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Genetics / Genomics JoVE (Open Access) Citable · DOI

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

DOI: 10.3791/58556-v
What you'll learn
  • ✓ Design and synthesize customized gRNA primers with appropriate overhangs for STAgR cloning
  • ✓ Perform Gibson assembly and bacterial transformation of multiplexed gRNA vectors
  • ✓ Screen and validate STAgR clones using colony PCR and sequencing
Protocol

Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

Difficulty
intermediate
Total time
~3–5 days (including bacterial growth and colony screening)
Biosafety
BSL-1

Steps

1
Design STAgR cloning primers with overhangs

Design primers encoding guide RNAs with compatible overhangs for seamless assembly. Calculate overhang sequences to enable directional concatenation of multiple gRNAs into a single vector.

▶ 00:34
2
Generate STAgR cloning fragments via PCR

Amplify gRNA sequences using designed primers to generate assembly-ready DNA fragments with defined overhangs. Purify PCR products for use in Gibson assembly.

▶ 01:22
3
Perform Gibson assembly and bacterial transformation

Combine gRNA fragments with linearized vector backbone in a Gibson assembly reaction, then transform competent bacteria. Plate transformed colonies on selective media.

▶ 03:56
4
Screen STAgR clones by colony PCR

Perform colony PCR on bacterial colonies to rapidly identify correct inserts. Confirm positive clones before proceeding to plasmid isolation and sequencing.

▶ 05:50
5
Analyze and validate multiplexed gRNA constructs

Sequence confirmed clones to verify correct assembly of all gRNA units and correct orientation within the vector. Document successful multiplexing of gRNA elements.

▶ 08:08
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