CRISPR-Cas Technology: S. thermophilus Bacterial Adaptive Immunity

This project delves into the applications of CRISPR-Cas technology, specifically focusing on its role in S. thermophilus bacterial adaptive immunity. The study provides a comprehensive overview of CRISPR-Cas systems, including their mechanisms and applications across various fields. It examines the architecture of S. thermophilus CRISPR and discusses the potential for gene editing and modification using this technology. The project explores the use of S. thermophilus in the CRISPR-Cas system, comparing its similarities to S. pyogenes. Factors affecting gene editing and novel methods for product amplification, such as the CRISPR array assembling method, are also discussed. Furthermore, the evolutionary relationship of S. thermophilus CRISPR loci and their diversity are analyzed, including different types of CRISPR systems. The project also includes an experimental plan designed to explore new applications of CRISPR-Cas technology in S. thermophilus.

Applications of CRISPR-Cas Technology in
S. thermophilus Bacterial Adaptive
Immunity

Application of CRISPR-Cas Technology
Lay out Summary
CRISPR stands for clustered regularly interspaced short palindromic repeats while Cas stands for
CRISPR-associated protein. CRISPR is a group of DNA chain that can be established within the
genes of prokaryotic creatures such as archaea and bacteria. The sequences involved are imitated
form DNA portions form viruses due to which prokaryotes are infected before and are used to
expose and shatter DNA from alike viruses all along consequent infections.
Analyzation of genes with the help of CRISPR CAS
CRISPR CAS is used to analyze genes with creatures.
Cas(termed as CRISPR-associated protein) is a type of enzyme CRISPR progressions as a
mentor cleave and recognize definite strings of DNA equivalent to CRISPR progressions. Cas
combines with CRISPR sequences in order to form the origin of a mechanism that is called
CRISPR-Cas9.
Together CRISPR-Cas constitutes a system termed as a prokaryotic immune system which
deliberates opposition to external genetic elements including those existing within phages and
plasmids which supports a system of acquired immunity.
Application in various fields
CRISPR is a very huge and widely used technology that has its application in various fields.
More than 1000 research paper had been published mentioning CRISPR. It is used to activate
static or unused genes of the human body, used to analyze ‘candida albicans’, to transform yeast,
etc.
Theme
The theme of the above research is that it focuses on putting the light on those areas where there
is a huge application of CRISPR CAS system, whether it is used in transforming yeasts or
altering genes.
2 | P a g e

Aims and objectives
● To analyze various types of gene editing
● To study about an architecture of S. thermophilus CRISPR 3 and S. pyogenes type II CRISPR-
Cas loci
● To examine a novel Streptococcus thermophilus locus which is termed as CRISPR 3
● To provide a new method in relation to CRISPR CAS technology
Timescale
Main Activities/ Stages
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6
Topic selection and its scope 
Secondary data sources
identification

Research proposal preparation 
Preparation of literature review 
Description of research methodology 
Analysing data 
Findings comparison 
Conclusion and recommendations  
Finalising and submission  
S. Thermophilus under CRISPR CAS
CRISPR-Cas locus provides a particular study about the immunity against external components
which are more variable amidst various prokaryotes. The main purpose of the above research is
to focus on terminating the editing of genome protocols found in Streptococcus thermophilus. At
first, those protocols and conditions are introduced that play a role in genome editing. Moreover,
categorization and variety related investigation S. thermophilus CRISPR-Cas help in
3 | P a g e

