CRISPR-Cas System in Translational Biotechnology

CRISPR-Cas System in Translational Biotechnology discusses applied and translational aspects of the CRISPR-Cas technology. The book bridges the gap between theoretical knowledge and practical solutions surrounding this emerging and impactful technology in several academic and industrial fields. It is split in five sections: CRISP-Cas fundamentals and advancements; CRISP-Cas in medical biotechnology; CRISP-Cas in environmental biotechnology; CRISP-Cas in food biotechnology; and biosafety, patents and commercialization of CRISP-Cas technology. Written by experts from diverse backgrounds, the content covers the subject and its impact in multiple fields.

It is a valuable resource for graduate students and researchers on bioinformatics, systems biology, and members of the biomedical field and biotechnology industry who are interested in learning more about CRISP-Cas system and its applications.

• Discusses applied aspects of CRISPR-Cas technology and state-of-the-art technological/translational advancements in the field
• Focuses on the CRISPR-Cas mediated genetic engineering for employment in various industries such as medical, agricultural, environmental and food
• Encompasses knowledge on CRISPR-Cas commercialization, potential markets and associated ethical challenges

• Language: English
• Publisher: Academic Press
• Release date: Nov 16, 2023
• ISBN: 9780323972284

Book preview

Section 1

CRISPR-Cas fundamentals and advances

Outline

Chapter 1 CRISPR-Cas9: chronology and evolution

Chapter 2 CRISPR: the Janus god of modern science

Chapter 3 Commercialization of CRISPR-Cas technology: issues and impact

Chapter 1

CRISPR-Cas9: chronology and evolution

Jignesh Mochi¹, Jaykumar Jani¹, Swati Joshi² and Anju Pappachan¹,    ¹School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat, India,    ²Indian Council of Medical Research-National Institute of Occupational Health (ICMR-NIOH), Ahmedabad, Gujarat, India

Abstract

The Clustered Regularly Interspaced Short-Palindromic Repeat (CRISPR) was discovered as an adaptive immune system in bacteria and archaea, being used for protection against viral infection. The CRISPR-Cas system is a complex network in which different types of protein complex, effector complex, and locus of genome play role in pre-CRISPR processing and interference. Since its discovery, it has become a crucial tool for biological research due to its promising and precis gene manipulation power. To corroborate its significance, Noble Prize in Chemical Sciences, 2020, was awarded to discoverer’s of this technique. Because of its easy tunability and success rate on the vast range of organisms, this technique is applicable not only for research but also has potential for clinical and agricultural improvements through genome editing of organisms. Rapid evolution in this technology is making it future ready; it has all the potential to become an important tool for future research. In this chapter, we present a brief introduction of CRISPR-Cas9 discovery, how this technique evolved for various biological applications, its limitation, and future aspects.

Keywords

CRISPR-Cas9; Cas proteins; guide RNA; genome editing; gene regulation; homology-directed repair (HDR); nonhomologous end joining

1.1 Introduction

The development of new methods for whole-genome sequencing makes sense for the design and implementation of large genome annotation projects. In present times, researchers or scientists are keen for converting this large amount of data into functionally and clinically relevant knowledge. It is a challenging issue for investigators to develop a valuable and reliable method to determine how genotype affects phenotype. One such powerful method is homologous recombination which is used for the inactivation of the target gene thus providing information on gene function (Capecchi, 2005). Still, the use of this method has some limitations, such as low efficiency of the gene inserted into the chromosomal target site, time-consuming and labor-intensive selection/screening approaches, and adverse mutagenic effects. Another method is RNAi for target gene knockdown providing an alternative to homologous recombination. The RNAi method is rapid, inexpensive, and high throughput; however, this method also suffers from certain limitations, such as incomplete gene knockout, unpredictable off-target effects, and temporary inhibition of gene function (McManus & Sharp, 2002). In the past decade, a new approach has been developed that enables manipulation of any gene in a variety of cell types and organisms. This newly developed technology is termed Gene Editing. This method is based on engineered chimeric nucleases that are composed of Zinc-finger nuclease (ZNF) that cut target DNA at any site and transcription activator-like effector nuclease (TALEN) proteins induced target DNA double-stranded break and this stimulates DNA repair mechanisms, leading to genome modification (Carroll, 2011; Gaj et al., 2013; Wyman & Kanaar, 2006). Both ZFNs and TALENs techniques have been widely used for Gene Editing in multiple organisms. There are still certain limitations; this approach is suffering, including introduction of off-target mutations and time- and labor-intensive construction of vector (Puchta, 2017; Tang et al., 2017). Therefore to overcome the limitation of the aforementioned strategies, scientists are drawn towards newly emerging gene-editing approaches, that is, Clustered Regularly Interspaced Short-Palindromic Repeat/CRISPR-associated nuclease (CRISPR-Cas system). CRISPR-Cas system is composed of CRISPR short sequence repeats which can be transcribed into CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), and Cas genes which encode Cas proteins possess endonuclease activity (Koonin et al., 2017). In this system, Cas protein cut the foreign DNA and then crRNA pair with the foreign DNA, which guides Cas protein to cleave target sequences of foreign DNA, thereby protecting the host (Makarova et al., 2011). There are different engineered and novel versions of CRISPR-Cas system to use; these will be discussed in upcoming sections of the chapter.

