Applications: Genome Editing Using CRISPR/Cas9

CRISPR/Cas9 Genome Editing: Transfection Methods

TRANSFECTION TIPS VIDEO: CRISPR/Cas9 Transfection
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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Joshua Chou, Ph.D.
Harvard School of Dental Medicine

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Overview of Genome Editing with CRISPR/Cas9

BACKGROUND

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.

EDITING MAMMALIAN GENOMES WITH CRISPR

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.

ADVANTAGES

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.

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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.

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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.

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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.

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Pros and Cons of DNA, RNA and Protein Formats for Genome Editing. Cas9 can be delivered as plasmid DNA for a simple, low-cost approach. Cas9 mRNA enables rapid gene expression, and eliminates the risk of insertional mutagenesis. Cas9/guide RNA ribonucleoprotein (RNP) complexes exhibit the most rapid pulse of genome editing activity and reduce the possibility of off-target cleavage events. Cas9 mRNA and RNP formats can also be efficiently delivered to cell types that are resistant to transfection with plasmid DNA.

Glossary of CRISPR TermsClick to expand

Term	Definition
Cas9	CRISPR Associated Protein 9 - Cas9 is an RNA-guided DNA endonuclease from the type II CRISPR system of Streptococcus pyogenes that has been adapted for use in genome editing applications.
Cas9 Nickase	A Cas9 mutant that cuts one strand of double-stranded DNA Cas9 has two endonuclease domains which together cleave both strands of DNA. Mutations in either of these domains convert Cas9 to a "nickase" which cleaves only a single strand in the DNA target. Two Cas9 nickases combined with gRNAs that target opposite DNA strands can be used to create DSBs with fewer off-target effects.
CRISPR	Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR refers to prokaryotic DNA elements involved in adaptive immunity which are characterized by clusters of identical repeats interspaced with non-identical segments called spacers. CRISPR has evolved to refer more generally to the use of Cas9 for genome editing.
crRNA	CRISPR RNA - One of two RNAs required to form a functional gRNA. The crRNA contains the sequence complementary to the DNA target and a segment of RNA that base pairs with the tracrRNA.
DSB	Double Strand Breaks result from endonucleolytic cleavage of both strands of DNA. This can be achieved through the use of wild type Cas9 or by employing two Cas9 nickases targeting opposite DNA strands.
Genome Editing	The creation of desired genetic modifications including gene insertions, deletions, or replacements through the use of targeted nucleases such as Cas9.
gRNA	Guide RNAs bind to Cas9 and direct the complex to a specific genomic location. Naturally occurring guide RNAs consist of two parts: a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). Alternatively, the crRNA and tracrRNA can be combined into a single chimeric oligonucleotide called a single guide RNA (sgRNA).
HDR	Homology Directed Repair is a mechanism of DNA repair that uses a homologous DNA template to rebuild sites of genomic damage. HDR can be leveraged in genome editing experiments to make precise genomic alterations by supplying the desired sequence for insertion flanked by segments of DNA that are homologous to the sequence surrounding the Cas9-induced DSB.
InDel	(Insertion/Deletion) Following creation of a DSB by Cas9, the cell initiates DNA damage repair. Repair by the error-prone NHEJ pathway can result in small insertions and/ or deletions at the site of cleavage. These indels can cause frameshift mutations or premature stop codons resulting in a genetic knock-out.
Mismatch Assay	A method for detection of indel mutations following Cas9 cleavage. Targeted genomic DNA is amplified by PCR. The PCR products are melted and reannealed to allow heteroduplexes to form between wild-type and mutant DNA. The hybridized products are then incubated with an enzyme that cleaves heteroduplexes but not perfectly matched DNA. The resulting DNA fragments are analyzed by electrophoresis to determine the percentage of cleavage events that results from indel formation.
NHEJ	Non-Homologous End-Joining is the predominant DNA DSB repair mechanism in mammalian cells. Unlike HDR, NHEJ directly ligates the ends of the DSB and does not require a homologous repair template. Researchers capitalize on the error-prone nature of NHEJ to create indels following targeted cleavage with Cas9.
Off-target Effects	Off-target effects refers to Cas9 cleavage events at genomic locations other than the intended site.
PAM	Protospacer-Adjacent Motif - In the naturally occurring prokaryotic CRISPR/Cas system, the DNA sequences recognized by gRNA are called protospacers. The PAM is a short sequence next to the target site that is required for Cas9 targeting both in prokaryotic adaptive immunity and in mammalian genome editing experiments.
sgRNA	Single Guide RNA, a chimeric RNA composed of crRNA and tracrRNA, connected by a short RNA linker.
Target Sequence	A 20 nucleotide genomic DNA sequence which base-pairs with gRNA and is cleaved by Cas9.
tracrRNA	Trans-Activating crRNA - One of two RNAs required to form a functional gRNA. The tracrRNA forms base pairs with the crRNA and is required for Cas9-mediated target cleavage.

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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

Applications: Genome Editing Using CRISPR/Cas9

CRISPR/Cas9 Genome Editing: Transfection Methods

Customer Testimonial

Overview of Genome Editing with CRISPR/Cas9

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