPR knockout experiment" → Scan a target gene sequence for PAM sites, extract candidate spacer sequences, and score them for on-target activity using Rule Set 2 or CRISPRscan algorithms.

• Python: custom PAM scanning with Bio.Seq, CRISPRscan scoring models

Find PAM Sites

from Bio.Seq import Seq
import re

def find_pam_sites(sequence, pam='NGG', guide_length=20):
    '''Find all PAM sites and extract guide sequences

    PAM patterns:
    - NGG: SpCas9 (most common)
    - TTTN: Cas12a/Cpf1 (5' PAM)
    - NNGRRT: SaCas9 (smaller, for AAV delivery)
    '''
    sequence = sequence.upper()
    guides = []

    # NGG PAM - guide is 20bp upstream of PAM
    if pam == 'NGG':
        for match in re.finditer(r'(?=(.GG))', sequence):
            pos = match.start()
            if pos >= guide_length:
                guide = sequence[pos - guide_length:pos]
                guides.append({
                    'sequence': guide,
                    'pam': sequence[pos:pos + 3],
                    'position': pos - guide_length,
                    'strand': '+'
                })

    # Also search reverse complement
    rc_seq = str(Seq(sequence).reverse_complement())
    for match in re.finditer(r'(?=(.GG))', rc_seq):
        pos = match.start()
        if pos >= guide_length:
            guide = rc_seq[pos - guide_length:pos]
            original_pos = len(sequence) - pos
            guides.append({
                'sequence': guide,
                'pam': rc_seq[pos:pos + 3],
                'position': original_pos,
                'strand': '-'
            })

    return guides