1.2 Discovery of CRISPR-Cas system

CRISPR-Cas system is an adaptive immune system that is present in bacterial and nearly all archaeal genomes (Koonin & Makarova, 2009). These systems evolved over billions of years and function to defend microbes from bacteriophage infection and other conjugating plasmids by targeting their DNA or RNA. The CRISPR-Cas discovery started in 1987 (Fig. 1.1), during this period, the 24 nucleotides repetitive sequences were identified with unknown functions in the Escherichia coli genome and which was the first instance of a CRISPR array (Ishino et al., 1987). The second instance comes from Haloferax mediterranei which contained 30–34 repeat nucleotide sequences spaced by 35-bp-long sequences called spacers (Mojica et al., 1993). During the period from 1987 to 1993, variable repeat sequences or spacer sequences were identified by scientists. The findings of repeated sequences in bacterial and archaeal genomes from bioinformatics studies by researchers drew their attention toward the presence of CRISPR array. In 2002, Jansen and colleagues reported the presence of conserved operon in the genome located near the DNA repair system (Makarova et al., 2002). These genes were associated with CRISPR genes, therefore, were termed CRISPR-associated (Cas) genes. In 2005–2006 two independent research studies observed spacer sequence in CRISPR, which was similar to bacteriophage sequence; these findings suggested that CRISPR-Cas system provided immunity against phages (Makarova et al., 2006; Mojica et al., 2005). After finding CRISPR-Cas, further research is continued in laboratories across the world. The CRISPR array mechanism was first identified as a protection mechanism against viral infections, and later it was used as a gene-editing technology to modify eukaryotic genomes. Thereafter, CRISPR-Cas system is applied in various fields of biology ranging from medicine to agriculture. Bikard and coworkers engineered Cas9 as a transcriptional repressor preventing binding of RNA polymerase (RNAP) to promoter sequences or hindering RNAP (Bikard et al., 2013). Studies are coming up proving that CRISPR system can be used in the field of gene regulation and synthetic biology for biotechnological applications. Scientific community is witnessing spectacular rise in interesting results coming out from studies on CRISPR-Cas system, suggesting that it has a long path to travel and uncover novel solutions to various unsolved problems in the field of biology.

Figure 1.1 Important milestones in the discovery and development of CRISPR-Cas platform.

1.3 Mechanism of CRISPR-Cas system

Before the research start on CRISPR-Cas systems, this system was functioned as immune system in bacteria and archaea, the latter is a key component of a new generation of the genome-engineering tools. The CRISPR-Cas mechanism of action consists of three main stages: adaptation, expression, and interference.

In the adaptation stage, a distinct complex of Cas proteins binds to a target DNA which is short motif known as a proto spacer-adjacent motif (PAM), and leaves out a portion of the target DNA, the protospacer. After duplication of the repeat at the 5′ end of the CRISPR array, the adaptation complex inserts the protospacer DNA into the array, so that it becomes a spacer. Some CRISPR-Cas systems employ an alternative mechanism of adaptation—namely, spacer acquisition from RNA, via reverse transcription by a reverse transcriptase encoded at the CRISPR-Cas locus.

At the expression stage, the CRISPR array is typically transcribed as a single transcript—the pre-CRISPR RNA (pre-crRNA)—that is processed into mature CRISPR RNAs (crRNAs), each containing the spacer sequence and parts of the flanking repeats. In different CRISPR-Cas variants, the pre-crRNA processing is mediated by a distinct subunit of a multiprotein Cas complex, by a single, multidomain Cas protein, or by non-Cas host RNases.

