CRISPR

What is CRISPR?

CRISPR stands for Clustered Regularly Interspaced Short Palendromic Repeats, which are DNA sequences found in prokaryotes to detect and destroy foreign (i.e. viral) DNA. Such sequences are important for the prokaryotic cell, as they act as a form of an immune system in conjunction with CAS-9, (CRISPR Associated Protein 9) an endonuclease that can recognize the foreign viral DNA and remove it. When a virus infects a prokaryotic cell containing the CRISPR sequences, the prokaryote can incorporate the viral DNA into its own genome via the CRISPR sequences. Once incorporated, the cell can transcribe the foreign DNA into a complementary RNA, which will act as a "guide" for the CAS-9 enzyme. In conjunction with its guide RNA, CAS-9 can recognize complementary DNA to its guide RNA (i.e. RNA that is complementary to the foreign viral DNA), and this combination can be used to therefore recognize and cleave said viral DNA if a similar virus were to infect the original prokaryotic cell a second time.

Why Is CRISPR/CAS-9 of Interest to Us?

CRISPR is of importance to our lab and many others due to the ability to manipulate the functionality of the CAS-9 enzyme to cut/remove genes of interest from a cell's genome. Ideally, our ultimate goal is to use CAS-9 in conjunction with guide RNA complementary to the CFTR gene to remove a dysfunctional CFTR gene, and ultimately replace it with a functional CFTR gene. If done correctly, this could theoretically cure Cystic Fibrosis if such a task could be performed in human lung cells. However, we have many steps to take before such a feat can be accomplished. The first step is to practice and perfect the introduction of CAS-9 into eukaryotic cells.

In order to perform our desired edits, there are different CRISPR systems to choose from. One of these is known as prime editing. Prime editing uses the same concept the CRISPR Cas9 system does in that a specific DNA sequence is targeted by an endonuclease by a guide RNA. However, instead of simply creating a double-stranded break, the protein complex involved in prime editing nicks one strand of the DNA and uses a reverse transcriptase to add a pre-designed DNA sequence where the nicked DNA is. DNA repair then occurs to allow the non-nicked strand to copy the edited strand, thus resulting in edited DNA. More information about prime editing can be found below.

Another option available to use is Base Editing. We already possess a base editing plasmid (pCMV-BE2). Base editing is similar to prime editing, except certain base editors can only perform certain edits. For example, Adenine base editors can only do Adenine-Guanine or Guanine-Adenine conversions. More information about base editing can be found below.

Prime Editing

Seeing as how there are >75,000 genetic variants that result in diseases like Cystic Fibrosis, the experimentation of genome editing technology has drastically increased in recent years. Techniques like the original CRISPR-Cas9 function by inducing a double strand break in the DNA, which allows for errors since indels (inserts and deletions) are prone to be added at the sites of editing. Base editing can avoid making double stand breaks in DNA, but is severely limited in the edits that can be made (can only carry out C→T, G→A, A→G, and T→C). Prime Editing is new technology synthesized by Liu et. al and described in their 2019 paper that is able to carry out multiple types of mutation and editing without creating double strand breaks; it is described as a "'search-and-rescue' genome editing technology that mediates target insertions, deletions, all 12 possible base-to-base conversions, and combinations thereof in human cells without requiring DSBs or donor DNA templates."

The original prime editing technology, called PE1, is made of a prime editing complex. This complex consists of a Cas9 nickase domain coupled with a reverse transcriptase domain, and a guide RNA specifically referred to as pegRNA. Addgene

e's Blog provides a simplified explanation of the mechanism of prime editing:

First, an engineered prime editing guide RNA (pegRNA) that both specifies the target site and contains the desired edit(s) engages the prime editor protein. This primer editor protein consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase part of the protein is guided to the DNA target site by the pegRNA. After nicking by Cas9, the reverse transcriptase domain uses the pegRNA as a template for reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Lastly, the editor guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process.

A great video that helps explain how prime editing works can be found here.

Prime Editing 3.0

Ever since Prime Editing's introduction, the system has been improved and appeared in three "versions". Prime editors 1, 2 and 3 (prime editing 1.0, 2.0, and 3.0). Prime editing 3.0 is the most current version and the version we plan to use for our edits. All three version of prime editing utizilize the editing scheme described in the above paragraph, but each version of prime editing increases the efficiency of the edits. All three versions of the prime editior are described in Liu's paper (linked below) However, a general summary of the differences is that prime editor 1 was the intial system. Prime editor 2 involved altering the resiudes involved in the reverse transcriptase to improve edit efficiency. Finally, prime editor 3 incorporates the use of an additional sgRNA after the heteroduplex is created. The sgRNA will guide the nickase to nick the unedited strand at a site nearby the edit, which will cause the cell's DNA repair machinery to remedy the heteroduplex but result in both strands afterwards containing the desired edit with a higher efficiency. Due to this, our system will require a second sgRNA as well as the prime editor 3.0 machinery described in the previous paragraph.

Design CRIPSR 3.0/Prime Editing Plasmids

Prime Editing paper

Image taken from Liu et al. (paper linked above)

Base Editing

There are two types of base editors: Cytosine Base Editors and Adenine Base Editors

Cytosine Base Editors (CBEs)

Can do C --> T (or G --> A) conversions

Adenine Base Editors (ABEs)

Can do A --> G (or T --> C) conversions

Base Editing Summary

Base Editors involve the use of a catalytically impaired Cas9 that cannot make DSBs, and a guide RNA. The guide RNA will lead the Cas9 to the target site (that is also nearby a PAM motif). The Cas9 enzyme will then localize a ssDNA deaminase to the targeted region. The binding via hybridization of the guide RNA to the DNA sequences causes a ssDNA R loop to form. The deaminase will then convert certain bases within the loop depending on the editor. "CBEs use cytidine deaminases to convert cytisines within the loop to uracils, which are read by polymerases as thymines. ABEs use laboratory made TadA* deoxyadenosine deaminases to convert adenosines within the R-loop to inosines, which are read as guanosines by polymerases."

After deamination, mismatch repair will occur. If the modified strand is used as the template, then the edit will be incorporated. If the unedited strand is used as the template, the edit will be erased. Most base editors will create a single-stranded nick in the non-edited strand as a way to signal to the cell/trick the cell that the unedited strand is the "damaged strand". This in turn increases the efficiency of the edit being incorporated into the genome, as the edited strand will more likely be used as the template for mismatch repair in this case.

Summary Taken from/adapted from the following reference: https://www.nature.com/articles/s41587-020-0561-9

Figure 3 in the above paper is a great image for understanding base editing.

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