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Experimental Overview of CRISPR Cas9 for Gene Knockout


 
 

Welcome to our training series on performing your own CRISPR Cas9 experiment for gene knockout. Each week we’ll send you new instructional material including decision-making tools, protocols, and troubleshooting advice on how to design and carry out your gene knockout experiment.

 
 

This Week We Will Learn


 
 

CRISPR-Crash-Course-check-icon_v2-08

How the CRISPR Cas9 system works

 

CRISPR-Crash-Course-check-icon_v2-08 Experimental overview

 

CRISPR-Crash-Course-check-icon_v2-08How to choose a Cas9 Nuclease

Cas9-character-holding-a-book-07
 
 

1. Introduction to CRISPR-Cas9


 
 

Watch this short video for an introduction to the CRISPR Cas9 system, or skip to the paragraph below.

CRISPR Cas9 technology has two main components: 1) a Cas9 endonuclease; and 2) a single guide RNA (sgRNA). The Cas9 nuclease possesses two DNA cleavage domains that can cut DNA, forming a double strand break (DSB). The sgRNA is an engineered single-stranded chimeric RNA, combining the scaffolding function of the bacterial tracrRNA with the specificity of the bacterial crRNA. The 20bp at the 5’ end of the sgRNA binds to a target site directly upstream of a protospacer adjacent motif (PAM) then Cas9 will create a precise DSB at the region.

This DSB will be repaired by the native repair mechanisms of the cell. If a knock-in is desired, the Homology-Directed Repair (HDR) pathway can be exploited to insert a sequence at this location. However, for the purposes of a simple gene knockout, we will focus on repair via Non-Homologous End Joining (NHEJ). This repair mechanism is error-prone, and often results in frameshift insertion or deletion (InDel) mutations that effectively knockout gene expression.

CRISPR-Repair-Mechanisms

Summary of two dsDNA cleavage repair pathways: NHEJ (Non-Homologous End Joining) and HDR (Homology Directed Repair).
 
 

2. CRISPR Knockout Protocol Overview


 
 
CRISPR-general-workflow
 
 

3. Choosing a Cas9 Nuclease for Knockout


 
 

While the most commonly used nuclease for knockout is wildtype spCas9 (originally from S. pyogenes), others have been proposed as alternatives. The most commonly used variants are outlined below:

 

WT spCas9

spCas9 Nickase

saCas9

Gene Length

4.1 kb

4.1 kb

3.3 kb

PAM Sequence

5’-NGG-3’

5’-NGG-3’

5’-NNGRRT-3’

sgRNA Requirement

1 sgRNA

2 sgRNAs

1 sgRNA

Off-Target Effects

***

*

**

 
spCas9 Nickase was designed to improve off-target mutagenic effects of CRISPR gene editing. The Cas9 Nickase is a mutant form of Cas9 with either a D10A or H840A mutation, which inactivates one of its cleavage domains. This mutant form results in the generation of a single stranded nick instead of a double stranded break at the target site. Since a single stranded break (or nick) is normally quickly repaired through the HDR pathway using the intact complementary DNA strand as the repair template, off-target effects of the Cas9 Nickase are minimized.

Cas9-Nickase-Mechanism

Cleavage of dsDNA by paired Cas9 Nickases.

 

saCas9 (from S. aureus) and spCas9 are able to cleave eukaryotic DNA in vivo with comparable efficiency, but saCas9 is approximately 1 kb small than spCas9. This allows it to be packaged effectively into adeno-associated virus (AAV) for delivery. AAV is a preferred method of gene delivery for in vivo studies due to its low immunogenicity and ability to selectively infect certain tissue types. We will go into more details about delivery methods in Week 3 of the training course.

As well, saCas9 opens up new possibilities for genome editing due to its different targeting capabilities compared to spCas9. spCas9 recognizes a PAM sequence of 5’-NGG-3’, while saCas9 recognizes 5’-NNGRRT-3’. A greater variety in PAM sequences available for use means an increased number of loci are available for genome editing. As well, because this sequence occurs less frequently in the genome than spCas9’s PAM sequence, saCas9 has a reduced risk of off-target cleavage.

>>> You can learn more about saCas9 by watching our video youtube

 

So, which nuclease should you use?

  • Use spCas9 if there is an appropriately placed PAM sequence and a low concern for off-target effects. This is the most commonly used Cas9, so there are many commercially available expression systems for it.
  • Use saCas9 if you wish to perform an in vivo experiment.  It is smaller, so it can be packaged inside AAV, which has low immunogenicity and is commonly used in gene therapy experiments. It may also be useful in circumstances where there are no spCas9 PAM sequences in the target region.
  • Use spCas9 Nickase if there is a large concern for off-target effects, and there are two PAM sequences in close proximity to each other within the target region. It is also useful for knock-in experiments.

 

>>> abm offers all of these nucleases and more. See our Cas9 proteins and expression vectors.

Remember that each Cas9 will require different sgRNA design considerations. We will go over sgRNA design in detail next week, as well as to how to determine whether a sgRNA will cut efficiently.

 
 

Test Your Knowledge!


 
 

Take our Week 1 Quiz, and graduate to becoming a Knockout Novice! 

 

How did you do? Share your results with #CRISPRCrashCourse: