#CRISPR guidelines part 3: Specificity of CRISPR/Cas9 cleavage.

Of the 23-base standard CRISPR genomic target sequence, the bases actually required for target recognition are the first 20 bases and the last 2 bases (...GG). Combined, this target is sufficiently long enough that most targets of interest will turn out to be unique in mammalian genomes.  However, Cas9 can tolerate mismatches, leading to concerns about off-target cleavage.

Off-target cleavage: Off-target cleavage events can occur and are well documented for CRISPR/Cas9.  The “seed region” of approximately 12 bases proximal to the PAM motif are most crucial for pairing and DNA cleavage, while mispairing in the distal bases can sometimes be tolerated [13]. The frequency of off-target CRISPR cleavage events is currently controversial, and is probably highly target- and system-dependent. The most current data relevant to mouse embryos is from Yang et al [7]. For 5 different guide RNAs designed to unique targets, they identified all potential off-target (OT) regions (N=47) in the mouse genome that had up to 3 or 4 mismatches within the 20 bp coding sequences of the guide RNAs. 6-10 mice or ES cell lines were screened per guide RNA. Of all the OT locations, mutations were induced at 3 of 47 OT sites screened. They noted that the only OT sites with detectable mutations had only 1 or 2 mismatches compared to the target. This correlates with the observation that multiple mismatches reduce CRISPR cleavage efficiency. A very useful online CRISPR target design tool is available that provides data on all off-target sites for predicted targets is found at: http://crispr.mit.edu.

There are a few reasons to believe that off-target cleavage issue seems is less of an overall concern for injected mouse embryos as compared to tissue culture-based CRISPR experiments, where off-target cleavage events have been studied in more detail [13]. Some of this reduced rate of off-target effects in mouse embryos may be due to the more transient expression of the CRISPR/Cas RNAs following embryo injections, as opposed to the longer duration of expression from transfected plasmids in cell culture. However it should be kept in mind that off-target cleavage may occur. Also note that, for mice, the potential effects of off- target mutations could potentially be removed by backcrossing the resulting mice to the parent strain.   

Mutations can be created using a “nickase” variant of Cas9 in which one of the two strand-specific DNA cleavage domains is inactivated by a single amino acid change [11, 14].   Single targets are not mutagenized at high efficiency since the single strand “nicks” are usually repaired in vivo by ligase.  However, by using two targets on opposite strands in fairly close proximity, NHEJ or HDR can be induced at moderate efficiency.  This scheme should reduce off-target mutations since off-target nicks will be isolated in the genome, and thus will usually be quickly repaired by ligase.  However, the complexity and constraints on target selection are increased in “paired-nickase” experiments.     

• CRISPR/Cas9 expression in mouse embryos:  In the Vanderbilt Transgenic Mouse / ES Cell Shared Resource (TMESCSR), we have successfully performed CRISPR/Cas9 mutagenesis in mouse embryos by injecting either (1) cytoplasmic injection of CRISPR/Cas9 RNAs or, (2) pronuclear injection of plasmid DNAs for transient expression of CRISPR/Cas.   We have recently adopted injection of the PX330 plasmid (Addgene #42230)[14], which can be easily modified to express customized guide RNAs for targets of interest. This allows easy customization of the reagent and a simple miniprep protocol to prepare the DNA for injection.    PX330 is a bifunctional plasmid that also expresses Cas9 mRNA.  

For those who are interested in making the RNAs themselves, we have used a Cas9 in vitro mRNA expression vector created by Dr. Wenbiao Chen here at Vanderbilt [15]. This Cas9 vector has been codon-optimized and incorporates nuclear localization signals, and is known to be highly functional in zebrafish and mouse embryos. Several other versions of Cas9 expression constructs are also available from Addgene. 

