A BRIEF DISCUSSION ON THE MECHANISM OF CRISPR-Cas SYSTEMS AND ITS APPLICATIONS

1. CRISPR-Cas guides the
future of Genetic
Engineering
PRESENTED BY ASHISH DUTTA
MSc MICROBIOLOGY 2nd
Semester

2.  This presentation is on the review paper written by Gavin J. Knott and
Jennifer A. Doudna Published in the journal SCIENCE on 31st August
2018 with DOI: 10.1126/science.aat5011.
 Clustered Regular Interspaced Short Palindromic Repeats(CRISPR) Cas
(CRISPR associated) Systems are a simple two component system that
allows to precisely edit any sequence in the genome of an organism.
 Crispr Cas is the most commonly used gene editing tool because of its
higher accuracy and precision compared to other nucleic acid
manipulating techniques like ZFN, TALENS and Meganucleases.
 Crispr Cas systems are naturally observed in many bacteria and
archaebacteria which provides them a RNA guided adaptive immunity
to foreign genetic elements by directing nucleases to bind and cut
specific nucleic acid sequences.

3.  Major terms associated
1. crRNA (Crispr RNA)- crRNA is the exact RNA sequence that we get from the
transcription of the CRISPR array. Crispr RNA contains the variable targeting
sequence and constitutes the first nucleotides in the single guide
RNA(sgRNA).
2. tracrRNA- Trans activating crispr RNA is made up of a long strech of bases
that are constant & provides the stem loop structure which is bound by the
CRISPR Nucleases .
3. sgRNA- The hybridization of crRNA(variable part) and tracrRNA(constant part)
forms a single chimeric unit called Single guide RNA(sgRNA) which directs the
Crispr nuclease.

4.  4. PAM- Protospacer Adjacent Motif is a short DNA sequence (usually 2-6 bp
long) that follows the DNA region targeted for cleavage by the CRISPR system. It is
located 2-6 nucleotides downstream of the DNA sequence targeted .There are
different PAM sequences they are nuclease specific. Eg- Cas9 targets the PAM
sequence 5’-NGG-3’.
. PAM is not captured along with the snippets of foreign DNA. It is PAM that saves
the bacterial genome from being targeted by its own nuclease .
 Crispr Cas mediated gene editing uses Cas nucleases guided by a single guide
RNA to to cleave the nucleic acid sequences.
 Crispr Cas mediated adaptive immunity consists of 3 steps

5.  Adaptation- In this step microbes capture snippets of foreign genetic elements
by help of Cas1 and Cas2 and incorporate them into their genomic CRISPR array.

6.  Expression- As a result of transcription the CRISPR array and associated cas
proteins are expressed,Cas proteins forms the Cas nuclease and CRISPR array
forms crRNA. crRNA binds to Cas Nuclease to form a surveillance complex and
provides specificity by base pairing with target nucleic acids.

7.  Interference- In this step the Cas effector nucleases target foreign genetic
elements complementry to their crRNA and makes the cut leading to
neutralization of the target and provides immunity to the host.

8.  Cas Nucleases- The diverse RNA programmable CRISPR Cas enzymes
 Cas nucleases are DNA & RNA targeting endonucleases,They possess several
properties for precise & efficient gene editing.
 Cas9 is the first and most widely used Cas nuclease for genetic engineering, it
was originally extracted from Streptococcus pyogenes (SpCas9).
 Specialty of cas nuclease is that it attaches with only the intended guideRNA
through specific recognition of crRNA and its interaction with tracrRNA.
 sgRNA binds to the target DNA adjacent to a PAM where correct nucleotide
sequence acts as a switch trigerring Cas nuclease to introduce the cut.

9.  Today Scientists have developed many programmable RNA guided homologs
and engineered variants of SpCas9, Now SpCas9 shares the spotlight with a
diversity of Cas9 homologs, DNA targeting Cas12 and RNA targeting Cas13.
 Types of Class 2 CRISPR Cas Systems
 Class 2 constitutes CRISPR Cas systems that uses a single large RNA guided Cas
nuclease for interference or cleavage. They are didived into 3 types.
 Class 2 type II- Targets dsDNA using effector nuclease Cas9. Cas9 binds to a DNA
sequence complementry to the sgRNA spacer adjacent to PAM. Cas9 recognizes
the correct base pairing and activates RuvC & HNH nucleases to cleave both
target and non target DNA strand.

10.  Class 2 type V- Cas12a is used to target ss & ds DNA. Cas12a binds to DNA
sequence complementry to the crRNA spacer adjacent to PAM. Cas12a
senses the correct base pairing to activate its RuvC nuclease for ssDNAase
activity cleaving the target and the non target DNA strands .

11.  Class 2 type VI- Cas13a targets ssRNA guided by a single crRNA. Cas13a
binds to a ssRNA sequence complementry to the crRNA spacer adjacent to
PFS(Protospacer Flanking Sequence). Cas13a senses the correct target
sequence & activate the HEPN nuclease for general ssRNAase activity.
 Type V and Type VI system exibits general multiple turnover nuclease activity
through correct base pairing to the guide RNA.

