The CRISPR page at CNB

National Centre for Biotechnology (CNB-CSIC) Madrid, picture by Lluis Montoliu

by  Lluís Montoliu (CNB-CSIC)

with help and comments from Davide Seruggia (Boston Children's Hospital) and Francis J.M. Mojica (Universidad de Alicante)

Lluís Montoliu's Lab Web Page

LAST UPDATED: 3 August 2017


Table of Contents for this Web page

What is CRISPR-Cas9?

Which are the basic papers describing the use of CRISPR-Cas9 technology?

Additional papers worth reading illustrating the many uses of CRISPR-Cas9 technologies

Brief history of CRISPR: Who discovered this system in bacteria?

Who is Who in the CRISPR-Cas World

NHEJ inhibitors: boosting the HDR pathway

Alternative methods to embryo microinjection to generate genome-edited mice with CRISPR-Cas9

Gene therapy protocols with CRISPR-Cas technologies

CRISPR-Cas attempts in human embryos

CRISPR approaches in genome-wide screenings

Which starting plasmids do I need to obtain/purchase to begin using CRISPR-Cas9 technology?

Other Cas9 proteins for genome editing experiments

The new Cpf1 nuclease and its derivatives

Other Class II Cas-like nucleases

Other sources of Cas9 for genome editing experiments

How do I choose the short DNA sequences from my favourite gene to design the sgRNA?

Where can I get full protocols for using CRISPR-Cas-related technologies?

Successful Knock-In strategies with CRISPR-Cas tools

CRISPR and gene drive approaches

Anti-CRISPR and Cas inhibitors

CRISPR and off-targets

CRISPR and Ethics

CRISPR Awards

Commercial resources for CRISPR-Cas-related reagents

Where can I discuss with other colleagues about the use of CRISPR-Cas9 techniques?

Global repositories of CRISPR publications & commentaries

Beyond CRISPR-Cas: Ago (Argonaute) No evidences of gene-editing activity

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What is CRISPR-Cas9?.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences and Cas (CRISPR-associated) proteins are the two elements of an ancient defence prokaryotic adaptive restriction system conserved in bacterial genomes. While CRISPRs represent the memory of the system, a repository of short, directly repeating nucleotide sequences flanked by short unique unique DNA fragments, acquired from previous infections, Cas proteins are the actual effectors, that are able to process the CRISPR sequences into small RNAs, and to cleave those infectious DNA molecules that perfectly match those CRISPR-derived RNAs. To translate a complex prokaryotic system into a simple genome editing tool, the crRNA (CRISPR RNA) and the tracrRNA (trans-activating crRNA) have been fused in a synthetic small guide RNA (sgRNA), composed by a hairpin RNA structure, resembling the tracrRNA, linked to a 20 bp sequence homologous to the target DNA. Out of all the CRISPR-associated proteins, Cas9 is the final effector, capable of complexing and cleaving both strands of a DNA molecule upon detecting a typical Watson&Crick homologous base pair match with the sgRNA. Therefore the CRISPR-Cas system is often referred as CRISPR/Cas9. Genome engineering by these RNA-programmable Cas9 nucleases have broad applications in biology, biomedicine and biotechnology. The microbiologist Emmanuelle Charpentier and the structural biologist Jennifer Doudna, along with the rest of their colleagues co-authoring the first seminal publications on this subject, are the ones to be credited for having investigated and brought this amazing prokaryotic tool to the attention and for the benefit of the eukaryotic world.
 

CRISPR-Cas technology and applications (video by Le Cong from Feng Zhang's lab at MIT/BROAD institute)

This video has been made possible thanks to Addgene and is part of this series

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Addgene

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Scheme illustrating the CRISPR-Cas9 mechanism of action (by Lluís Montoliu ©)

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Scheme illustrating the two repair pathways occurring after CRISPR-Cas9 action (by Lluís Montoliu ©)

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Which are the basic papers describing the use of CRISPR-Cas9 technology?.

There are already many papers, reviews and reports describing the CRISPR-Cas9 technology. These few papers below are our recommended "starting set" of "MUST-READ" papers, listed as they were published, worth reading before starting using this technology. Of course, additional publications appear constantly and, hence, it is advisable to regularly review the most recent literature on this subject.

Seruggia et al. NAR 2015: Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis

Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis

Seruggia et al. Nucleic Acids Research (2015)

Additional papers worth reading illustrating the many uses of CRISPR-Cas9 technologies.