• TALENs as alternatives to CRISPR/Cas: TALENs also can direct DNA cleavage at desired targets, and so share many conceptual and outcome similarities to CRISPR/Cas [1]. The primary advantage TALENS afford compared to CRISPR/Cas is more flexibility in target site choice. The primary disadvantages are twofold: first, TALENs appear usually to have lower targeting efficiency than CRISPR/Cas reagents; second, a new TALEN vector has to be designed and created for each target, which increases the time and cost. Customized TALEN vectors can be purchased from commercial vendors, who also usually provide design assistance. Although the TMESCSR does not provide TALEN design assistance, we can perform pronuclear injections with TALEN reagents provided by the investigator; please contact the TMESCSR if you wish to pursue this.

Bibliography

1.            Menke, D.B., Engineering subtle targeted mutations into the mouse genome. Genesis, 2013. 51(9): p. 605-18.

2.            Jinek, M., K. Chylinski, I. Fonfara, M. Hauer, J.A. Doudna, and E. Charpentier, A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21.

3.            Wang, H., H. Yang, C.S. Shivalila, M.M. Dawlaty, A.W. Cheng, F. Zhang, and R. Jaenisch, One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell, 2013. 153(4): p. 910-8.

4.            Li, D., Z. Qiu, Y. Shao, Y. Chen, Y. Guan, M. Liu, Y. Li, N. Gao, L. Wang, X. Lu, and Y. Zhao, Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol, 2013. 31(8): p. 681-3.

5.            Fujii, W., K. Kawasaki, K. Sugiura, and K. Naito, Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res, 2013. 41(20): p. e187.

6.            Shen, B., J. Zhang, H. Wu, J. Wang, K. Ma, Z. Li, X. Zhang, P. Zhang, and X. Huang, Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Research, 2013. 23(5): p. 720-3.

7.            Yang, H., H. Wang, C.S. Shivalila, A.W. Cheng, L. Shi, and R. Jaenisch, One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell, 2013. 154(6): p. 1370-9.

8.            Hsu, P.D., D.A. Scott, J.A. Weinstein, F.A. Ran, S. Konermann, V. Agarwala, Y. Li, E.J. Fine, X. Wu, O. Shalem, T.J. Cradick, L.A. Marraffini, G. Bao, and F. Zhang, DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 2013. 31(9): p. 827-32.

9.            Hou, Z., Y. Zhang, N.E. Propson, S.E. Howden, L.F. Chu, E.J. Sontheimer, and J.A. Thomson, Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci U S A, 2013. 110(39): p. 15644-9.

10.            Wang, T., J.J. Wei, D.M. Sabatini, and E.S. Lander, Genetic screens in human cells using the CRISPR-Cas9 system. Science, 2014. 343(6166): p. 80-4.

11.            Ran, F.A., P.D. Hsu, C.Y. Lin, J.S. Gootenberg, S. Konermann, A.E. Trevino, D.A. Scott, A. Inoue, S. Matoba, Y. Zhang, and F. Zhang, Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 2013. 154(6): p. 1380-9.

12.            Fu, Y., J.D. Sander, D. Reyon, V.M. Cascio, and J.K. Joung, Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol, 2014. 32(3): p. 279-84.

13.            Fu, Y., J.A. Foden, C. Khayter, M.L. Maeder, D. Reyon, J.K. Joung, and J.D. Sander, High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol, 2013. 31(9): p. 822-6.

14.            Cong, L., F.A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P.D. Hsu, X. Wu, W. Jiang, L.A. Marraffini, and F. Zhang, Multiplex genome engineering using CRISPR/Cas systems. Science, 2013. 339(6121): p. 819-23.

15.            Jao, L.E., S.R. Wente, and W. Chen, Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A, 2013. 110(34): p. 13904-9.

#CRISPR guidelines part 2: CRISPR target choice considerations.

The target should typically have the sequence motif N₂₁GG. The first 20 bases form the “protospacer” and they must to be incorporated as part of the guide RNA that will localize the Cas9 DNAse to the target site. These bases are required to pair with the target complementary DNA strand. The CRISPR/Cas9 mechanism can apparently utilize almost any sequence for the unspecified bases. Although some target sequences can apparently exhibit varying cleavage efficiencies, the cleavage efficiency at a specific site cannot currently be predicted based on the characteristics of the target sequence. Therefore, there may be occasions where targets need to be tested empirically.