understanding the connection among Streptococcus. Also, its ability to differentiate external
segments and spacer source investigation also signifies a new way of phase opposition.
The system of CRISPR-Cas was brought into being in thermophilic archaea for the very first
time and was utilized to combat external infections related to DNA (Garneau et al., 2010). It
constitutes Cas and CRISPR protein, the anterior is a part of a common group of short-layoff
palindromes which consist of various duplicative progressions and the following one is a type of
protein which is correlated with helicase activity, nuclease, and CRISPRs genes (Chylinski et al.,
2014). The structure of the system of CRISPR-Cas is more alike RNA interference. There are a
total of three stages included in its immune system namely interference, adaption and expression
(Choi and Lee, 2016). Moreover, the new system of classification that mainly includes 3 types
was suggested by Makarova et al. (2011).
Background
CRISPR CAS is a widely used/applied technology which helps in many ways to cure some
serious diseases including cancer, or altering genes. The above research focuses on providing a
context to the use of S. Thermophilus with the help of CRISPR CAS system. CRISPR CAS
changed the history of revolution after its foundation and now it plays a major role in genome
editing.
Themes
Use of S. Thermophilus
A very important and most beneficial lactic acid bacteria termed as Streptococcus thermophilus
is broadly adopted in the generation of dairy products. Additionally, its use in the field of
CRISPR Cas system appears to be one more popular area of research. After the continuing
process of evolution and immunity, S. thermophilus CRISPR Cas loci provides abundant
varieties. In accordance with dissimilar genetic location, duplicative progressions, and Cas
genes, they are divided into 4 major types- CRISPR 1, CRISPR 2, CRISPR 3, and CRISPR 4.
All of these are further enclosed in 3 types of CRISPR Cas system. The sequences of the
CRISPR Cas system differ from part to part due to the reason that there is a difference in the
amount of space.
4 | P a g e

Application of S. thermophilus CRISPR Cas system in the field of gene editing
When the CRISPR system was compared with intensely small recombineering adeptness which
is assumed to be 10-4 to 10-6 (Sharan et al., 2009) and tedious expansion of classical methods of
homologous recombination, it could undoubtedly clarify the mutant architecture due to the
reason that it has high efficiency of editing (nearly about 50 to 100%) and chances to elite dual-
crossover cases in single step (Chen et al., 2017).
The start of the CRISPR Cas system is from progression definite identification and object DNA
loci cleavage. The termination of the system is with permanent mutations over DNA restoration
system (Selle and Barrangou, 2015). Currently, the system of CRISPR-Cas associated with S.
pyogenes (SpCas9) is broadly enforced as an appliance in gene alteration. Nevertheless, weak
points of the above system are recorded as its additional studies and considerable application
(Hsu et al., 2013). Due to the same reason, the focus of researchers is more on the S.
thermophilus CRISPR Cas system that provides inferior targeting capability and lesser Cas
portion (Xu et al., 2015). In the opion of Müller et al., (2016) the main process of this system
contains the building of working vector that contains the progression of StCas9 system and
interpretation analysis in the host cell.
Basis of similarities between StCas9 and SpCas9
Various researches propose that the cleavage activities shown by StCas9 system are very similar
to that of SpCas9 system and workability of gene editing in plants, human and microorganisms
(Xu et al., 2015; Müller et al., 2016; Steinert et al., 2016). The Cas9 protein is one of the
essential parts of the StCas system. As the director of probing action, the compositions of Cas9
include a recognition(REC) lobe and a nuclease (NUC) (Anders et al., 2014; Nishimasu et al.,
2014). Before, the PAM-definite probing of destination DNA dual strand is mainly due to RuvC,
NUC alike nuclease and PI domains. And the further is accountable for identification of sgRNA
destination DNA complex (Chen et al., 2017). St1Cas9 and St3Cas9 are included in the StCas9
system that further present image of S. thermophilus CRISPR Cas system in CRISPR 1 and
CRISPR 3 loci respectively. St1Cas9 addresses 5′-NNAGAAW while St3Cas9 addresses 5′-
5 | P a g e

