Crispr cas: A new tool of genome editing

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CRISPR cas : A new genome editing tool

-: Major Guide :-Dr. Rukam S. Tomar

Assistant ProfessorDept. of Biotechnology

JAU, Junagadh

-: Minor Guide :-Dr. M. K. MandaviaProfessor and Head

Dept. of BiochemistryJAU, Junagadh

-: Speaker :-

Abhay A. PalaRegd. No.: J4-01390-2014

M.Sc. (Plant Mol. Biology & Biotechnology)Dept. of Biotechnology

JAU, Junagadh

Content…

1. Introduction 2. History3. CRISPR in bacteria 4. Classification of CRISPR5. General structure of cas9 protein6. Mechanism of CRISPR cas97. Applications 8. Data base of CRISPR 9. Case studies 10. Conclusion11. Future aspects

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Introduction…• Genome editing, or genome editing with engineered nucleases (GEEN)

is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, or "molecular scissors”.

• The nucleases create specific double-strand breaks (DSBs) at desired locations in the genome and harness the cell’s endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and non-homologous end-joining (NHEJ).

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Why genome editing? To understand the function of a gene or a protein, one interferes with

it in a sequence-specific way and monitors its effects on the organism.

In some organisms, it is difficult or impossible to perform site-specific mutagenesis, and therefore more indirect methods must be used, such as silencing the gene of interest by short RNA interference (siRNA).

But sometime gene disruption by siRNA can be variable or incomplete.

Nucleases such as CRISPR can cut any targeted position in the genome and introduce a modification of the endogenous sequences for genes that are impossible to specifically target using conventional RNAi.

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Comparison between traditional and modern genome editing technologies

Mutagen Chemical(e.g., EMS) Physical (e.g., gamma, X-ray or fast neutron radiation)

Biological (ZFNs, TALENs or CRISPR/ Cas)

Biological- Transgenics (e.g., Agro or gene gun)

Characteristics of genetic variation

Substitution and Deletion Deletion and chromosomal mutation

Substitution and Deletion and insertion

Insertions

Loss of function Loss of function Loss of function and gain of function

Loss of function and gain of function

Advantages Unnecessary of knowing gene function or sequences

Unnecessary of knowing gene function or sequences

Gene specific mutation Insertion of genes of known functions into host plant genome

Easy production of random mutation

Easy production of random mutation

Efficient production of desirable mutation

Efficient creation of plants with desirable traits

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Disadvantages Inefficient screening of desirable traits

Inefficient screening of desirable traits

Necessity of knowing gene function and sequences

Necessity of knowing gene function and sequences

Non specific mutation Non specific mutation

Prerequisite of efficient genetic transformation

Prerequisite of efficient genetic transformation

Other features Non transgenic process and traits

Non transgenic process and traits

Transgenic process but non transgenic traits

Transgenic process and traits

Mutagen Chemical(e.g., EMS) Physical (e.g., gamma, X-ray or fast neutron radiation)

Biological (ZFNs, TALENs or CRISPR/ Cas)

Biological- Transgenics (e.g., Agro or gene gun)

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1987• Researchers find CRISPR sequences in Escherichia coli, but do not

characterize their function.

2000• CRISPR sequence are found to be common in other microbes.

2002• Coined CRISPR name, defined signature Cas genes

2007• First experimental evidence for CRISPR adaptive immunity

2013• First demonstration of Cas9 genome engineering in eukaryotic cell

HISTORY

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CRISPR – Cas systems• These are the part of the Bacterial immune system which detects

and recognize the foreign DNA and cleaves it.1. THE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

loci 2. Cas (CRISPR- associated) proteins can target and cleave invading DNA in a

sequence – specific manner.A CRISPR array is composed of a series of repeats interspaced by

spacer sequences acquired from invading genomes.

Components of CRISPR

1. Protospacer adjacent motif (PAM)

2. CRISPR-RNA (crRNA)

3. trans-activating crRNA (tracrRNA)

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Different CRISPR-Cas system in Bacterial Adaptive Immunity

Class 1- type I (CRISPR-Cas3) and type III (CRISPR-Cas10) uses several Cas proteins and the crRNA

Class 2- type II (CRISPR-Cas9) and type V (CRISPR-Cpf1) employ a large single-component Cas-9 protein in

conjunction with crRNA and tracerRNA.

