What is gRNA Sequence Checker?

CRISPR gRNA sequence checker — Evaluates a 20-nt guide RNA against: (a) PAM (NGG) presence, (b) GC% within 40–60%, (c) poly-T (TTTT) avoidance — U6 promoter transcription terminator, (d) self-complementarity (hairpin) risk, (e) GC distribution, 5' G start. Outputs a composite score.

When should I use gRNA Sequence Checker?

(1) Quickly pre-validate gRNA candidates from external tools (CRISPOR/CHOPCHOP) (2) Verify sequence integrity before ordering synthesized oligos (3) Remove low-quality candidates from multi-gRNA libraries (4) Spot Cas9 activity risks (poly-T, hairpin) early

Can you show me an example of gRNA Sequence Checker?

Example 1) 5'-GAGTCCGAGCAGAAGAAGAA-3' (PAM: GGG) → Length: 20 ✓, GC: 55% ✓ → Poly-T: none ✓, Hairpin: weak ✓ → 5' G start ✓ → Score 5/5 (excellent) Example 2) 5'-AAATTTTAAATGCGCATATA-3' (PAM: AGG) → GC: 25% ✗ (too low) → Poly-T: TTTT detected ✗ (U6 terminator!) → Score 1/5 (redesign needed)

gRNA Sequence Checker

CRISPR guide RNA 서열 검증 및 분석

Score = f(GC%, poly-T, self-complementarity, PAM)

gRNA SEQUENCE

PAM TYPE

RESTRICTION SITE CHECKS

BbsI(GAAGAC)— Golden GateBsaI(GGTCTC)— Golden GateBsmBI(CGTCTC)— Golden Gate (NEB)EcoRI(GAATTC)— General cloningBamHI(GGATCC)— General cloning

CUSTOM FORBIDDEN MOTIFS

도구 가이드

정의

CRISPR gRNA 서열 검증기 — 입력 20-nt guide RNA 서열을 다음 기준으로 평가합니다: (a) PAM 위치(NGG) 확인, (b) GC% 40~60% 범위, (c) poly-T(TTTT) 회피 — U6 promoter transcription terminator, (d) self-complementarity(hairpin) 위험, (e) GC 분포·5' G 시작 여부 등. 종합 score 출력.

목적

(1) 외부 도구(CRISPOR/CHOPCHOP)에서 도출한 gRNA 후보 빠른 1차 검증 (2) 합성 oligo 발주 전 sequence integrity 확인 (3) Multi-gRNA 라이브러리에서 low-quality candidate 제거 (4) Cas9 활성 저하 risk factor(poly-T, hairpin) 사전 발견

사용법

① gRNA spacer 서열 입력 (20 nt — PAM 제외) ② 옵션: PAM 함께 입력(23 nt 전체)하면 PAM 위치 자동 확인 ③ 자동 평가 출력: • Length check (20 nt) • GC% (권장 40~60%) • Poly-T 존재 여부 (4개 이상 연속 T) • Self-complementarity (hairpin) 검사 • 5' G 시작 여부 (U6 promoter 효율 ↑) • Overall score (1~5)

예시

예 1) 5'-GAGTCCGAGCAGAAGAAGAA-3' (PAM: GGG) → Length: 20 ✓, GC: 55% ✓ → Poly-T: 없음 ✓, Hairpin: 약함 ✓ → 5' G 시작 ✓ → Score 5/5 (excellent) 예 2) 5'-AAATTTTAAATGCGCATATA-3' (PAM: AGG) → GC: 25% ✗ (낮음) → Poly-T: TTTT 검출 ✗ (U6 terminator!) → Score 1/5 (재설계 필요)