NGGNG (Garneau et al., 2010; Magadán et al., 2012). Thus, the destination capability shown by
both the system is lightly dissimilar. The efficiency presented by St3Cas system is higher than
the efficiency presented by St1Cas system (Müller et al., 2016).
Nevertheless, both the systems can be utilized in the modification of genome in accordance with
existing researches (Xu et al., 2015; Müller et al., 2016; Steinert et al., 2016).
Factors affecting gene editing
In addition, there are various important factors that affect editing capability and activities of
StCas9 system. First of all, there is sgRNA that consist of tracrRNA (trans-activating RNA) and
crRNA (CRISPR RNA) that possesses its usual stem-loop architecture which can improve the
synergy among sgRNA and Cas9. But ring and stem have authority over its action. For example,
a stem-circle structure in 3′ end of tracrRNA assumes crucial jobs in action of StCas9. What's
more, its inward lumps are fundamental for the elements of StCas9 while the difference inside
circle arrangements has no impact. (Xu et al., 2015). As noted, crRNA is the structured spacer
following up on the target DNA strand to give explicit focusing on. tracrRNA is in charge of
Cas9 blend and its 3′ locale is fundamental to StCas9 action (Wiedenheft et al., 2012; Xu et al.,
2015). Moreover, the measurements of sgRNA and Cas9 is firmly identified with the askew
effectiveness of StCas9 framework while abbreviated tracrRNA could raise its indicating
capability. Also, PAM and its close-by bases are critical for explicit acknowledgement of target
pieces. Particularly, G in PAM a respectable halfway point is fundamental for the action of
StCas9. Taking everything into account, StCas9 framework cannot exclusively be generally
utilized in the editing of genes yet in addition present increasingly dynamic application
prospects.
New method for amplification of products
Moreover, a new method termed as CRISPR array assembling method which is based on spacer
S. thermophilus has been described. Along these lines, this can be utilized as a leap forward of
helpful multiplex focusing on. First of all, tracrRNA tape, cas9 part, and CRISPR pioneer
arrangement together with CRISPR eliminator ought to be gotten by amplifying of PCR.
6 | P a g e

After that, the products that are going to be amplified are put together in a suitable direction with
specific constraints endonucleases area. With respect to multiplex focusing on, the procedure of
DR (direct rehash)- Spacer direction is embraced rather than sgRNA direction. Legitimate
preliminaries ought to be incorporated for enhancing of intrigued grouping containing required
DR arrays and explicit spacers right off the bat. Especially, when structuring groundworks,
thought of holding relating limitation destinations is important. Through specifically toughening
oligonucleotides, the first DR exhibit can be essentially gathered into essential CRISPR vector.
After that, primers are utilized to assemble and extend consequent spacers and DR progressions
into CRISPR location to construct an artificial structure of Spacer-dr (Gou et al., 2007).
Assortments of explicit vectors going for various target genes can be remade by along these
lines. At long last, the following recombinant plasmid is moved into beneficiary cells to get
target freaks, and phenotype perception and sequencing are completed to measure its altering
productivity.
Saddling chemically dormant Cas9, whose endonucleolytic movement has been dispensed with
yet recognizable proof and restricting capacities are as yet held, together with specific
interpretation aspects forestalling or encouraging the banding of RNA polymerase (RNAP) and
its advertiser, a proficient translation guideline can be accomplished (Bikard et al., 2013).
The evolutionary relationship of S. thermophilus CRISPR Loci and their diversity
In accordance with CRISPR progression instruction of various acknowledged genome exertions
written in CRISPR directory, the category of CRISPR loci in most of the S. thermophilus strains
are of different types (more than one). The main four categories of CRISPR system can be
classified by different allocation or arrangements and DR of Cas genes. In addition, there are
various distributions of CRISPR Cas system in S. thermophilus ND07.
Universally, the dynamic idea of CRISPR loci may demonstrate important for composing and
near investigations of strains and microbial populaces. Likewise, CRISPRs give basic
experiences into the connections among prokaryotes and their surroundings, quite the
coevolution of viral and host genomes.
CRISPR4 chiefly works through antiviral guard intervened by Cascade superfamily. What's
more, its cas2 stop codon agrees with halfway grouping of the recurrent component. There is no
7 | P a g e

novel spacer expansion when remote DNA attacking, however huge up-managed articulation of
quality encoding Cascade complex can be watched, particularly cas7 (Young et al., 2012).
CRISPR 4 experiences a one of a kind interpretation process and assumes unmistakable jobs
amid phage opposition. Novel spacers are adept to assume increasingly vital jobs on opposing
phages. Of note, spacer erasure has been identified in bacteriophage-insensitive S. thermophilus
freaks (BIMs) amid phage opposition process. What's more, the erasure dependably happens at
the contiguous position with expansion, suggesting the probability that cancellation occurs by
virtue of homologous recombination between DR arrangements (Deveau et al., 2009).
The above figure represents the features of CRISPR loci
Ordinarily, bunched, routinely interspaced short palindromic rehashes (CRISPRs, white boxes)
are gone before by a pioneer arrangement (black box) which at is AT-rich however generally not
rationed. The number of rehashes can differ considerably, from at least two to a couple of
hundred. Rehash length, be that as it may, is confined to 23 to 50 nucleotides. Rehashes are
isolated by correspondingly measured, non-dreary spacers (hued boxes) that share arrangement
personality with parts of plasmids and bacteriophage genomes and determine the objectives of
CRISPR impedance. A lot of CRISPR-related (cas) qualities promptly goes before or pursues the
rehashes. These qualities are monitored, can be ordered into various families and subtypes, and
encode the protein apparatus in charge of CRISPR action.
Methodological approach
There are various methods used in the above research in order to find a relevance including the
main title of the research. A method named “array addressing “ has been used in the above
section which is based upon spacer S. Thermophilus.
8 | P a g e