Zetsche et al., (2015)

functioning of type II CRISPR system

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Different Cas proteins and their functionProtein Distribution Process Function

Cas1 Universal

Spacer acquisition DNAse, not sequence specfic, can bind RNA; present in all Types

Cas2 Universal

Spacer acquisition specific to U-rich regions; present in all Types

Cas3 Type I signature

Target interference DNA helicase, endonuclease

Cas4 Type I, II

Spacer acquisition RecB-like nuclease with exonuclease activity homologous to RecB

Cas5 Type I

crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE

Cas6 Type I, III

crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE

Cas7 Type I

crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE

Cas8 Type I

crRNA expression Large protein with McrA/HNH-nuclease domain and RuvC-like nuclease; part of CASCADE

Cas9 Type II signature

Target interference Large multidomain protein with McrA-HNH nuclease domain and RuvC-like nuclease domain; necessary for interference and target cleavage

Cas10 Type III signature

crRNA expressionand interference

HD nuclease domain, palm domain, Zn ribbon; some homologies with CASCADE elements

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Action of CRISPR in bacteria

The CRISPR immune system works to protect bacteria from repeated viral attack via three basic steps:

(1) Adaptation

(2) Production of cr RNA

(3) Targeting

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Structure of cas9 protein

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Structure of crRNA

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Versatile Nature of CRISPR Technology

Jeffry et al., 2014

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CRISPR/Cas9-based knock-out of phytoene desaturase gene (PDS) in Populus tomentosa

Combining crRNA and tracrRNA into sgRNA was the crucial step for the development of CRISPR technology

22Joung et al., 2012

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What makes CRISPR system the ideal genome engineering technology

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Examples of crops modified with CRISPR technology

CROPS DESCRIPTION REFERNCESCorn Targeted mutagenesis Liang et al. 2014Rice Targeted mutagenesis Belhaj et al. 2013Sorghum Targeted gene modification Jiang et al. 2013bSweet orange Targeted genome editing Jia and Wang 2014Tobacco Targeted mutagenesis Belhaj et al. 2013Wheat Targeted mutagenesis Upadhyay et al. 2013, Yanpeng et

al. 2014PotatoSoybean

Targeted mutagenesisGene editing

Shaohui et al., 2015Yupeng et al., 2015

Harrison et al., 2014

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Genome editing tool

Transformation method

Crops Targeted genes

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General protocol for CRISPR

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RECENT ADVANCES

Discovery of new version of Cas9Engineered Cas9 with altered PAM specificity

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Cpf1 (CRISPR from Prevotella and Francisella 1) at Broad Institute of MIT and Harvard, Cambridge.

CRISPR-Cpf1 is a class 2 CRISPR systemCpf1 is a CRISPR-associated two-component RNA programmable DNA

nucleaseDoes not require tracerRNA and the gene is 1kb smaller Targeted DNA is cleaved as a 5 nt staggered cut distal to a 5’ T-rich PAMCpf1 exhibit robust nuclease activity in human cells

Zetsche et al., (October 22, 2015)

New Version of Cas9:

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Cpf1 makes staggered cut at 5’ distal end from the PAM

Organization of two CRISPR loci found in Francisella novicida .The domain architectures of FnCas9 and FnCpf1 are compared

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DNAi-Targeted DNA degradation

Brian J. et al., 2015

Once an engineered organism completes its task, it is useful to degrade the associated DNA to reduce environmental release and protect intellectual property.

Here is a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA.

This enables engineered regions to be obscured when the cell enters a new environment.

DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR array defines the DNA target. When the genome is targeted, this causes cell death, reducing viable cells by a factor of 10^8

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Application in Agriculture Can be used to create high degree of genetic variability at precise locus in the

genome of the crop plants.

Potential tool for multiplexed reverse and forward genetic study.

Precise transgene integration at specific loci.

Developing biotic and abiotic resistant traits in crop plants.