At the interference stage, the crRNA, which typically remains bound to the processing complex (protein), serves as a guide to recognizing the protospacer (or a closely similar sequence) in the invading genome of a virus or plasmid, which is then cleaved and inactivated by a Cas nuclease (or nucleases) that either is part of the effector or is recruited at the interference stage. The above summary is a brief, oversimplified description of the CRISPR-Cas functionality that inevitably omits many details. These can be found in recent reviews on different aspects of CRISPR-Cas biology.

1.4 Classification of CRISPR-Cas system

The vast diversity of the CRISPR-Cas system classified into two classes (I–II) which are based on locus organization and gene conservation; this system also further subdivided into six types are based on the phylogenetic, composition of the genome, and structural and functional diversity of Cas protein. Class I CRISPR-Cas systems, consisting of types I, III, and IV, and 16 subtypes with multisubunit crRNA–effector complexes for interference (Table 1.1). Class II CRISPR-Cas systems, consisting of types II, V, and VI, and 17 subtypes, only with a single subunit of a crRNA–effector protein. The classification of type I and II systems is based on the identification of all C as genes in each CRISPR-Cas locus and then determining the unique signature genes that would allow the assignment of these loci to types and subtypes.

Table 1.1

1.4.1 Class I CRISPR-Cas system

This system is defined by types I, III, and IV with multisubunit crRNA–effector complex.

1.4.1.1 Type I

Type I contain a unique signature gene Cas3 and is further subdivided into seven subtypes (I-A, I-B, I-C, I-D, I-E, I-F, and I-U) (Koonin et al., 2017). The Cas3 gene encodes a single-stranded DNA helicase protein which stimulates to unwind double-stranded DNA and RNA–DNA duplexes by using ATP molecules. The other HD family endonuclease domain (located at N-terminal domain of Cas3 protein) is fused to the helicase domain of Cas3, performing the cleavage of the target DNA (Sinkunas et al., 2011). The type I system is usually encoded by a single operon that contains the Cas1, Cas2, and Cas3 genes together with the genes for the subunit of cascade or effector molecules, such as Cas5, Cas6, Cas7, and Cas8 (Fig. 1.2). Each Cas gene is presented in type I as a single copy (Haft et al., 2005).

Figure 1.2 Class I CRISPR-Cas system and its types: (A) organization of Cas protein in class I CRISPR-Cas system and (B) organization of operon for each type of class I system. CRISPR array is indicated with square and rectangle and represents repeats and spacer sequences, respectively.

1.4.1.2 Type III

Like type I, type III also possesses the signature gene Cas10, which encodes a Palm domain which is a multidomain that is similar to the core domain of polymerases and cyclases (Anantharaman et al., 2010). Type III system, Cas10 protein forms a large subunit complex with effector proteins. Recently the Cas 10 crystal structure has been solved and has been identified four distinct domains (Cocozaki et al., 2012): the N-terminal cyclase-like domain which is similar to RRM fold as the palm domain, a helical domain containing the Zn-binding site, and alpha-helical domain at C-terminal which similar to the thumb domain of DNA polymerase (A-Family) and Cmr5, a small alpha-helical protein. Type III each locus encodes the small subunit of effector complex protein from Cas5 gene and other several paralogous proteins from Cas7 gene. The Cas10 is also fused to an HD family nuclease domain that is different from the HD domains of type I CRISPR-Cas systems and contains a circular permutation of the conserved motifs of the domain (Makarova et al., 2006). Recently, Type III systems have been categorized into two subtypes, III-A and III-B which are similar but could be different by the presence of distinct genes encoding small subunits of effector complex, such as csm2 in III-A and cmr5 in III-B, respectively. The subtype III-A loci contain Cas1, Cas2, and Cas6 genes (Makarova et al., 2013) that have been responsible for DNA targets (Marraffini & Sontheimer, 2008), whereas subtype III-B systems lack Cas genes and hence depend on other CRISPR-Cas systems present in the same genome. The type III-B systems have been shown to target RNA (Hale et al., 2009, 2012). The complexity and organization of type III systems are much more diverse than type I systems, this diversity is due to gene duplications and deletions, domain insertions and fusions, and the presence of additional uncharacterized domains. Both type III variants are typically present in a genome along with other CRISPR-Cas systems.