Moreover, the data used to conduct the research is secondary. The main advantage of using a
secondary data is that you do not have to perform a particular set of instructions again and again.
The values for them are provided in general. The experimental plan included in the above section
defines a way in which a data should be collected, analysed, used and presented.
A new method
A new method of addressing CRISPR termed as array assembling has been discussed in the
previous section. The method is based upon spacer S. thermophilus.
Thusly, this can be used as a jump forward of accommodating multiplex concentrating on.
Above all else, tracrRNA tape, cas9 part, and CRISPR pioneer game plan together with CRISPR
eliminator should be gotten by enhancing of PCR.
Purpose of the study
The main purpose and focus of this research are spread on various technologies of gene editing,
growth-related connection, and anti-phage actions of S. thermophilus.
The study provides a new method that can be further useful in the field of CRISPR CAS
technology.
Explanation of the method used
From that point forward, the items that will be enhanced are assembled an appropriate way with
explicit imperatives endonucleases region. Concerning multiplex concentrating on, the system of
DR (direct repeat)- Spacer course is grasped instead of sgRNA bearing. Real fundamentals
should be fused for improving captivated gathering containing required DR exhibits and express
spacers directly off the bat. Particularly, while organizing foundations, thought of holding
relating impediment goals is essential. Through explicitly toughening oligonucleotides, the first
DR show can be basically assembled into basic CRISPR vector. From that point forward,
preliminaries are used to amass and broaden subsequent spacers and DR movements into
9 | P a g e

CRISPR area to develop a counterfeit structure of Spacer-dr. Combinations of express vectors
going for different target qualities can be changed by thusly. Finally, the accompanying
recombinant plasmid is moved into recipient cells to get target monstrosities, and phenotype
recognition and sequencing are finished to gauge its adjusting profitability.
Saddling artificially lethargic Cas9, whose endonucleolytic development has been shed yet
conspicuous evidence and confining limits are up 'til now held, together with explicit
understanding angles thwarting or empowering the banding of RNA polymerase (RNAP) and its
promoter, a capable interpretation rule can be cultivated.
Types of CRISPR Cas elements
There are a total of four types of CRISPR Cas elements. CRISPR 1 and CRISPR 3 are associated
with Csn type-2 A. both the CRISPR loci have an important aspect in interference and
adjustment stages. The division and mechanism of Cas progressions in CRISPR 3 and CRISPR 1
loci are comparably moderate having the extent of Cas genes in efforts of their duplicative spacer
areas. Nevertheless, the progression comparison is small and similar. It presents 33.6% identity
in Cas 1 genes and 41.3% identity in Cas 2 genes. In addition, to which, all of them exhibit the
strong capability of the combination of unique spacers. There are various experiments that
conclude the same thing.
With the help of vitro experiments, S. thermophilus can normally acquire spacers from plasmic
DNA bacteriophages into its CRISPR 3 and CRISPR 1 (Garneau et al., 2010). This appearance
can perform a better process of spacer combination as well as it contributes to a deep recognition
of external DNA. moreover, CRISPR 1 is extensively dispersed in S. thermophilus.
When it comes to CRISPR 2, it is included in the system of Csm type-3 A. it is very dissimilar in
both of the duplicating progressions and dispersion of Cas genes, having an identity of 27.78% in
the sequences of DR and Cas genes situated together with the duplicating spacer allocation. Plus,
with respect to CRISPR 2 locus, high extent of Cas however low level of rehash spacer locale
result in its bad capacity of coordinating latest spacers. Dispersion of CRISPR loci demonstrates
that CRISPR2 is most likely a bit of deteriorated part started from the gram-positive precursor
(Horvath et al., 2008).
10 | P a g e

