Molecular Tricks @ NovoHelix

molecular tricks @ NovoHelix

The laboratory wet bench can be a very humbling space, even for veteran and experience-tested scientists. Throughout our careers we have used many resources to retool failed experiments including discussions at professional meetings, shadowing an experienced colleague, re-reading time-tested protocols from Molecular Cloning by Sambrook and Russel or Manipulating the Mouse Embryo, thumbing through the NEB product catalog to better understand enzymatic activities, downloading detailed protocols from journals such as Methodsin Enzymology or Nature Protocols or simply watching YouTube or JOVE videos, or using crowd-sourced tools like Tg-List, EmbryoMail or CRISPR-Google Groups. Collectively, these resources can provide new insights that help reshape experimental designs to test old and new hypotheses.

NovoHelix is at the interface of many scientific disciplines including applied genetics, embryology, epigenetic reprogramming, genome engineering, regenerative and reproductive medicine, and basic molecular biology. As we have both succeeded and fumbled along our scientific endeavors, we thought it might be helpful to provide expert knowledge where we can. This Molecular Tricks @ NovoHelix section is meant to disseminate a tidbit of this ‘devil-in-the-details’ knowledge. We understand that there are many approaches to solving a problem, and we will regularly update this site with tips and tricks from our scientists, common pitfalls and failures, and comment on methods and protocols from preprint and peer-reviewed manuscripts. Let’s get experimenting!

Index

Name

Description

Website

head-to-tail knock-in insertions

Skryabin and colleagues show that long-range PCR validation is insufficient to screen for a common CRISPR-mediated knock-in error, namely head-to-tail insertions of DNA repair templates.

At NovoHelix we have observed this head-to-tail insertion phenomena across multiple projects and have developed strategies to collapse the multi-copy arrays into a single useable targeted knock-in. While these gene-editing errors have been observed by the University of Muenster team and known to the NovoHelix scientists since 2013, many newcomers to CRISPR have overlooked this common error and will need to re-validate knock-in animal models generated by CRISPR. We are excited that these multi-copy knock-in arrays have been independently observed and reported, and as a direct consequence we hope that the animal modeling community can devise approaches to mitigate these knock-in errors.

The final peer-reviewed manuscript is available at Science Advances:

A preprint of the article has been published on bioRxiv:

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recombineering counter-selection tools

Recombineering allows synthetic biologists to introduce targeted mutations in bacterial artificial chromosomes (BACs). To date, most researchers have utilized counter-selection approaches to introduce targeted point mutations, but counter-selection suffers from limited robustness due to frequent escapees and many clones often have to be screened and validated. Bird and colleagues have devised an ingenious solution to circumvent counter-selection when introducing targeted point mutations: positive selection with a synthetic intron cassette containing a selectable marker and adjacent to the intended mutation site. They call this technique ESI (Exogenous/Synthetic Intronization) mutagenesis and demonstrate that the cells receiving the BAC transgene efficiently splice out the synthetic intron.

Check out their ESI mutagenesis preprint publication on bioRxiv:

For a review of recombineering counter-selection techniques from the labs of Don Court, George M. Church or Francis Stewart, see the following publications:

tolC, SDS, colicin E1, vancomycin:

tetA-sacB cassette, tetracycline, sucrose, fusaric acid:

bacterial toxin-antitoxin system, namely ccdB counter-selection with ccdA antidote:

optimized P. luminescens rpsL gene, rpsL-neo cassette, streptomycin:

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IRES syntax

One can swap out the 2A peptide sequence with an internal ribosomal entry site (IRES); however, one would need to introduce a stop codon for the newly incorporated IRES. While the encephalomyocarditis virus (EMCV) IRES is GC-rich, I have PCR amplified and subcloned multiple versions of this IRES sequence without difficulty by using an engineered family B polymerase such as Phusion or Kapa HiFi and T5 flap endonuclease mediated isothermal assembly (aka Gibson assembly). Sanger sequence the final construct to ensure no mutations were PCR introduced (recommended not to skip this step).

Depending on the experimental design, one should plan to introduce the preferred IRES (viral bases 273-845; rather than the minimal IRES viral bases 400-836). One should also be familiar with the EMCV IRES A6 or A7 bifurcation loop. Ann Palmenberg's group explains the bias between the upstream or downstream cistron regarding the bifurcation loop and provides an illustration for clarity, so please review the paper:Bochkov & Palmenberg, 2006 Biotechniques.