1.4.1.3 Type IV

Type IV system is functionally uncharacterized and found in several bacterial genomes and often on a plasmid, as can be characterized by the AFE_1037-AFE_1040 operon in Acidithiobacillus ferrooxidans ATCC 23270. This system is similar to subtype III-B loci which lack Cas1 and Cas2 genes and is not closest to a CRISPR array and in many other cases, this CRISPR array is not detectable in a genome. Type IV systems contain a partially degraded large subunit, Csf1, Cas5, and Cas7 which form multisubunit crRNA–effector complex (Makarova et al., 2011). The csf1 is the signature gene for this system. Type IV CRISPR-Cas systems have two distinct variants, one is a DinG family helicase, whereas the second which lacks DinG but typically contains a gene which for a small α-helical protein, presumed a small subunit 33 (White, 2009). Type IV systems could be mobile components that, similar to subtype III systems, use crRNAs from different CRISPR arrays once these become available (Vestergaard et al., 2014).

1.4.2 Class II CRISPR-Cas system

This system contains type II, type V, and type VI CRISPR-Cas systems and 17 subtypes. The distinctive characteristic of these types of systems is that their effector complexes involved a single, large, multidomain protein, such as Cas9 which is bonded with crRNA.

1.4.2.1 Type II system

Type II CRISPR-Cas systems are different in terms of the number of the gene from the class I system, types I and III. The Cas9 gene is a signature gene for type II (Fig. 1.3). The Cas9 gene encodes a multidomain protein that performed the functions of the crRNA–effector complex with target DNA cleavage (Louwen et al., 2014). The Cas9 protein contains two nuclease domains, one is RuvC-like nuclease and the second is the HNH nuclease domain that is responsible for target DNA cleavage (Jinek et al., 2012). In addition to the Cas9 gene, there are two additional genes presented in the CRISPR-Cas locus, such as Cas1 and Cas2 genes. Type II CRISPR-Cas loci contain also other genes that encode tracrRNA, an RNA that is partially complementary to the CRISPR array (Chylinski et al., 2013). Type II CRISPR-Cas systems are classified into three subtypes, II-A, II-B, and II-C (Fonfara et al., 2014).

Figure 1.3 Class II CRISPR-Cas system and its types: (A) organization of genetic, structural, and functional parts of Cas protein in class II CRISPR-Cas system and (B) organization of operon for each type of class II system. CRISPR array is indicated with square and rectangle, representing repeats and spacer sequences, respectively.

Type II-A systems contain one additional gene, known as csn2, which is a signature gene for type II-A. The Cns2 gene has two distinct variants like long and short variants and plays a role in spacer acquisition (Chylinski et al., 2014). The Csn2 proteins form a ring-like homotetramer structure containing P-loop ATPase fold, but the ATP-binding site is not activated (Lee et al., 2012). This protein directly binds to linear double-stranded DNA through ring structure.

Type II-B does not contain csn2, but it contains the Cas4 gene, which is similar to type I systems. The Cas4 proteins have 5′ single-stranded DNA exonuclease activity (Zhang et al., 2012).

Type II-C locus possesses three protein-coding genes, such as Cas1, Cas2, and Cas9, and is the most common type II CRISPR-Cas system in bacteria (Koonin & Makarova, 2013).

1.4.2.2 Type V system

This system is discovered by the presence of the cpf1 (Cas12) gene adjacent to Cas1, Cas2, and CRISPR arrays in bacterial and archaeal genomes (Schunder et al., 2013). Cpf1 is a large protein that contains a RuvC-like nuclease domain which is similar to Cas9 protein and the TnpB protein which is a class of IS605 family transposons, However, Cpf1 lacks the HNH nuclease domain (Shmakov et al., 2015). The nuclease activity of the RuvC-like domains of Cpf1 is responsible for cleaving the target DNA (Gasiunas et al., 2012). A type V system contains 10 subtypes and contains a single effector protein Cas12 (cpf1).

1.4.2.3 Type VI system

The type VI systems contain Cas13 effector protein that possesses two HEPN RNase domains. The HEPN superfamily consists of diverged RNases that play important roles in various defense-related functions in both prokaryotes and eukaryotes (Anantharaman et al., 2013). The HEPN RNase function as a toxin domain of numerous bacterial and archaeal toxin-antitoxin modules. The discovery of the Cas13 effectors containing HEPN domains prompted the prediction of type VI RNA targeting CRISPR-Cas systems (Smargon et al., 2017).