Potential tool for developing virus resistant crop varieties.

Can be used to eradicate unwanted species like herbicide resistant weeds, insect pest.

Potential tool for improving polyploid crops like potato and wheat.

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Some pitfalls of this technology

Proper selection of gRNA Use dCas9 version of Cas9 protein Make sure that there is no mismatch within

the seed sequences(first 12 nt adjacent to PAM)

Use smaller gRNA of 17 nt instead of 20 nt Sequence the organism first you want to

work with Use NHEJ inhibitor in order to boost up

HDR

Solutions

Off target indelsLimited choice of PAM sequences

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How to avoid off-target effects?

- Optimization of Injection conditions (less cas9/sgRNA)

- Bioinformatics : Find a sgRNA target for less off-targets

“CRISPR Design” (http://crispr.mit.edu)

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sgRNA designing tools

Optimized CRISPR Design (Feng Zhang's Lab at MIT/BROAD, USA)

sgRNA Scorer (George Church's Lab at Harvard, USA)

sgRNA Designer (BROAD Institute)

ChopChop web tool (George Church's Lab at Harvard, USA)

E-CRISP (Michael Boutros' lab at DKFZ, Germany)

CRISPR Finder (Wellcome Trust Sanger Institute, Hinxton, UK)

RepeatMasker (Institute for Systems Biology) to double check and avoid selecting target sites

with repeated sequences

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Case Studies

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Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice

Case study 1

Yang et al. (2014) USA

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MATERIALS AND METHODS1. Construction of plasmids expressing Cas9 and guide RNAs -pUbi-Cas9 binary vactor which is synthesise by GeneScrept and cloned into pENTR4

2. Transient gene expression in rice protoplasts -Isolation and transfection of rice mesophyll protoplasts were carried out.

3. Agrobacterium-mediated rice transformation -Agrobacterium tumefaciens strain EHA105 by electroporation.

4. Detection of large fragment deletion by T7E1

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Structure of Binary Vector

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(A) Schematic of a cluster of five diterpenoid genes within an ~170kb region on rice chromosome number 4.

(B) Agarose gel electrophoresis image of the PCR products amplified from DNA.

(C) Sequences of large DNA segment deletions induced by sgRNAs.

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Callus Line of T0 plant

(B) Callus lines containing the ~245kb deletions identified with PCR approach and confirmed with sequencing of amplicons.

(C) DNA sequence changes in the representative plants generated from three callus lines (#16, 17 and 21) with the Cas9/ sgRNA induce large deletions (dashed lines) in one chromosome and small nucleotide changes (dashed lines for deletions and lower case letters for insertions) in the homologous chromosome.

Targeted genome modifications in soybean withCRISPR/Cas9

Jacobs et al.(2015) USA

Case study-2

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MATERIALS AND METHODS

• Plant material

• Plasmid construction of cas9 and sgRNA

• Hairy root transformation of soybean by A. rhizogenes (strain K599) • GFP imaging via Olympus MVX10 microscope with a GFP filter

• sequencing and analysis

Schematic showing the targeted GFP sequences.

(B)C9 + GFP 5' target events

• Wild-type sequences are in green, deletions are shown as dashes, and SNPs are shown in orange.

(C) C9 + GFP 3' target events

• Wild-type sequences are in green, deletions are shown as dashes, and SNPs are shown in orange.

Modification efficiency for hairy root events

1. 01gDDM1- 78-80% indel frequency

2. 11gDDM1- 87-90% indel frequency

3. 01gDDM1 +11gDDM1 – 21-23% indel frequency

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Conclusion… Genome editing tools provide new strategies for genetic manipulation in plants and are likely

to assist in engineering desired plant traits by modifying endogenous genes.

Genome editing technology will have a major impact in applied crop improvement and commercial product development .

CRISPR will no doubt be revolutionized by virtue of being able to make targeted DNA sequence modifications rather than random changes.

In gene modification, these targetable nucleases have potential applications to become alternatives to standard breeding methods to identify novel traits in economically important plants and more valuable in biotechnology as modifying specific site rather than whole gene.

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