These are additional papers we have found useful and interesting to read to illustrate the many uses of the new CRISPR-Cas9 technology for genome edition purposes. This list is non-exhaustive, nor it represents necessarily the best papers published. It is simply a list of recent publications which have been selected according to the novelty of the technical or scientific approaches reported. 

Francis Mojica presenting "the origins of the CRISPR-Cas systems" in a seminar at CNB-CSIC, Madrid, on 14 October 2015, picture by Lluis Montoliu 

Francis Mojica (University of Alicante) presenting "the origins of the CRISPR-Cas systems"

in a seminar at CNB-CSIC, Madrid, on 14 October 2015, picture by Lluis Montoliu

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Francis Mojica (University of Alicante) -right- with Lluis Montoliu

Madrid, 14 October 2015, picture by Lluis Montoliu

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.Brief history of CRISPR: Who discovered this system in bacteria?.

There are the first published publications and books describing CRISPR in bacteria and archeas, eventually leading to the current scenario where CRISPR are applied universally.


Who is Who in the CRISPR-Cas World.

This is a non-exhaustive collection of pictures with some of the many researchers that have contributed significantly with their studies to our current understanding of the CRISPR-Cas systems in prokaryotes and their applicaton for genome editing purposes in eukaryotes.

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Francis Mojica

University of Alicante, Spain

 

Rodolphe Barrangou

North Carolina State Univ, Raleigh, USA

 

Philippe Horvath

DuPont Nutrition and Health, France

 

Luciano Marraffini

The Rockefeller Univ, New York, USA

 

John van der Oost

Wageningen University, The Netherlands

 

Emmanuelle Charpentier

MPI for Infect. Biol., Berlin, Germany

 

Jennifer Doudna

Univ California Berkeley, CA, USA

 

Virginijus Siksnys

Vilnius University, Lithuania

 

Feng Zhang

BROAD-MIT, Cambridge, MA, USA

 

George Church

Harvard Med School, Boston, MA, USA

 

Rudolf Jaenisch

Whitehead Inst, Cambridge, MA, USA

 

J. Keith Joung

Mass Gen Hosp, Charlestown, MA, USA

 

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NHEJ inhibitors and other strategies for boosting the HDR pathway.

There are the first published publications reporting the use of a variety of NHEJ inhibitors in order to boost the HDR pathway when using a CRISPR-Cas9 approach, in order to improve the efficiency and success of knock-in/genome edition/substitution features versus the standard, default and far more effective NHEJ repair system, usually leading to the accumulation of indels.

Alternative methods to embryo microinjection to generate genome-edited mice with CRISPR-Cas9.

These articles reported the use of embryo electroporation as a valid alternative method to pronuclear/citoplasmic microinjection of CRISPR-Cas9 reagents. The main difference is whether embryos are first obtained, their zona pellucida digested before electroporation and then subsequently transferred into recipient pseudopregnant females, as described by Qin et al. 2015; Hashimoto&Takemoto 2015; Qin et al. 2016, or whether the electroporation step is done in situ, in oviduct, as in the GONAD method (Takahashi et al. 2015), thus avoiding both the microinjection and the embryo-transfer steps. A new article has been added combining ultrasuperovulation with IASe reagent, cryopreservation methods, IVF and microinjection of thawed fertilized oocytes with CRISPR-Cas reagents

Gene therapy protocols with CRISPR-Cas technologies.

These are some of the relevant articles published reporting the use of CRISPR-Cas tools in gene therapy procedures, in vitro (on cells) and in vivo (on animals). This is one of the fastest growing applications of CRISPR-Cas approaches.

CRISPR-Cas attempts in human embryos i.

These are the first attempts of using CRISPR-Cas technology in human embryos, logically encountering similar results as reported in other mammalian species, that is: mosaicism and potential off-targets. Nothing new, but important to be aware of, in order to carefully think before considering the serious use of CRISPR-Cas in human embryos, an approach currently not recommended. Articles commenting these approaches are also added to this list.

CRISPR approaches in genome-wide screenings

These are some of the relevant articles published reporting the use of CRISPR-Cas tools in genome-wide screening applications.

Which starting plasmids do I need to obtain/purchase to begin using CRISPR-Cas9 technology?.