1.5 Limitations of CRISPR technology

Even with its wide use in target-specific genome editing, CRISPR technology carries a few limitations which prevent its application and commercialization. Such limitations are pictorially presented in Fig. 1.4

Figure 1.4 Major limitations of current CRISPR-Cas9 system.

1.5.1 CRISPR-Cas9 off-target effects

One of the major limitations was reported from the group that developed CRISPR technology and that is the off-target effect (Cho et al., 2014). CRISPR's off-target effect is manifested when the Cas9 nuclease cleaves other sites than its programmed site, this effect is mainly observed in the case of class 2 type II S. pyogenes (Koonin et al., 2017). The Cas9 nuclease cleaves after the third nucleotide corresponding to PAM sequence. Detailed analysis of CRISPR cleavage mechanism confirmed that the sgRNA mismatch can be tolerated up to five base pairs since the complex is less stringent after the fifth base from the PAM sequence. This stringency results in multiple off-target binding; it was also observed that mismatch also depends on the amount of Cas9 used (Kuscu et al., 2014). Various efforts have been made by the researcher to reduce the off-target effect. Investigation suggests a strait forward method, that is, optimization of gRNA and Cas9 amount and proportion. In addition to the amount of Cas9 delivery, direct delivery of gRNA and Cas9 into the cell also decreases to off-target effects (Fu et al., 2013). A study of Cas9 protein suggests the presence of a different variant of Cas9 among the different organisms. This variation of Cas9 protein has expanded the scope of CRISPR technology because different Cas9 protein has different catalytic activity and variation in PAM motif which suggests easy tunability of Cas9. Such an example is Cas12a, also known as Cpf1 use to generate knock-out mice without any off-target effect (Ao et al., 2018; Makarova et al., 2018; Wright et al., 2016). Other than Cas9 protein, sgRNA can also be optimized to reduce the off-target effect. The seed region is the five base pair sequence present at a proximity of a PAM motif, which is responsible for guiding the Cas9 complex to its target (Cencic et al., 2014). While the distal sequence is needed for Cas9 activity. Current understanding of sgRNA and its composition on its binding specificity and off-target effect drawn various conclusions which are; the uracil (U) rich seed sequence has shown to increase Cas9 specificity; however, the sequence with high GC content decreases Cas9 activity (Wang et al., 2014) and G is more favorable than C immediately after the PAM motif. At the 5th position after the PAM motif, C is more favoured, 9th–10th position after PAM A is more preferred while at the 18th position after PAM C is unfavorable. Consideration of these criteria will reduce off-target effects (Gagnon et al., 2014; Kuscu et al., 2014; Wang et al., 2014). Alteration in sgRNA sequence has a significant effect on target specificity. Shortening of 3ntd at 5′ end of sgRNA complementary to the target site or addition of two G-ntd to 5′ end increases specificity and decreases off-target effect by 5000-fold. Various reports suggest the use of the D10 mutation variant of Cas9 instead of wild type to increase target specificity (Tong, Whitford et al., 2019). D10 mutant version paired with two sgRNA that each cleaves only one stared, this reduces 50- to 1500-fold off-target activity in cell lines and mouse zygotes. Furthermore, the fusion of catalytically inactive Cas9 with FokI (fCas9) nuclease domain has >140 higher specificity than WT Cas9 and >fourfold than paired nickases (Guilinger et al., 2014; Wyvekens et al., 2015).

1.5.2 DNA damage and apoptosis

As it is discussed previously, Cas9 protein creates DSB upon the CRISPR activity and it is well documented that in many cases this DSB induces apoptosis rather than gene editing in the host. Experimental evidence in human pluripotent stem cells suggests activation of P53 in response to DSB created by CRISPR edit (Ihry et al., 2018). Furthermore, deletion of a huge fragment of DNA through CRISPR edits, such as spanning kilobytes and rearrangements of the gene, is the accidental consequence of on-target gene editing that has been reported in many cases; this can be the main concern for clinical applications of this technology (Kosicki et al., 2018; Moses et al., 2019). In such a case use of the nuclease-inactivated Cas9 variant is helpful as it can perform gene manipulation without creating DSB in the gene (Moses et al., 2019).

