What is a CRISPR Cas system?


The CRISPR-Cas systems found in bacteria and archaea are versatile small RNAs for adaptive defense and regulation

Bacteria and archaea have evolved various defense and regulatory mechanisms allowing them to react to various stressful situations caused by the environment, such as a virus attack. The recently discovered versatile CRISPR-Cas functions as a prokaryotic immune system. This system confers resistance to exogenous genetic elements such as plasmids and phages by providing a form of acquired immunity. The CRISPR Cas system has two novel features that allows the host to specifically incorporate short sequences from invading genetic elements such as a virus or plasmid into a region of its genome that is distinguished by clustered regularly interspaced short palindromic repeats (CRISPRs). Next, these sequences are transcribed and precisely processed into small RNAs to guide a multifunctional protein complex (Cas proteins) to recognize and cleave incoming foreign genetic material. This CRISPR Cas system is thought to be an adaptive immunity system which uses a library of small noncoding RNAs as a powerful weapon against fast-evolving viruses and is also used as a regulatory system by the host cells.

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Commonly used abbreviations for the CRISPR Cas systems

Cas:

CRISPR-associated genes are located in the vicinity of CRISPR array and are necessary for the silencing of invading nucleic acid.

Cas9t:

Cas9–crRNA–tracrRNA ternary complex, which functions as an RNA guided DNA endonuclease and mediates site-specific DNA cleavage.

Clustered regularly interspaced short palindromic repeat (CRISPR):

An array of short conserved repeat sequences interspaced by unique DNA sequences of similar size called spacers. They often originate from phage or plasmid DNA. CRISPR array together with Cas genes form the CRISPR Cas system, which functions as an adaptive immune system in prokaryotes.

CRISPR RNA (crRNA):

small RNA molecule generated by transcription and processing of the CRISPR array. crRNA is composed of a conserved repeat fragment(s) and a variable spacer sequence, which matches the complimentary sequence in the invading nucleic acid.

Homologous repair (HR):

Error-free DNA repair pathway that seals the broken DNA molecule using a homologous sequence (template).

Non-homologous end joining (NHEJ):

A pathway that repairs DNA double strand breaks (DSB) in the absence of a homologous template; usually leads to small insertions or deletions.

Protospacer adjacent motif (PAM):

A short conserved nucleotide stretch located in the vicinity of a protospacer in the target DNA and necessary for DNA cleavage by Cas9t.

Protospacer:

A fragment in the target DNA, which matches a spacer sequence in the CRISPR array.

Single guide RNA (sgRNA):

RNA hairpin obtained by connecting crRNA and tracrRNA into a single molecule.

Transcription activator-like effector nuclease (TALEN):

An artificial nuclease obtained by fusing Xanthomonas transcription activator-like effector (TALE) DNA binding domains to the nonspecific nuclease domain.

Trans-acting CRISPR RNA (tracrRNA):

Trans-encoded small RNA molecule, forms a duplex with a repeat fragment of crRNA.

Triple helix forming oligonucleotide (TFO):

An artificial oligodeoxynucleotide, which binds to the polypurine sequences of the double-stranded DNA forming DNA triple helix.

Zinc finger nuclease (ZFN):

An artificial nuclease created by fusing zinc finger motifs, which serve as DNA recognition modules, to a nonspecific DNA cleavage domain of the FokI restriction endonuclease.

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Since Lin et al. in 1985 showed that DNA added to mouse L cells by the calcium phosphate method can be inserted into the genome of those cells by homologous recombination, many attempts have been made to target genes specifically, and several methods for this task have been developed over the years. Moreover, greater advancements in genome editing technologies have been made in the recent decade. Targeted genome editing, which enables the generation of site-specific changes in the genomic DNA of cellular organisms, has now become an important goal for genome engineers. Furthermore, for stem-cell based gene therapies improved methods that allow the efficient and side-specific editing of human stem cells are needed. Zinc-finger nuclease (ZFNs) and transcription activator-like effector nucleases (TALENs) based methods have already been successfully applied to genomic engineering in human pluripotent stem cells (hPSCs). ZFNs and TALENs fuse a DNA-binding domain to a DNA cleavage domain to create DSBs in specific genomic sequences. Unfortunately, applications of these methods are relatively laborious and time consuming. Since its discovery in 1987, the CRISPR Cas system has been intensively studied for its use to successfully edit the genome in mammalian cells. The clustered, regularly interspaced short, palindromic repeat (CRISPR) is a component of an immunity system found in prokaryotes containing an array of short, conserved repeat sequences interspaced by unique DNA sequences of similar size called spacers. This system often originates from phage or plasmid DNA. The CRISPR array together with cas genes form the CRISPR Cas system. The cas (CRISPR-associated) genes are located in the vicinity of the CRISPR array and are necessary for the silencing of invading nucleic acid. Cas9t (Cas9–crRNA–tracrRNA) is a ternary complex that functions as an RNA-guided DNA endonuclease and mediates site-specific DNA cleavage. The CRISPR Cas system functions as an adaptive immune system in prokaryotes. The CRISPR-associated endonuclease is directed by small RNAs to cleave foreign sequences of nucleic acids that penetrate a prokaryotic cell.