There are currently many plasmids with multiple Cas9 variants and diverse templates for preparing the synthetic small guide RNA (sgRNA). We routinely use and recommend start using the following plasmids, all available through Addgene. All these plasmids do not contain the T7 promoter in their backbones. Hence, this feature must be added at the 5' of the oligonucleotide used to amplify the DNA region to be transcribed into RNA. In contrast, the following two plasmids do contain the T7 promoter in their backbones. Hence, they can be used for direct in vitro RNA transcription.

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Addgene


Other Cas9 proteins for genome editing experiments.

Cas9 was originally obtained from Streptococcus pyogenes, but it exists in many other bacteria. In addition, new variants, with novel properties, are being isolated and characterized.

The new Cpf1 nuclease and its derivatives.

There are other CRISPR-Cas-like systems not using Cas9 as the effector-nuclease protein. One of the first ones being characterized was Cpf1, a single RNA-guided Endonuclease described by Feng Zhang's lab in 2015. Here we will be listing relevant publications describing the new properties of this new nuclease as they are released. Cpf1 has been known re-named to Cas12a.

Other Cas-like nucleases

Additional CRISPR-Cas-like systems not using Cas9 as the effector-nuclease protein. C2c2 nuclease has been now re-named to Cas13a.

Graphic illustrating the evolution of CRISPR publications in PubMed (by Lluís Montoliu ©)


 

Other sources of Cas9 for genome editing experiments.

Cas9 is normally provided to cells as an expression vector, or, to embryos, as purified in vitro-transcribed RNA from any of the existing plasmids. However, there are additional sources for Cas9 worth taking them into account, such us the use of Cas9 mice (constitutively or conditionally expressing a Cas9-expressing cassette), Cas9 lentiviral vectors or Cas9 purified protein itself.
  • Cas9 ubiquitous expression in mice: Rosa26-Cas9 knockin mice (JAX mice #024858). These CRISPR/Cas9 knockin mice constitutively express CRISPR associated protein 9 (cas9) endonuclease and EGFP in a widespread fashion under the direction of a CAG promoter. When used in combination with single guide RNAs, they allow editing of single or multiple mouse genes in vivo or ex vivo. (see also JAX mice #026179).
  • Cas9 conditional expression in mice: Rosa26-LSL-Cas9 knockin mice (JAX mice #024857). These CRISPR/Cas9 knockin mice have Cre recombinase-dependent expression of CRISPR associated protein 9 (cas9) endonuclease and EGFP directed by a CAG promoter. When used in combination with single guide RNAs and a Cre source, they allow editing of single or multiple mouse genes in vivo or ex vivo.
  • Lentivirus carrying Cas9-expressing units: several Cas9 lentiviral vectors available in this Addgene web page.
  • Cas9 purified protein: several vendors already offer Cas9 purified protein (i.e. NEB*, ToolGen) (*= apparently without NLS)

How do I choose the short DNA sequences from my favourite gene to design the sgRNA?.

There are several CRISPR-Cas9 web sites providing extremely useful services and helping you to design the sgRNA complementary to the expected target site within your favourite gene or genomic location. These are the ones we currently use and recommend using:
  • 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)
  • WTSI Genome Editing (WGE) portal  (Wellcome Trust Sanger Institute, Hinxton, UK)
  • CRISPR Finder (Wellcome Trust Sanger Institute, Hinxton, UK)
  • CRISPR Scan (Antonio Giraldez' lab at Yale University, USA)
  • RepeatMasker (Institute for Systems Biology) to double check and avoid selecting target sites with repeated sequences
  • WatCut (University of Waterloo) to design silent mutations that are helpful for genotyping edited alleles
  • Tide (NKI) Free web tool for the easy quantitative assessment of genome editing
  • ZifIt (Zinc Finger Consortium) to design target sequences for ZFNs, TALENs and CRISPRs
  • CRISPOR (TEFOR French Infrastructure) CRISPOR is a program that helps design, evaluate and clone guide sequences for the CRISPR/Cas9 system
  • CrispRGold is a sgRNA design tool that searches for optimal sgRNA candidates in given genes or sequences. CrispRGold is described in this publication.

Breaking-Cas is a most flexible new guide designing web tool, developed at CNB. This program is useful for predicting guide RNAs for SpCas9, SaCas9, AsCpf1, FnCpf1, C2c2 and other newer nucleases, like NgAgo, using all genomes (>1000) available from Ensembl, and allowing to place any given PAM sequence at 5' or 3', or no PAM at all (for Ago), any length of target sequence, any experimental data regarding frequencies of nucleotides per site, etc...

