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Advancing genome editing with Cas fusion proteins

CRISPR alleviates many historical challenges to genome editing, and research is advancing rapidly with new Cas fusion proteins

Genome editing is the process of altering the sequence of a targeted region of DNA in any genome. Several methods of genome editing were developed in past decades, but all of these previously discovered methods presented many challenges. Despite these challenges, the groundwork was laid for new developments such as clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. CRISPR has been found to be easy, fast, and inexpensive, and it holds much promise for basic and translational research applications. Building on earlier discoveries, new approaches such as base editing, as well as prime editing with pegRNA, are leading the way to even more precise and efficient genome editing.

Genome editing: a brief history

Genome editing is the precise targeting and changing of a chosen genomic DNA sequence. Genome editing techniques are used for targeted replacement, insertion, or deletion of DNA. The terms “gene editing” and “genome engineering” are often used interchangeably with “genome editing.” Before the introduction of CRISPR technology, genome editing in multicellular eukaryotic systems was performed using ineffective, inaccurate, slow, or inefficient methods involving viral vectors, meganucleases, oligonucleotide‐directed mutagenesis, zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs).

For several decades, basic molecular biology techniques have enabled scientists to introduce specific mutations in receptive cell types, especially bacteria and yeast. Such mutagenesis is relatively straightforward, because both bacteria and yeast can easily be “transformed”—caused to take up foreign DNA. Yeast cells undergo homologous recombination even when there is no double-strand break (DSB) in their DNA to be repaired [1], whereas bacteria take up and incorporate plasmids into their genomes [2]. Many important discoveries have been enabled using such mutagenesis processes in bacteria and yeast, but these organisms do not translate well as comparative models of the human body.

In experiments with mammalian or other animal cells [3], harsh chemicals and bombardment with radiation have been used for several decades to induce mutations. However, a specific change at a precise location in a genome cannot be created this way. Transgenic mice, that is, mice with a genomic fragment from a different organism (e.g., a promoter region of a human gene) inserted randomly into their genome, were first developed in the 1980’s. Many transgenic mice were developed to study either promoter regulation or overexpression of genes. In these cases, the transgene introduced would often include either a general or a tissue-specific promoter along with a protein-coding sequence. The development of knockout mice was a significant advance over the next few years, for which the Nobel Prize was awarded in 2007 to Mario R. Capecchi, Martin J. Evans, and Oliver Smithies. Rather than using chemicals or radiation, the procedures they developed for producing knockout mammals involved using complex molecular biology techniques to introduce the desired mutations in mouse embryonic stem cells. These cells were used to generate live mice with precise, specific mutations. The same techniques were also shown to work in rats and other live mammals. Although generating such a knockout or other mutant animal was challenging and expensive, it was easier than introducing a specific desired mutation in the genome of an immortalized mammalian cell line in culture, which often was impossible.

ZFNs and TALENs: fusion proteins that led the way in specific genome editing

Two protein-based systems, ZFNs and TALENs, were the most specific approaches to mutagenesis developed before CRISPR. ZFNs and TALENs are engineered endonucleases composed of DNA-binding domains fused to FokI, an endonuclease that cleaves DNA (Table 1). The DNA-binding domains must be individually designed to target specific DNA sequences within the genome, allowing the FokI to cleave DNA at the desired location. FokI must dimerize to cleave DNA, so two TALENs or two ZFNs are required for cutting a specific site in the genome. In other words, when using either ZFNs or TALENs, two entire protein sequences must be designed and introduced into the cells to be edited. These two proteins flank and thus target a genomic site. This requirement supports specificity but increases cost and complexity of design. Indeed, since ZFNs can be designed to be very specific, they were the most convenient and promising method for targeting the genome to create a DSB at a specified location for several years. TALENs, developed later than ZFNs, also proved to have good specificity. Some TALENS and ZFNs have been used in clinical trials [4, 9].

  • The primary similarity between ZFNs and TALENs is that both are large proteins. They must be custom-designed with protein repeat domains arranged in a specific order to recognize a specific DNA sequence.
  • The primary difference between ZFNs and TALENs is that each protein repeat domain of a ZFN recognizes a group of three DNA base pairs, whereas each protein repeat domain of a TALEN recognizes only a single DNA base pair. This makes TALENs more flexible and easier to design than ZFNs. However, TALENs, like ZFNs, are still expensive and time-consuming to produce.

Table 1. Comparison of genome editing methods

 CRISPR-Cas systems  TALENs  Zinc finger nucleases
 
  • Requirement: design of a short RNA sequence; commercially available protein
  • Inexpensive
  • Quick setup
  • Editing efficiency up to 100%
  • Flexibility for many applications including screening an entire genome
 
  • Requirement: design of a large protein
  • Expensive
  • Slow to set up
  • May allow > 80% editing efficiency [5]
  • Often limited to single-target applications
 
  • Requirement: design of a large protein
  • Expensive
  • Slow to set up
  • Typically <30% editing efficiency [6]; may reach >80% [7,8]
  • Often limited to single-target applications

CRISPR-Cas systems

CRISPR-Cas genome editing methods provide the simplest route to editing genomic DNA. The technology uses a Cas endonuclease rather than FokI to generate double-strand breaks in DNA, because unlike Fok I, Cas endonucleases have the inherent property of being easy to direct to a target site. They do not have to be fused to other proteins for targeting purposes. Indeed, Cas endonucleases require only an easily designed and quickly produced short guide RNA to specify the DNA target sequence to be cleaved, unlike ZFNs and TALENs, which require scientists to develop and produce entire new protein sequences for every DNA target sequence to be modified. The most commonly used Cas endonucleases are Cas9 and Cas12a. CRISPR technology makes genome modification in cells or organisms easier, faster, more efficient, and much less expensive than previous genome editing methods. Some CRISPR-Cas systems have been used in clinical trials [4] (all IDT CRISPR-Cas products are for research use only unless otherwise indicated in writing).

