Cell Biology JoVE (Open Access) Citable · DOI

CRISPR Guide RNA Cloning for Mammalian Systems

Name: CRISPR Guide RNA Cloning for Mammalian Systems
Uploaded: 2026-06-30
Duration: 6 min 48 s
Description: The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

DOI: 10.3791/57998-v

What you'll learn

✓ Design and prepare sgRNA primers for CRISPR mammalian cloning
✓ Ligate annealed oligonucleotides into expression vectors efficiently
✓ Transform and validate CRISPR constructs in E. coli

Protocol

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

Difficulty

intermediate

Total time

~2-3 days (primer design, ligation overnight, E. coli culture 12-16 hrs, PCR analysis)

Biosafety

BSL-1

Steps

Prepare primers and expression vectors

Design sgRNA-specific oligonucleotide primers and select appropriate mammalian expression vectors. Prepare all reagents and materials needed for downstream cloning steps.

▶ 00:25

Ligate annealed gRNA oligonucleotides

Anneal complementary oligonucleotides and ligate them into the prepared expression vector using standard ligation protocols. Incubate overnight to maximize ligation efficiency.

▶ 02:05

Transform ligation product into E. coli

Introduce the ligated plasmid into chemically competent or electrocompetent E. coli cells. Plate on selective media and incubate overnight for colony growth.

▶ 02:59

Perform PCR analysis of clones

Conduct colony PCR screening to identify successful CRISPR construct clones. Analyze PCR products by gel electrophoresis to confirm correct insert size and orientation.

▶ 04:03

Verify CRISPR guided RNA cloning results

Review PCR analysis results and confirm successful cloning of sgRNA constructs. Document positive clones for downstream mammalian cell transfection experiments.

▶ 05:35

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