Several research groups have recently shown that the CRISPR Cas system can be modified to allow for the direct cleavage of a desired target sequence in mammalian cells. The use of this system allows researchers to induce specific genes, following double strand breaks induction and non-homologous end joining. Furthermore, donor sequences could also be introduced by homologous recombination. Since CRISPR Cas only requires the design of a new RNA guide sequence and not of new enzymes, it is much simpler and cheaper to use in comparison to ZFNs and TALENs. The flood of recent publications suggests that the CRISPR Cas method is remarkably efficient. However, as with all new technologies, caution is advised. A few challenges still remain that need to be addressed before this technique can mature. One such challenge is the potential high frequency of off-target mutagenesis that CRIPR Cas may induce in human cells. Furthermore, the 20-base pair target sequence must be followed by a protospacer adjacent motif (PAM). This could be a barrier for mutation corrections at specific genomic locations. As is always the case when new scientific methods are developed, further studies are needed to show or prove that this system will become the genome editing method of choice, used as a routine tool in future stem cell research.

Genome editing tools that are currently available rely on the double-strand break (DSB) repair pathways of the cell. A DSB occurring in DNA triggers a natural process of DNA repair either by ‘error-prone’ non-homologous end joining (NHEJ) or by homologous recombination (HR). Molecular tools that can generate DSBs at specific sites within the genome are ideal gene editing tools. 

Characteristics of the ideal gene editing tool

The ideal tool for this type of gene editing should meet the following criteria:

(1)    High frequency of desired sequence changes in the target cell population;

(2)    No off-target cleavage; and

(3)    Rapid and efficient assembly of nucleases that target any site on the genome at low cost.

The next table shows a few currently available genome editing tools.

Gene Editing Tools

Tool

Modules

Cleavage Module

Target site length, bp

Reprogramming

Targeting Frequency

Specificity

Others

HEase

 

 

Target recognition

domain

Nuclease

domain

 

14–40

 

Complicated: requires

protein engineering

Low

 

Off-targeting

reported

 

High cleavage

efficiency

ZFN

Zinc finger domains

FokI nuclease

domain

 

18–36

Complicated: requires

domain shuffling,

assembly, and protein

engineering,

from 10 weeksa

High

Off-targeting

reported

 

Sequence bias,

some variants

show toxicity

 

TALEN

TALE domains

FokI nuclease

domain

 

24–59

Relatively easy:

requires domain

shuffling, assembly,

and protein engineering,

from 4 weeksb

High, nearly

every sequence

 

Systematically not determined

 

Large protein size

 

TFO

conjugate

 

TFO

(+ restriction

Endo-nuclease)

 

Restriction

endonuclease

 

4–8 + TFO

Relatively easy: requires only DNA oligo but

includes a complicated

chemistry step

Low, restricted

by RE and

TFO sequences

 

Systematically not determined

 

Slow equilibrium

 

Cas9t

RNA

(+ PAM)

Cas9

20 + PAM

(2–5)

Easy and fast: requires only sgRNA

High, depends

on PAM

Off-targeting

reported

Multiplexing

possibilities

 

a  According to manufacturer’s information (http://www.sigmaaldrich.com/life-science/zinc-finger-nuclease-technology/custom-zfn.html).

b  According to manufacturer’s information (http://www.cellectis-bioresearch.com/products/talen-basic). (Source: Gasinuas et al. 2013

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Remaining open questions for CRISPR Cas that need to be addressed as of December 2013 are:

For the CRISPR Cas system, the following questions still need to be addressed:

1.         Can Cas9 be targeted to any desired DNA sequence in the genome?

2.         What is the role of the chromatin state on Cas9 cleavage?

3.         How is the PAM recognized by the Cas9 complex?

4.         How can Cas9 specificity be improved and off-target cleavage minimized?

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Selected References

Bassett, A.R., Liu, J.-L., CRISPR/Cas9 and genome editing in Drosophila, Journal of Genetics and Genomics (2014), doi: 10.1016/j.jgg.2013.12.004.

Cong, L, Ran, FA, Cox, D, Lin, S, Barretto, L, Habib, N et al. (2013). Multiplex genome engineering using CRISPR/Cas Systems. Science, e-pub ahead of print 3 January 2013. 