12. Applications of Cas mediated Genome editing
 Because of the availability of programmable RNA guided nucleases present in a
naturally evolved system the applications of CRISPR Cas extends beyond
precision gene editing.
 Genome wide Screening to identify genes involved in a particular phenotype.
 Identification and validation of potential drug targets includes targeting high
value targets, high value biomarkers etc.
 Agricultural applications of CRISPR Cas have produced modified crops.
 To treat genetic diseases personalized to a patient’s disease etiology. Eg- Gene
editing to correct mutations or induce defective exons in Duchenne Muscular
Dystrophy(DMD) in mouse models.

13.  To treat neurological disorders by inactivation of defective genes associated
with Huntington’s disease & Amyotrophic lateral sclerosis. Scientists have also
succeeded to eliminate an entire chromosome in aneuploid human pluripotent
stem cells.
 To inactivate retroviruses in pig models.
 In advanced immunotherapies against cancer by using CRISPR to engineer T
cells, Therapies using Chimeric antigen receptor (CAR)T cells & checkpoint
inhibitors (including antibodies that antagonize programmed cell death
protein1(PD1) have resulted in impaired tumor clearance in Subcutaneous
xenograft model.
 To cure Sickle cell Anaemia by using Cas9.

14.  To tackle the challenges associated with NHEJ and HDR DNA repair system
scientists have developed Cas nucleases fused to single base editors.
 Eg- nickaseCas9(nCas9) carriers a single base editor to target locus
facilitating base conversion without dsDNA break.
 A recently developed deaminase enables nCas9 editing to catalyze A-T to
G-C transitions,now its possible to create any of the 4 possible transition
mutation at a specific locus.
 Transcriptional regulation with dCas9
 By decoupling DNA binding from the enzymatic activity by mutation in
nuclease domain we get catalytically deficient Cas9. This has revolutionized
functional genetic screening by enabling specific, rapid multiplxed gene
knockout even in immune cells and neurons.

15.  This advancement in dCas9 allows for genomic perturbation without DNA
damage, Eg- dCas9 fused to TET1(demythylase) targeted to the deregulated
FMR1 locus reserved the phenotype of fragile X syndrome in mouse models
and neurons.
 Gain of function in defective or non-functional genes, Eg- dCas9 targets the
gene activation system to treat Type1 diabetes, acute kidney injuries &
murine muscular Dystrophy.
 Post transcriptional engineering with RNA targeted Cas
 Cas nucleases are used to transiently perturb the transcriptome through
direct RNA targeting by using programmable RNA guided nucleases & a PAM
presenting oligonucleotide(PAMmer)

16.  Cas9 RNA targeting is used to (i) Eliminate pathogenic RNA foci.
(ii) To Rescue mRNA splicing defects (iii) In protein production to attenuate
polyQcontaining protein production from RNA’s with trinucleotide CAG repeats.
 Cas13a RNA targeting is used for RNA interference and editing mammalian
cells and also for modulating in vivo splicing.
 Programmable Nucleic acid Imaging
 To image repetitive genomic loci in live cell using dCas fused to fluorescent
reporters.
 High resolution live cell imaging for single nucleotide polymorphism.
 RCas9 to track RNA in live cells.

17.  Nucleic acid detection and diagnostics
 Cas13 as a tool for detecting RNA transcript of interest in a pool of RNA by
detecting its RNase activity.
 SHERLOCK(Specific High Sensitivity Enzymatic Reporter Unlocking) was
developed as a platform for incorporating pre amplification of the input
material to create a tractable paper based assay with improved sensitivity.
 SHERLOCKv2 is used to detect dengue & zika virus ssRNA.
 DETECTR(DNA endonuclease-targeted CRISPR trans reporter) is developed
and coupled with isothermal pre amplification can accurately detect
Human papillomavirus.
 Detection of transcript using CRISPR Cas is rapid & readily adaptable in the
clinic and is less expensive.

18.  Specificity & delivery of CRISPR Cas
 Unintended binding modifications & cleavage of nucleic acids pose a
challenge to all technologies for genetic manipulation.
 Considerable advances have been done to improve specificity, to achieve
correct gene regulation and to reduce off target binding.
 Vehicles to deliver the Cas payload remains a major obstacle in the
presence of immune responses to sgRNA & Cas9 in humans.
 In lab we have a lot of options for delivering the Cas payload but
unfortunately many of these options can’t be used in clinical settings.
 One strategy to solve this problem is Direct injecting of nanoparticles
containing Cas nuclease and sgRNA.

19.  Cas9-sgRNA nananoparticles injected directly corrects the causative DMD in
mice models.
 The success of CRISPR Cas based therapeutics will depend on future
development of suitable vehicles for delivering the Cas payload.
 Conclusion
 CRISPR Cas based technologies provide an accessible & adaptable means to
alter, regulate and to visualize genomes.
 CRISPR Cas tools have vastly accelerated the pace of research from
understanding the genetics of previously unstudied organisms to discovering
genes that directly contribute to the disease.

20.  Many Cas9 based clinical trials are in progress which results will guide
future use of CRISPR in somatic cell editing both ex vivo and in patients.
 Agricultural applications of CRISPR Cas are already creating products for
various markets.
 This ever expanding reportire of application firmly places the CRISPR-Cas
Toolkit at the cutting edge of genome editing and more broadly Genetic
Engineering