The CRISPR Basics Handbook

CRISPR, ZFNs, and TALENS all require optimization, but this is easier with CRISPR

ZFNs and TALENs are large proteins, so in the context of a cell, adjacent modules within these proteins exhibit crosstalk, and their context affects their specificity [10]. Thus, scientists need to screen many possible designs just to edit a single genomic site. Some comparable screening is necessary with CRISPR too. Scientists usually need to use a handful of CRISPR guide RNA sequences (about 3 or 4) to test-target genomic sites within proximity of each other. This is because different guide RNAs are not equally efficient at directing Cas enzymes to cut the specified target. Testing several guide RNA sequences allows identification of guides that have higher targeting efficiency at the target site and fewer off-target effects. However, it is much easier, less time-consuming, and cheaper to design a few guide RNA sequences than to screen for efficiency and site-specificity of several ZFNs or TALENS. Furthermore, when an empirically-designed web-based tool such as the IDT Alt-R gRNA Design Tool is used, the testing process to determine the most efficient gRNA sequence is much faster than manually selecting a sequence.

Advanced CRISPR techniques that use Cas fusion proteins

CRISPR base editing

CRISPR “base editing” involves a Cas enzyme fused with an enzyme that directly changes one base to another within single-stranded DNA (ssDNA). These enzymes are known as nucleobase deaminases and can be classified either as cytosine base editors (CBE) or adenine base editors (ABE) [11]. The CBEs convert C-G base pairs to T-A base pairs; the ABEs convert A-T to G-C. Usually, a Cas9 nickase, rather than wild-type enzyme, is fused to the CBE or ABE. This is so that one strand will be cut while the other strand (which is temporarily single-stranded in the targeted region due to Cas-mediated unwinding) undergoes exchange of one base for another. Then cellular machinery will repair the cut region to match the strand with the newly replaced base. The base editing approach allows remarkable specificity.

CRISPR prime editing

Another high-specificity advance in genome editing is the use of prime editing, a form of CRISPR that also uses a fusion protein of Cas9 nickase with another enzyme. In this system, the enzyme fused to the Cas is a reverse transcriptase. This fusion protein is used with an unusually long guide RNA, known as prime editing guide RNA or pegRNA. The pegRNA encodes the new sequence, allowing transcription to introduce the desired mutations. Prime editing works in a complementary fashion to the base editor systems, depending on the location of the desired edits [12].

Fusion proteins with Cas enzymes: current and future prospects

In addition to changing a DNA sequence by knocking out genes, inserting, or mutating DNA, CRISPR genome editing techniques have been modified to produce other effects on genomic DNA. Additional applications of CRISPR fusion proteins include gene silencing, known as CRISPR interference (CRISPRi); and transportation of transcription activators (CRISPRa), fluorescent proteins, and epigenomic modifier enzymes to specific sites in the genome. Creative new approaches to using sequence-specific endonucleases, in particular the Cas family of enzymes, continue to be introduced. Sequence specificity of targeting is allowing research and development into many new solutions in industry, medicine, and agriculture.

RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

References

  1. Gardner JM, Jaspersen SL. Manipulating the Yeast Genome: Deletion, Mutation, and Tagging by PCR. Springer New York; 2014:45-78.
  2. Holmes RK, Jobling MG. Genetics. In: Baron S, ed. Medical Microbiology. Galveston: University of Texas Medical Branch at Galveston; 1996.
  3. Clark DP, Pazdernik NJ, McGehee MR. Mutations and Repair. In: Clark DP, Pazdernik NJ, McGehee MR, eds. Molecular Biology (Third Edition): Academic Cell; 2019:832-879.
  4. Li H, Yang Y, Hong W, et al. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduction and Targeted Therapy. 2020;5(1).
  5. Knipping F, Osborn MJ, Petri K, et al. Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification. Mol Ther Methods Clin Dev. 2017;4:213-224.
  6. Bjurstrom CF, Mojadidi M, Phillips J, et al. Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases. Mol Ther Nucleic Acids. 2016;5:e351.
  7. Chan Y-S, Takeuchi R, Jarjour J, et al. The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive. PLoS One. 2013;8(9):e74254.
  8. Esvelt KM, Smidler AL, Catteruccia F, et al. Concerning RNA-guided gene drives for the alteration of wild populations. Elife. 2014;3:e03401.
  9. Paschon DE, Lussier S, Wangzor T, et al. Diversifying the structure of zinc finger nucleases for high-precision genome editing. Nat Commun. 2019;10(1):1133.
  10. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262-1278.
  11. Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet. 2018;19(12):770-788.
  12. Anzalone AV, Randolph PB, Davis JR, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576:149–157.

Published Mar 9, 2021