Bochkov YA, Palmenberg AC. Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. Biotechniques. 2006 Sep;41(3):283-4, 286, 288 passim. PubMed PMID: 16989088.

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Drop dialysis for embryo genome engineering

General considerations for drop dialysis of CRISPR repair template DNA (ssDNA or dsDNA)

drop dialysis volume. The dialysate should be around 200x – 500x larger than the sample volume. I have dialyzed against

18.2 megaohms resistant water (type I water),
microinjection buffer [10 mM Tris, pH 7.5; 0.1 mM EDTA], or
BAC microinjection buffer [10 mM Tris, pH 7.5; 100 mM NaCl, 0.1 mM EDTA] depending on the application.

drop dialysis time. For most DNA desalting applications prior to embryo manipulation, the bulk of the salt in the sample is dialyzed within 2 hours. The sample ‘saltiness’ can be measured by a conductivity probe. I invested in a microscale conductivity probe because I prepare pre-implantation embryo media and human pluripotent stem cell media; for batch testing, I use the conductivity and osmometer readings to test lots prior to culture and manipulation. While you can let the dialysis proceed overnight to reduce low-concentration impurities, or alternatively, change the buffer after 2 hours with new buffer and proceed another 2 hours.

stirring. An autoclaved small white PTFE stir bar can be used to reduce drop dialysis time. Place the stir bar on low in the center of a petri dish and stir. While this stirring is generally unnecessary, in a time-crunch prior to an afternoon microinjection session, I have done it without issue. Conductivity/’saltiness’ dropped approximately 25% faster over the course of 2 hours.

DNA size. Let’s consider the DNA size in terms of DNA mass and DNA length because the membrane filters often use a molecular weight cut-off (MWCO) or a pore size (micron) dimension that reflects either mass or length, respectively.

Dickerson et al, 1982 provide DNA length measures.

1 bp ≈ 0.33 nm
1,000 bp = 1 kb ≈ 0.33 µm

Dickerson RE, Drew HR, Conner BN, Wing RM, Fratini AV, Kopka ML. The anatomy of A-, B-, and Z-DNA. Science. 1982 Apr 30;216(4545):475-85. doi: 10.1126/science.7071593. PMID: 7071593

In general, select membrane pore that is to be retained.

DNA length. 1 kb = 0.33 µm/0.05 µm porosity = 6.6x bigger.
DNA mass. The average weight of a base pair (bp) is 650 daltons, by extension 1 kb = 650 kDa. For a 1 kb repair template, the selection of a membrane with less than ~200 kDa MWCO is sufficient. The size of the DNA molecule is inversely related to dialysis equilibrium, and larger pores will reduce dialysis equilibrium timescales.

Membrane chemistry and size. Common dialysis filter membranes used/recommended for embryo manipulation include Millipore/Sigma Cat Nr. VMWP02500 with chemistry mixed cellulose esters (MCE), diameter 25 mm, and porosity 0.05 µm.

DNA volume: membrane diameter recommendations:

DNA volume less than 25 ul à use 13 mm diameter (Cat Nr. VMWP01300/porosity 0.05 µm or VSWP01300/porosity 0.025 µm)
DNA volume less than 200 ul à use 25 mm diameter (Cat Nr. VMWP02500)
DNA volume less than 1 ml à use 45 mm diameter (Cat Nr. VMWP04700)

Tip: For those of us that have big clumsy hands, use sterile forceps to assist in placing the filter shiny side up on the dialysate buffer.

Q: Can I substitute the common protein-nonbinding polyethersulfone/PES filter membrane with porosity 0.05 µm with a mixed cellulose esters (MCE) filter membrane for drop dialysis?

A: Not recommended. I have empirically tested and DNA drop spreads out by capillary action and makes it difficult to recover after a couple of hours of drop dialysis. A slide-a-lyzer dialysis unit with a PES membrane with appropriate porosity should work acceptably.

Consumables. Ensure that all consumables are RNAse-free and non-toxic to the embryo.

See Luis Montoliu’s YAC protocol, Step 19:

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