CRISPR/Cas9 Genome Editing: Transfection Methods

This video covers the basic mechanism of CRISPR/Cas9 genome editing and introduces the key points affecting researchers' decisions about whether to deliver Cas9 and guide RNA in plasmid, RNA or RNP format.

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Harvard School of Dental Medicine

Overview of Genome Editing with CRISPR/Cas9


Bacteria and archaea exhibit chromosomal elements called clustered regularly interspaced short palindromic repeats (CRISPR) that are part of an adaptive immune system that protects against invading viral and plasmid DNA. In Type II CRISPR systems, CRISPR RNAs (crRNAs) function with trans-activating crRNA (tracrRNA) and CRISPR-associated (Cas) proteins to introduce double-stranded breaks in target DNA. Target cleavage by Cas9 requires base-pairing between the crRNA and tracrRNA as well as base pairing between the crRNA and the target DNA (See figure CRISPR/Cas9 Genome Editing). Target recognition is facilitated by the presence of a short sequence called a protospacer-adjacent motif (PAM) that conforms to the sequence NGG.


The bacterial CRISPR/Cas9 system has been adapted to serve as a versatile platform for RNA-directed genome editing in mammalian cells. The Cas9 endonuclease can be programed by a dual RNA (crRNA and tracrRNA), or the core components of these RNAs can also be combined into a single hybrid guide RNA. Once the Cas9 has cleaved the target DNA, two endogenous repair mechanisms, non-homologous end joining (NHEJ) and homology-directed repair (HDR), are triggered in response to the DNA break. The features of these DNA break repair pathways can be exploited to generate gene knock-outs or introduce defined modifications at the site of cleavage. NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function. HDR requires homologous DNA as a template for repair and can be leveraged to create a limitless variety of modifications specified by the introduction of donor DNA containing the desired sequence flanked on either side by sequences bearing homology to the target.


The simplicity of using of a noncoding RNA guide to target DNA for site-specific cleavage provides a distinct advantage over alternative genome editing technologies such as ZFNs and TALENs. Using the CRISPR/Cas9 strategy, retargeting the nuclease complex only requires introduction of a new RNA sequence and there is no need to reengineer the specificity of DNA-binding proteins.

RNA-programmed DNA Cleavage by Cas9

CRISPR/Cas9 Genome Editing. The Cas9 endonuclease (blue) is targeted to DNA by a guide RNA which can be supplied as a two-part system consisting of crRNA and tracrRNA or as a single guide RNA, where the crRNA and tracrRNA are connected by a linker (dotted line). Target recognition is facilitated by the protospacer-adjacent motif (PAM). Cleavage occurs on both strands (scissors) 3 bp upstream of the PAM.

Multiple Genomic Alterations are Possible Following Cleavage of Target DNA by Cas9

Multiple Genomic Alterations are Possible Following Cleavage of Target DNA by Cas9. Variable length insertions and/or deletions (indels) can result near the DNA break due to mistakes in DNA repair by the endogenous non-homologous end joining (NHEJ) pathway. These indels frequently result in disruption of gene function. Alternatively, by supplying a DNA repair template, researchers can leverage the homology-directed repair (HDR) pathway to create defined deletions, insertions or modifications.

CRISPR Multiple Delivery Methods Data

DNA, RNA and Protein Formats for CRISPR Genome Editing. TransIT-X2® Dynamic Delivery System was used to deliver Cas9 pDNA/gRNA and Cas9 protein/gRNA (RNP complex). TransIT®-mRNA was used to deliver Cas9 mRNA/gRNA.  A T7E1 mismatch assay was used to measure cleavage efficiency at 48 hours post-transfection. For more details, see our methods for delivery of CRISPR/Cas9 DNA, RNA and RNP.

Glossary of CRISPR TermsClick to expand

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Brochure: CRISPR/Cas9 Genome Editing
Poster: Optimization of DNA, RNA and RNP Delivery Methods for Efficient CRISPR/Cas9 Mediated Cell Engineering
White Paper: Optimization of DNA, RNA and RNP Delivery Methods for Efficient CRISPR/Cas9 Mediated Cell Engineering

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