 


Where can I get full protocols for using CRISPR-Cas-related technologies?.

There are different publications and web resources where you can find detailed protocols for using the CRISPR-Cas9 techniques. These are some of the ones we recommend using:

Successful Knock-In strategies with CRISPR-Cas tools.

There are different publications where diverse strategies resulting in succcessful Knock-In mutant alleles in animals have been reported.
 

CRISPR and gene drive approches.

CRISPR tools have been also engineered to trigger gene drive strategies, in which the inserted cassette encodes both for the Cas9 nuclease and for the guide RNAs that target the cassette to a given genomic locus, thus resulting in biased non-mendelian inheritance. This approach has been mostly explored for pest control in insects known to act as vectors for infectious diseases, such as malaria, yellow fever and zika.

Anti-CRISPR and Cas inhibitors.

CRISPR-Cas tools are extremely efficient. Anti-CRISPR proteins and Cas-inhbitors, as proteins or small molecules, are beginning to be discovered and are expected to provide a new source for controlling CRISPR activity. Some of the recent developments are described here.

CRISPR and off-targets

CRISPR-Cas tools can trigger off-target mutations, depending on many parameters, most importantly the RNA guide used. The existence and relevance of these potential off-target altered genomic sequences is often reported and a matter of discussion. Some of these papers are summarized here.

CRISPR and Ethics.

CRISPR experiments can be associated with several Ethics issues, including its application to animals, to human beings, to human embryos and to wild-type species in the environment. A number of Ethics debates have been triggered over the past years around these issues, some of these documents are listed here:
 

CRISPR Awards.

The following list is a NON-exhaustive compilation of Prizes awarded to several key researchers in this field for their various contributions to our current knowledge on the CRISPR systems and CRISPR tools for gene editing purposes

Scheme illustrating the most advanced CRISPR-Cas9 reagent: the RiboNucleoProtein (RNP) (by Lluís Montoliu ©)


Commercial resources for CRISPR-Cas-related reagents.

The following list is a NON-exhaustive compilation of companies producing CRISPR-Cas-related reagents commercially, fulfilling the need for intermediate reagents (i.e. longer DNA oligonucleotides) of final reagents (i.e. RNAs) ready-to-use for CRISPR-Cas applications. If you know additional companies marketing similar products, please do let us know.
  • Dharmacon (GE) you can order crRNA, tracrRNA, sgRNA, RNA for Cas9, Cas9 vectors and Cas9 protein
  • Sigma you can order sgRNA, sgRNA-expressing and Cas9-expressing vectors and Cas9 RNA, positive and negative controls available
  • ThermoFisher you can order sgRNA, Cas9 RNA or protein or expressing vectors for all reagents
  • PNA Bio you can order sgRNA, Cas9 RNA or protein or expressing vectors for all reagents
  • NEB purified NLS Cas9 protein
  • IDT you can order long complementary oligos for reconstituting templates for in vitro transcription for producing sgRNA and Cpf1 products
  • gBlocks (IDT) you can order entire DNA-based sgRNA sequences ready-to-use for in vitro transcription

Where can I discuss with other colleagues about the use of CRISPR-Cas9 techniques?.

There are several lists, forum and google groups where the use of CRISPR-Cas9 techniques is regularly discussed. These are the ones we use:

Global repositories of CRISPR publications & commentaries.

There are several repositories of CRISPR publications and commentaries, including:

Beyond CRISPR-Cas: Ago (Argonaute)-no evidences of gene editing activity  .

These are the first papers reporting a new (what would have been possibly the fourth, after ZFN, TALEN and CRISPR) promising gene editing tool: Ago (Argonaute), also from prokaryotes, a DNA-guided DNA endonuclease. Current evidences collected world wide do not confirm the results initially reported in Nature Biotechnology by Gao et al. (2016) and do not support any proof of gene editing activity that can be attributed to Ago. Using Gaetan Burgio's last sentence in his latest preprint : "Together our findings indicate that NgAgo is unsuitable as a genome editing tool." Eventually, the original paper in Nature Biotechnology has been retracted by the authors on 2 August 2017.

Scheme illustrating the pressumptive Ago mechanism of action (by Lluís Montoliu ©)


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