Advancing Enzyme-Directed Evolution with CRISPR Technology    

Directed evolution is a powerful forward engineering approach that is heavily relied upon in protein engineering, which is essential for producing variations with enhanced performance or novel traits. Creating molecular variety at the DNA level and choosing potential proteins from a large pool of variations are the two steps in this process. However, for screening and validation, the majority of protein engineering approaches used today mostly rely on in vitro methods or microbial expression systems. The existing screening techniques may not be optimal for many protein types needing phenotypic selection in human or mammalian cells. CRISPR technology has the potential to select and diversify proteins in mammalian cells.

Introduction to CRISPR/Cas base editing enzymes

Directed evolution has already been used by nature to successfully create antibody libraries in mammalian cells. This is accomplished via somatic hypermutation, which is brought on by enzyme activation-induced cytidine deaminase (AID) in the immunoglobulin gene areas of B lymphocytes, and VDJ recombination in certain gene loci of B lymphocytes.

Figure 1. A protein-encoding gene is inserted into a Cas9-expressing mammalian cell, possibly with additional features like a selection marker and modifications to inhibit expression. (Griesbeck O. et al., 2021)

A technique that is adaptable to several types of heterologous cells and is comparable to somatic hypermutation in B lymphocytes has been developed using CRISPR/Cas technology. Local point mutations are induced in mammalian cells by recruiting AID to different protein-coding areas by the use of catalytically inactive Cas9 (dCas9) variants. The rate of mutation is similar to B lymphocyte somatic hypermutation. Multiplexed guide RNAs (gRNAs) can be used to simultaneously target different protein areas, like GFP.

Targeted protein diversification is accomplished by CRISPR/Cas9 through the induction of double-strand breaks and homologous recombination repair (HDR). Although it requires some prior knowledge of the parental protein (e.g., from earlier mutation studies, biophysics, or structural insights), this method can produce highly regulated and diversified mutation libraries.

Technological Advances

A cytidine deaminase called AID transforms cytidine into uridine, which in turn causes other bases to be replaced using cellular repair processes. AID-induced mutations, however, are restricted to codons that have C-G pairings. Adenine deaminases for directed evolution targeting A-T pairings and more cytidine deaminases with varying accuracy and mutation consequences have been discovered in recent years. Through the combination of dCas9 or nCas9 nickase with cytidine and adenine editors, a dual base editing system has been developed that permits the simultaneous alteration of A-T and C-G pairs in living cells. Over time, this technique is being improved and used to study protein mutation in mammalian and plant cells.

Technological Challenges

Finding the ideal editing window mutation rate to generate the most beneficial protein variants is essential for optimizing protein engineering. To guarantee the ideal arrangement of guide RNAs covering protein-coding areas, while taking into account potential off-target effects, the most appropriate base editors and their combinations for protein engineering must also be determined. One limiting factor is still the basic sequence requirements of CRISPR/Cas nucleases for PAM (protospacer adjacent motif). For Cas9 to attach to target sequences and activate the enzyme, it needs a PAM sequence of 5'-NGG. The distribution or scarcity of accessible non-coding guide sites (NGG sites) in the coding region will lead to an unsatisfactory arrangement of single guide RNAs (sgRNAs) and inadequate mutation outcomes. Combining Cas9 with Cas12a variants with different PAM requirements can significantly improve the arrangement of sgRNAs, resulting in more comprehensive mutations.

By creating molecular variety at the DNA level and choosing potential proteins from it, directed evolution is a successful engineering technique that enhances protein function or adds new features. While CRISPR technology offers a new pathway for protein modification in mammalian cells, traditional protein engineering methods generally rely on in vitro techniques or microbial expression systems for screening and validation. A new chapter in the identification of a variety of proteins that need to be investigated in the context of mammalian cells could be heralded by the use of CRISPR/Cas tools for directed evolution in mammalian cells.

Accelerate the Emergence of Innovative Achievements

The CRISPR technology from Creative Biogene's goal is to give you the most advanced and dependable resources possible so you can enhance protein function and add new features. You can quickly and precisely carry out protein-directed evolution with our CRISPR/Cas technology. We can offer you specialized solutions whether you need to create mutant libraries, add insertion-deletion mutations, or carry out precise genome editing. We are eager to work with you to further your research objectives and open up new vistas in enzyme-directed evolution technology.

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