The last 3 bases of the N₂₁GG motif (that is, ...NGG) are the PAM (protospacer- adjacent motif). The last GG dinucleotide is required by the S. pyogenes Cas9 for efficient target recognition and interacts directly with the Cas9 protein. Deviations from this motif significantly reduce efficiency of cleavage [8]. In the near future, Cas9 variants may be engineered that utilize different PAM motifs. Finally, some Cas9 proteins from other bacterial species can apparently have different PAM recognition motifs [9], so the array of possible CRISPR/Cas targets may increase as alternate Cas variants become available.

• Are there required sequence characteristics within the 20-base base-pairing region? To efficiently synthesize the guide RNA, the RNA polymerase will usually prefer certain bases in the first 1 or 2 bases of the protospacer sequence.  Note that these are defined by the type of RNA polymerase used to synthesize the guide RNA, not the CRISPR/Cas9 mechanism itself.  For in-vitro synthesized guide RNAs, T7 RNA polymerase will be typically employed.  T7 RNA Pol “prefers” GG as the first two bases.    If the guide RNA is to be expressed inside the target cell from an injected (or transfected) DNA plasmid, the vector will often contain the human U6 snRNA promoter for this purpose.  This promoter is transcribed by RNA Pol III which prefers G as the first base.

Other than that, there are apparently few strict limits on the sequence content of the protospacer.  However, efficiency of guide RNA loading onto Cas9 is optimized when the last 4 protospacer bases are purines and U is avoided (T in the target sequence) [10].  Also, avoid targets with stretches of 4 or more T’s in a row as they can impair guide RNA transcription.  TTTTT is the termination signal for RNA Pol III.

•  Can the protospacer sequence be lengthened to improve efficiency or specificity?  No.  [11]

• Can the protospacer be shortened?  Surprisingly, the protospacer can apparently be shortened by trimming off 1-3 bases from the 5’ end, with negligible loss in cleavage efficiency and reduction in off-target cleavage [12].   

• Where in the target does Cas9 cleave the DNA?  The Cas9-mediated strand cleavage sites are within the protospacer, close to the PAM motif.  Cas9 normally cleaves both strands.   The precise cleavage site on either strand can vary slightly [2] but map within the colored regions below:

5’---NNNNNNNNNNNNNNNNNNNNGG---------5’

3’---NNNNNNNNNNNNNNNNNNNNCC---------3’

Green/Magenta: Sites of cleavage by Cas9, from Jinek et al. Science 337, 816 (2012).   Gray: PAM sequence.

• Identifying potential CRISPR targets in genes of interest: The PAM motif is reasonably frequent in mammalian DNA. Many or most genes will have enough targets to be amenable to some form of mutagenesis with this motif, so we suggest it as a starting point for target searching in your gene of interest. Also, a CRISPR target design tool is available: http://crispr.mit.edu.

In the near future, S. pyogenes Cas9 variants may be created that utilize different PAM motifs (but not as of early 2014). Finally, some Cas9 proteins from other bacterial species can apparently have different PAM recognition motifs [9], so the array of possible CRISPR/Cas targets may increase as alternate Cas variants become available.

Next post: Specificity of CRISPR/Cas9 cleavage.

Bibliography

1.            Menke, D.B., Engineering subtle targeted mutations into the mouse genome. Genesis, 2013. 51(9): p. 605-18.

2.            Jinek, M., K. Chylinski, I. Fonfara, M. Hauer, J.A. Doudna, and E. Charpentier, A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21.

3.            Wang, H., H. Yang, C.S. Shivalila, M.M. Dawlaty, A.W. Cheng, F. Zhang, and R. Jaenisch, One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell, 2013. 153(4): p. 910-8.