In the case of CRISPR 4, it belongs to the system of Cse type 1-E and is present in few strains
only. CRISPR 4 experiences an exclusive inscription process and performs different roles
meanwhile phage opposition. The percentage of repeating progression is 28% only diversified
Cas modules are dispersed crucially over the duplicative spacer area. There is no novel spacer
expansion when outside DNA attacking, however huge up-controlled articulation of quality
encoding Cascade complex can be watched, particularly cas7. Hence, CRISPR 4 primarily
performs over antiviral armament interposed by the Cascade family.
Conclusion of dispersion
It is concluded that the dispersion of Cas 1 and Cas 2 has concentrated authorities over
procurement of attacking progression. They are capable of forming a DNA complex termed as
Cas 1- Cas 2 protospacer when nominating a specific external DNA. Also, both the clusters
possess amid all the system of CRISPR Cas and introduce preservation that can be used in the
conditional genomic analysis. It has been determined that the new complex formed (Cas 1- Cas
2) behaves as an integrating agent novel spacer in CRISPR locus. Moreover, the latest spacers
are likely to aggregate into the end which is leading the duplicative spacer area (5’ area) although
latent spacers are suitable to be removed from the tail end of the duplicative spacer area (3’ end).
The occurrence of integration and deletion process can be simultaneous. Hence, the CRISPR
leading end displays high uncertainty while the CRISPR tail end shows high equality amid
various strains. Research determined that regularity among CRISPR and Cas protein is in or by
comparison extreme in similar type. This consideration signifies coevolution and interrelation of
mutual support among them. Also, there are critical similarities of various Cas protein amidst
different species, for instance, Cas 7 and Cas 9 all of which play an important part in protecting
external DNA. The research states that CRISPR 1 and CRISPR 4 are present in a few species
only while CRISPR is commonly found in most of the Streptococcus species.
Impact of the above system
11 | P a g e

Current researches regarding acquired immunization specified by CRISPR Cas system have
provided the fact that applying an efficient accession over S. thermophilus can protect it from
phages attack. The growth of S. thermophilus and reproduction is adversely affected by the S.
thermophilus phages and lead to a vast commercial breakdown in dairy production eventually. S.
thermophilus phages are divided into 2 parts namely pac and cos type in accordance with their
DNA cluster structure. The cos type part of S. thermophilus phages contains Sfi21, Sfi19, 7201,
and DT1 whereas the pac type phages contain Sfi11, 2972, and O1205. All of them are general
bacteriophages with the entire genome arranged and show huge similarities, which could help in
the improvement of the anti-phage capability of CRISPR Cas system.
The CRISPR Cas system frames a wall in bacteria that cannot be destroyed or broken, operating
through interrupting and cutting off external genes. When it is exposed to phage, a large group of
strains combines to form a progression from attacking phage as spacers. Moreover, the latest
spacer extension is discovered in CRISPR 1 and CRISPR 3 only. Although, there is no novel DR
progression acquired by CRISPR 2 and CRISPR 4. CRISPR 4 Cas exhibited biochemical actions
in vitro. After reduplication of the CRISPR locus consisting of extended DR progressions and
additional processing, the individual abandoned crRNA with a different architecture integrates
with Cas complex in order to design CRISPR effect nucleoprotein complex (crRNP). The main
function of crRNP is to work as endonuclease directed by crRNA. In the course of time, the
anchor part is invulnerable to the re-attacking phage by the way of sequence particular gap of
phage DNA (Gao, 2018).
The similarities among CRISPR spacer progressions and current progressions in GenBank
directory are examined and determined. First of all, most of the spacers are similar to S.
thermophilus bacteriophages genome. To the greatest extent, a single spacer can show
similarities with various phages and numerous spacers could oppose the equivalent phage.
Moreover, after studying the coevolution amid phages and hosts, it can be proposed or concluded
that, after disclosure to phage violation, new spacers are added to S. thermophilus CRISPR loci
as well as mutations or alterations may also take place in phage genome. Also, more recently
combined spacers arise from the classifying string of the managing genome (Luo, Leenay, and
Beisel, 2016).
12 | P a g e