Giedrius Gasiunas and Virginijus Siksnys;  RNA-dependent DNA endonuclease Cas9 of the CRISPR system:Holy Grail of genome editing? Trends in Microbiology November 2013, Vol. 21, No. 11, 562-567. 

Gesner EM, Schellenberg MJ, Garside EL, George MM, Macmillan AM; Structure of Thermus Thermophilus Cse3 Bound to an RNA Representing a Product Complex. Nat.Struct.Mol.Biol. (2011) 18 p.688. PDB ID: 3QRR]

Woong Y Hwang, Yanfang Fu, Deepak Reyon, Morgan L Maeder, Shengdar Q Tsai, Jeffry D Sander, Randall T Peterson, J-R Joanna Yeh & J Keith Joung;  Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature Biotechnology 31, 227–229 (2013). doi:10.1038/nbt.2501

Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337:816–821. [PubMed]

Matthijs M Jore, Magnus Lundgren, Esther van Duijn, Jelle B Bultema, Edze R Westra, Sakharam P Waghmare, Blake Wiedenheft, Ümit Pul, Reinhild Wurm, Rolf Wagner, Marieke R Beijer, Arjan Barendregt, Kaihong Zhou, Ambrosius P L Snijders, Mark J Dickman, Jennifer A Doudna, Egbert J Boekema, Albert J R Heck, John van der Oost & Stan J J Brouns; Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nature Structural & Molecular Biology 18, 529–536 (2011). doi:10.1038/nsmb.2019.

Wei Li, Fei Teng, Tianda Li & Qi Zhou; Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nature Biotechnology 31, 684–686 (2013). doi:10.1038/nbt.2652.

Dali Li, Zhongwei Qiu, Yanjiao Shao, Yuting Chen, Yuting Guan, Meizhen Liu, Yongmei Li, Na Gao, Liren Wang, Xiaoling Lu, Yongxiang Zhao & Mingyao Liu;  Heritable gene targeting in the mouse and rat using a CRISPR-Cas s ystem. Nature Biotechnology 31, 681–683 (2013). doi:10.1038/nbt.2661.

F.-L. LIN, K. SPERLE, AND N. STERNBERG; Recombination in mouse L cells between DNA introduced into cells and homologous chromosomal sequences. Proc. Natl. Acad. Sci. USA. Vol. 82, pp. 1391-1395, March 1985.

Mali, P, Yang, L, Esvelt, KM, Aach, J, Guell, M, Di-Carlo, JE et al. (2013). RNA-guided human genome engineering via Cas9. Science, e-pub ahead of print. 3 January 2013.

Daisuke Mashiko, Yoshitaka Fujihara, Yuhkoh Satouh, Haruhiko Miyata, Ayako Isotani & Masahito Ikawa; Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Scientific Reports (2013) 3, Article number: 3355 doi:10.1038/srep03355.

David J. Segal and Joshua F. Meckler; Genome Engineering at the Dawn of the Golden Age Annu. Rev. Genomics Hum. Genet. 2013. 14:135–58. First published online as a Review in Advance on May 20, 2013. The Annual Review of Genomics and Human Genetics is online at genom.annualreviews.org 

Michael Spilman,Alexis Cocozaki,Caryn Hale,Yaming Shao,Nancy Ramia,Rebeca Terns,Michael Terns,Hong Li,and Scott Stagg; Structure of an RNA Silencing Complex of the CRISPR-Cas Immune System. Molecular Cell 52, 146–152, October 10, 2013. 

Raymond H.J. Staals,Yoshihiro Agari,Saori Maki-Yonekura,Yifan Zhu,David W. Taylor,Esther van Duijn,Arjan Barendregt,Marnix Vlot,Jasper J. Koehorst,Keiko Sakamoto,Akiko Masuda,Naoshi Dohmae,Peter J. Schaap,Jennifer A. Doudna,Albert J.R. Heck,Koji Yonekura,John van der Oost,and Akeo Shinkai; Structure and Activity of the RNA-Targeting Type III-B CRISPR-Cas Complex of Thermus thermophilus. Molecular Cell 52, 135–145, October 10, 2013. 

Hui Yang,Haoyi Wang,Chikdu S. Shivalila,Albert W. Cheng,Linyu Shi,and Rudolf Jaenisch; One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering. Cell 154, 1370–1379, September 12, 2013.

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Categories: Cellular Reprogramming, CRISPR Cas, DNA, DNA Editing, DNA Structure, DNA Synthesis, Epigenetics, Gene Editing, Gene Expression, Gene families, Genetics, Genome, In Vitro Transcription, LincRNA, lncRNA, Long noncoding RNA, Molecular Probes, non-coding RNAs, Regulatory RNA, RNA, RNA Editing, RNA Structure, RNA Synthesis, RNA World

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1 reply

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