4.            Li, D., Z. Qiu, Y. Shao, Y. Chen, Y. Guan, M. Liu, Y. Li, N. Gao, L. Wang, X. Lu, and Y. Zhao, Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol, 2013. 31(8): p. 681-3.

5.            Fujii, W., K. Kawasaki, K. Sugiura, and K. Naito, Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res, 2013. 41(20): p. e187.

6.            Shen, B., J. Zhang, H. Wu, J. Wang, K. Ma, Z. Li, X. Zhang, P. Zhang, and X. Huang, Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Research, 2013. 23(5): p. 720-3.

7.            Yang, H., H. Wang, C.S. Shivalila, A.W. Cheng, L. Shi, and R. Jaenisch, One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell, 2013. 154(6): p. 1370-9.

8.            Hsu, P.D., D.A. Scott, J.A. Weinstein, F.A. Ran, S. Konermann, V. Agarwala, Y. Li, E.J. Fine, X. Wu, O. Shalem, T.J. Cradick, L.A. Marraffini, G. Bao, and F. Zhang, DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 2013. 31(9): p. 827-32.

9.            Hou, Z., Y. Zhang, N.E. Propson, S.E. Howden, L.F. Chu, E.J. Sontheimer, and J.A. Thomson, Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci U S A, 2013. 110(39): p. 15644-9.

10.            Wang, T., J.J. Wei, D.M. Sabatini, and E.S. Lander, Genetic screens in human cells using the CRISPR-Cas9 system. Science, 2014. 343(6166): p. 80-4.

11.            Ran, F.A., P.D. Hsu, C.Y. Lin, J.S. Gootenberg, S. Konermann, A.E. Trevino, D.A. Scott, A. Inoue, S. Matoba, Y. Zhang, and F. Zhang, Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 2013. 154(6): p. 1380-9.

12.            Fu, Y., J.D. Sander, D. Reyon, V.M. Cascio, and J.K. Joung, Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol, 2014. 32(3): p. 279-84.

13.            Fu, Y., J.A. Foden, C. Khayter, M.L. Maeder, D. Reyon, J.K. Joung, and J.D. Sander, High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol, 2013. 31(9): p. 822-6.

14.            Cong, L., F.A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P.D. Hsu, X. Wu, W. Jiang, L.A. Marraffini, and F. Zhang, Multiplex genome engineering using CRISPR/Cas systems. Science, 2013. 339(6121): p. 819-23.

15.            Jao, L.E., S.R. Wente, and W. Chen, Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A, 2013. 110(34): p. 13904-9.

#CRISPR guidelines part 1: Introduction

1. Introduction

Over the last few years, zinc finger nucleases and TALENS have been developed and used as new tools to allow targeted mutagenesis in a wide variety of cells and model organisms, including mice [1]. Beginning in early 2013, several labs also independently achieved remarkably high efficiencies of gene targeting in animal cells using the CRISPR/Cas9 system. These tools exploit the ability of the Cas9 protein to cleave DNA targets specified by a “guide RNA” containing a 20-base match to the genomic target [2]. Co-expressing the guide RNA with Cas9 in mouse embryos can efficiently generate mutations in the target sequence. It is clear that the CRISPR/Cas system will be widely developed and used as a targeted mutation system in human cells and in mice, due to its superior efficiencies and advantages over similar targeted cleavage systems (e.g. TALENs). However, TALENS may offer targeting specificities in some instances where CRISPR/Cas9 may not. Therefore it is not clear that CRISPR/Cas9 will completely supplant TALENS. Here we provide an overview of the CRISPR/Cas9 system and its application to targeted genome editing in mouse embryos.

Inducing mutations in mouse genes using CRISPR/Cas9: In 2013, several groups reported remarkably high efficiencies of CRISPR/Cas mutagenesis in mouse embryos following injection of CRISPR guide RNAs and Cas9 mRNA into 1-cell mouse embryos [3-7]. A frequently observed result was that up to half or more of the liveborn pups carried mutations in one or both of the target alleles. Moreover, the high efficiency of single- gene targeting allows multiplexing of two, three or even more targets in the same injection, potentially allowing several genes to be targeted at once.