1.5.3 Immunotoxicity

Despite the DNA damage immunotoxicity of CRISPR and its components limits its implication on human trials. An article by Charleswarth et al. suggests that half of the human population under the study possesses preexisting antibodies for Cas9, that is, anti-Cas9 antibodies (Charlesworth et al., 2019). In addition, it was shown that the adeno-associated virus (AAV) vector used for the delivery of the CRISPR and its components in the host has a binding affinity for the MSC-I and II which suggests strong immune resonance. The ortholog of Cas-9 such as Spcas9 and cjCas9 has extensive editing activity and no immune response in mice. However, in human SpCas9 and SaCas9 shows an immune response which leaves CjCas9 as the only possible option for human clinical trials. Though more experimental evidence is needed before reaching to any conclusion. Future studies may identify other orthologs of Cas9 having high catalytic activity and low or no immune response compared to currently available options and may serve as useful for human clinical trials for gene therapy (Moreno et al., 2019).

1.5.4 Delivery of CRISPR system to hosts

The delivery vehicle for the CRISPR and its components greatly influences the efficiency and immunotoxicity in a host organism. The viral-based vector and delivery system is generally used for the transfer CRISPR system but it also raises the risk for immunotoxicity. Currently, AAV-based vector system is a key tool for the delivery (Xu et al., 2019). Next, the delivery method also influences the efficiency of the CRISPR system. The main delivery method used is electroporation/nucleofection or microinjection, each of which has its own advantages and limitations (Wilbie et al., 2019). Furthermore, the cellular system under study either ex vivo or in vivo also has an impact on CRISPR activity. The ex vivo delivery method ensures the safety of the patient and good control of edited cells (Zhang et al., 2020). However, limitations include handling of the cells outside the native microenvironment, retention of in vivo functions of the cells, and grafting cell in the patient. Furthermore, this method limits its use on certain cell types, such as hematopoietic stem and progenitor cells (HSPCs) and T-cells (Baylis & McLeod, 2017; Yu et al., 2016). For the in vivo applications, the CRISPR components can be delivered through intravenous injections or can be locally injected to specific tissue types. Limitations of this method are degradation of CRISPR components by circulatory proteases and nucleases, opsonization by opsonins, and clearance by the mononuclear phagocytic system. In addition to that, delivery vehicle having >1 nm diameter limits its bypass through vascular endothelium. Further advancements are needed to tackle the limitation of the current delivery techniques (Tong, Moyo, et al., 2019).

1.6 Applications of CRISPR technology

As mentioned earlier CRISPR-based system is largely being used and optimized for gRNA-directed mutation generation at a specific location in a given genome. The disruption in a gene will help to evaluate the function of a specific gene in an organism. However, in a new approach dead Cas9 protein is used instead of WT Cas9, since dCas9 has no nuclease activity it does not cleave DNA. This system maintains specificity to bind with specific DNA thus fusion of different additional proteins with dCas9 expands CRISPR applications which include site-specific modification (base flipping), regulation of gene expression, and DNA modification (acetylation, methylation, phosphorylation, etc.) (Ferreira et al., 2019; Xu & Qi, 2019). These various gene-modulating methods can be used for metabolic engineering, animal model preparation, biotherapy, and understanding the function of various genes. These applications are briefly discussed in the following section and major applications are presented in Fig. 1.5. Li et al. used CRISPR-based system to screen genes responsible for thermotolerance in Saccharomyces cerevisiae (Li et al., 2019). Likewise, Lee et al. targeted 99.7% (4,565) genes from Vibrio natriegens to identify the minimum required set of genes for the growth (Lee et al., 2019). Furthermore, CRISPR technology has opened up a new path to engineer industrial microorganisms for various applications. Several methods were proposed as a laboratory-based adaptive evolutionary system to induce programmed mutation and, genome shuffling to generate useful microbes, such as thermostable strains of S. cerevisiae. The method called EvolvR induces mutation in the genome up to 7,770,000-fold within a selectable window, this method uses engineered polymerase and nickase with a much higher mutation rate compared to wild type (Jakočiūnas et al., 2018). CasPER is a Cas9-mediated Protein Evolution Reaction; this method uses error-prone PCR and Cas9 to integrate mutation in the genome with efficiency up to 98%–99% (Jakočiūnas et al., 2018). Another method is CREATE, a CRISPR-enabled trackable genome engineering, this method links each guide RNA to homologous repair cassettes combined with both of these edits loci in the gene. These also function as barcodes to track genotype and phenotype relationships (Garst et al., 2017).

Figure 1.5 Broad biological applications of CRISPR-Cas technology.

1.6.1 Role of CRISPR in gene