Single guide RNAs (sgRNAs) are a critical component of CRISPR/Cas9 genome editing—success of the experiment often depends on sgRNA design. With so much relying on the sequence, it's common practice to test multiple sgRNAs for a single modification. Cloning of sgRNAs is often done with Type IIS restriction enzymes and ligase, a method that is time-consuming and inflexible. Restriction sites for Type IIS enzymes usually need to be engineered into vectors, and ligase is generally inefficient with large vectors. Given these factors, Type IIS-based approaches require multiple cloning steps to generate your final construct.
In-Fusion Cloning greatly improves and streamlines the genome editing workflow. This seamless technology avoids the bottlenecks of the usual methods and easily accommodates the substantial cloning needs of CRISPR/Cas9 experiments.
Advantages include:
- Single-step, ligase-free reactions
- Restriction site-independent cloning
- Direct cloning into large expression vectors
- Seamless multi-insert cloning
- High-throughput capabilities
These features show particular utility for sgRNA/Cas9 cloning. With a more efficient workflow, you can easily generate many plasmids at once, as may be needed to test multiple sgRNAs for a single modification or for the paired nickases approach. Additionally, the capability for seamless multi-insert cloning is ideally suited to cloning more than one sgRNA in a single vector for multiplex genome editing.
Explore the CRISPR cloning resources below or choose your In-Fusion Cloning kit now.
In-Fusion Cloning has been used for seamless cloning of sgRNA constructs for CRISPR/Cas9 vector systems.
- Development of CRISPR as an antiviral strategy to combat SARS-CoV-2 and influenza. Read now »
- Microhomology-assisted scarless genome editing in human iPSCs. Read now »
- A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants. Read now »
- Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system. Read now »
- MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Read now »
- The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation. Read now »
- Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Read now »
- Efficient genome editing in the oomycete Phytophthora sojae using CRISPR/Cas9. Read now »
- Targeted mutagenesis of guinea pig Cytomegalovirus using CRISPR/Cas9-mediated gene editing. Read now »
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