Mutagenic effects of CRISPR/Cas9-mediated cleavage: CRISPR/Cas9-mediated cleavage of the target gene results in both DNA strands being cleaved within the target sequence. Cas9 is a double-stranded DNA endonuclease that depends on interaction with the guide RNA for DNA cleavage. The resulting double-stranded break at the target site is usually repaired by the non-homologous end-joining (NHEJ) DNA repair pathway. This usually results in loss of a few, to several hundred, nucleotides around the cleavage site, although insertions are sometimes observed. Thus, when CRISPR/Cas9 is targeted to gene coding regions it efficiently creates mutations that are often deleterious and/or effectively null alleles. However, the resulting mutations could be in-frame; obviously, position within the gene may affect the severity of mutations in a gene-dependent manner. Thus, a variety of mutations may be generated by simple CRISPR/Cas9-targeting.

CRISPR/Cas9 can facilitate precise genome editing: If a homologous DNA molecule is also present (a homology donor molecule), the cleaved DNA strands can be repaired using homology-directed-repair (HDR) instead of the NHEJ pathway (see Figure; reviewed in [1]. This enables precisely engineered sequences to be introduced at or very close to the target site. In mouse embryos, this has been accomplished by co-injecting the CRISPR guide RNA and the Cas9 mRNA along with a long single-stranded DNA oligonucleotide having at least 60 bases of homology on either side of the target site [3, 7]. A novel sequence (e.g. LoxP site, altered restriction site, peptide tag, or SNP variant) is designed into the oligo between the homologous arms. Cleavage can also facilitate targeted integration of longer molecules, e.g. GFP-style reporter cassettes.

Next post: CRISPR target choice considerations

Bibliography1.            Menke, D.B., Engineering subtle targeted mutations into the mouse genome. Genesis, 2013. 51(9): p. 605-18.2.            Jinek, M., K. Chylinski, I. Fonfara, M. Hauer, J.A. Doudna, and E. Charpentier, A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21.3.            Wang, H., H. Yang, C.S. Shivalila, M.M. Dawlaty, A.W. Cheng, F. Zhang, and R. Jaenisch, One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell, 2013. 153(4): p. 910-8.4.            Li, D., Z. Qiu, Y. Shao, Y. Chen, Y. Guan, M. Liu, Y. Li, N. Gao, L. Wang, X. Lu, and Y. Zhao, Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol, 2013. 31(8): p. 681-3.5.            Fujii, W., K. Kawasaki, K. Sugiura, and K. Naito, Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res, 2013. 41(20): p. e187.6.            Shen, B., J. Zhang, H. Wu, J. Wang, K. Ma, Z. Li, X. Zhang, P. Zhang, and X. Huang, Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Research, 2013. 23(5): p. 720-3.7.            Yang, H., H. Wang, C.S. Shivalila, A.W. Cheng, L. Shi, and R. Jaenisch, One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell, 2013. 154(6): p. 1370-9.

Vanderbilt TMESCSR core #CRISPR guidelines 4/22/14

I've updated some CRISPR guidelines, mainly for users of the Vanderbilt transgenic mouse core (TMESCSR), but they are generally applicable.  The full guidelines are available at the core's website.  I'll post portions of the guidelines on this blog over the next few days.

Quantifying Genome-Editing Outcomes with SMRT Sequencing tinyurl.com/nx9l6sb #CRISPR

Been thinking recently about the most practical way to thoroughly assess off-target mutagenesis… Seems like a good job for a medium-throughput-scale next-gen sequencing.   The workflow could be 1. identify the off-targets, to a depth of ~ 4 mismatches; these will number in the dozens at least.  2. PCR all off-targets from CRISPR'd sample.  3. NGS, but we probably won't need millions of reads; thousands will do, especially for live founder mice and certainly for testing their progeny.   This recent paper is a good example of quantifying editing at the on-target site, but could obviously be adapted to analyzing off-targets too:

Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing

Ayal Hendel, Eric J. Kildebeck,  Eli J. Fine, Joseph T. Clark, Niraj Punjya, Vittorio Sebastiano,Gang Bao, Matthew H. Porteus

Cell Reports, Volume 7, Issue 1, p293–305, 10 April 2014

Direct application of Cas9 protein + CRISPR RNA to cells

Here's the two recent papers in Genome Research describing transfer of Cas9 protein + guide RNA directly to cells, with efficiency and reduced off-target effects.

Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA.

Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H.
Genome Res. 2014 Apr 2. [Epub ahead of print]

Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.

Kim S, Kim D, Cho SW, Kim J, Kim JS.
Genome Res. 2014 Apr 2. [Epub ahead of print]

4 currently available tools for finding CRISPR off-targets

I was aware of 2 but heard about 2 more yesterday...  3 of these are web-based and 1 is a download/local install.   Of these. I've used the Zhang lab CRISPR design tool and ZiFit (see my Feb 18, 2014 post).   I have not yet tried Cas-OFFinder nor CasOT.   In alphabetical order:

1.  Cas-OFFinder

Reference: Bioinformatics. 2014 Feb 17. [Epub ahead of print]Cas-OFFinder: a fast and versatile algorithm that searches for potential off-targetsites of Cas9 RNA-guided endonucleases.   Bae S, Park J, Kim JS.

   This tool apparently allows searching with different PAM motifs that are recognized by Cas9 from species other than S. pyogenes.  Those variant Cas9's aren't widely used yet, but I imagine it's just a matter of time.

2. CasOT   (No web tool, local install only)
Reference: Bioinformatics. 2014 Jan 21. [Epub ahead of print]
CasOT: a genome-wide Cas9/gRNA off-target searching tool.  Xiao A, Cheng Z, Kong L, Zhu Z, Lin S, Gao G, Zhang B.

3.  Zhang lab (MIT) CRISPR design site

For identification of standard 23-base targets, scored by a metric that minimizes likelihood of off-target cleavage.  Also for locating paired targets for paired-nickase approach.    

4.  ZiFit
From the Zinc Finger Consortium; originally for zinc finger & TALEN target screening, adapted for CRISPR .  Allows identification of shorter targets for the truncated-target approach.

Vanderbilt CRISPR interest group

The meeting today for Vanderbilt folks interested in CRISPR was well attended!  Thanks for coming to all who did.  Also many thanks to Drs. Ian Macara and Ron Emeson.  As we mentioned, for Vanderbilt labs interested in exploring cooperative projects to push CRISPR methods forward in the VUMC TMESCSR (transgenic core), please contact me.  

Comparison of injecting DNA vs RNA / pronucleus vs. cytoplasm

 The CRISPR guide RNA and Cas9 mRNA can be directly injected, or, transiently expressed from DNA plasmids.  Here's a head-to-head comparison of the resulting efficiencies when injecting DNAs vs. RNAs into mouse embryos, and whether to go into the pronucleus or the cytoplasm.

Bottom line:  RNA injection into the cytoplasm is the most efficient way for generating both efficient mutagenesis and numbers of live pups.   However, plasmid DNA works too.   For me, the main concern with RNA is the reliability of the RNA synthesis to provide consistency across injections.

Sci Rep. 2014 Mar 28;4:4513.  

Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering.

Horii T, Arai Y, Yamazaki M, Morita S, Kimura M, Itoh M, Abe Y, Hatada I.

CRISPR/Cas9-mediated knock-ins in zebrafish

This paper demonstrates targeted insertion by non-homologous-end-joining - and thus, not by homologous recombination - in fish embryos, and with germline transmission.  It's still unclear to me how efficient this will be to generate germline transmission in mice, but this is a promising step.

Genome Res. 2014 Jan;24(1):142-53. doi: 10.1101/gr.161638.113. Epub 2013 Oct 31.
Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.
Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F.