A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Summary

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

Abstract

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

Introduction

Recent technological advances have radically transformed and accelerated the ability to precisely engineer genomes. In particular, the CRISPR-Cas9 system, which relies on the RNA-guided endonuclease Cas9 to induce a double strand break (DSB) near the target sequence of interest, has been extensively used to accurately engineer the genome of the majority of model organisms used in biomedical research^1,2,3,4. Significantly, the use of CRISPR-Cas9 has unlocked genome editing even in difficult species like C. elegans⁵. Regardless of species, generating point mutations with the CRISPR-Cas9 based genome editing system relies on three core components: 1) Cas9 endonuclease, 2) a single guide RNA (sgRNA) that directs the Cas9 endonuclease to a target sequence, and 3) a user designed homology-directed repair (HDR) template containing the desired edit(s) of interest².

There are several methods that can be used to introduce the targeting sgRNA and Cas9 nuclease into cells including plasmid, RNA, and viral-based delivery methods⁶. Recently, direct delivery of pre-complexed sgRNA-Cas9 Ribonucleoproteins (RNPs) has emerged as a powerful and efficient tool in CRISPR-Cas9-based genome editing⁷. The direct delivery of pre-complexed CRISPR-Cas9 RNPs has several distinct advantages, namely: 1) RNPs bypass the need for cellular transcription and translation, 2) RNPs are rapidly cleared, which may increase specificity by reducing available time for off-target cleavage, and 3) RNPs contain no foreign DNA/RNA elements which circumvents the introduction of non-native sequences into the host genome through random integration. Together, these attributes likely provide a short-lived burst of on-target CRISPR editing while minimizing off-target effects.

We describe a simple and efficient protocol for introducing site-specific genomic changes in C. elegans. This protocol includes targeting sgRNA and single stranded oligonucleotide (ssODN) HDR template design, sgRNA-Cas9 RNP complexing and delivery, and a genotyping strategy for the unequivocal identification of properly edited animals. Using this strategy, not only can the desired site-specific changes be recovered, but other non-specific indel mutations may also be recovered. Thus, our strategy permits the generation of an allelic series using a single strategy, where both mono-allelic, bi-allelic, and indel mutants can be generated in the F₁ generation.

Protocol

All animal care and experimental procedures followed the guideline from the National Institutes of Health and the Institutional Animal Care and Use Committee (IACUC) at the University of Michigan. Use RNase-free solutions and pipette tips throughout the protocol. Clean the working area, pipettes, tubes, and centrifuge with RNase Decontamination solution following the manufacturer guidelines (see Materials Table).

1. sgRNA Target Selection

1. Using a web browser, open the webpage: http://crispor.tefor.net⁸
   1. Input ~60 base pairs (bp) of sequence flanking the desired edit of interest (i.e. 30 bp 5' and 3' of desired edit).
   2. Select the Caenorhabditis elegans genome from the pulldown menu.
   3. Select the Protospacer Adjacent Motif (20 bp-NGG - SpCas9, SpCas9-HF1, eSPCas9 1.1) from the pulldown menu.
   4. Click submit. In the results page, choose the top ranked target sequence closest to the edit of interest. If there are no suitable target sites within the flanking 60 bp input sequence, expand the query sequence up to 100 bp (i.e. 50 bp either side of the desired edit).
2. From an available source, e.g. synthego, obtain a 20-mer target sgRNA with the following specifications: 3 nmol; no modifications.
3. Upon receipt, centrifuge lyophilized sgRNA containing tube at >12,000 x g for 1 min at room temperature. Add 60 µL of nuclease-free TE to the tube, and gently re-suspend by pipetting up and down 15 - 20 times using a P200 pipette. The final concentration is 50 µM.

2. Homology-directed Repair Template Design

1. Design an ssODN HDR template containing: 1) the mutation of interest, 2) a unique in-frame restriction endonuclease site, 3) a silent mutation of one or both of the Gs within the NGG PAM sequence, and 4) 50 bp 5' and 3' homology arms flanking the first and last mutation. (Figure 1C)^9,10.
   1. If mutation of the NGG PAM sequence is not possible, introduce 5 - 6 silent mutations within the sgRNA target recognition sequence to prevent sgRNA-mediated cleavage of the ssODN HDR template.
2. From an available source, e.g. idtdna, obtain an "Ultramer DNA oligonucleotide" with the following parameters: Scale: 4 nmol; Formulation: None; Purification: Standard Desalting.
3. Upon receipt, centrifuge lyophilized ssODN containing tube at >12,000 x g for 1 min at room temperature. Gently re-suspend ssODN to a final concentration of 100 µM in nuclease-free ddH₂O by pipetting up and down 15 - 20 times using a P200 pipette.

3. Design Genotyping Primers

1. Design a primer set flanking the genome modification to generate a ~400 - 700 bp PCR amplicon¹¹.
2. Optimize PCR cycling conditions to produce a single and specific amplicon¹¹.

4. Prepare Injection Mix

1. Centrifuge Table 1 reagents at maximum speed for 2 min at 4 °C.
2. In the order listed, add Table 1 reagents to a nuclease-free PCR tube.
3. Mix thoroughly by gently pipetting up and down 10 times with a P20 pipette.
4. Incubate injection mix for 10 min at room temperature.

5. Injection Protocol

1. Load injection micropipette with approximately one-half of the injection mix¹². Save remaining injection mix in case the injection needle clogs or breaks.
2. Break micropipette tip so that ~20 - 30 p.s.i. produces a gradual flowing solution¹².
   NOTE: Larger diameter tips negatively affect animal health and decrease F₁ progeny yield.
3. Inject 10 - 15 young adult worms in both gonadal arms if possible¹². If not, injection into one gonadal arm is sufficient.
4. Allow injected animals (P₀) to recover for 1 - 2 h at room temperature on an OP50-seeded 35 mm nematode growth media (NGM) plate. Subsequently, single injected P₀ animals to individual OP50-seeded 35 mm NGM plates using a platinum wire worm pick¹².

6. Screen P₀ Plates and Single mCherry(+) F₁s

1. Two days post-injection, identify P₀ plates containing F₁ progeny expressing mCherry in the pharynx using a fluorescent stereomicroscope (Figure 1D, day 2 - 3). Choose three P₀ plates that contain the most mCherry(+) F₁ animals.
2. From each of the three selected P₀ plates, single 8 - 12 mCherry(+) F₁ animals for a total of 24-36 mCherry(+) F₁s using a worm pick (Figure 1D, day 2 - 3).
   NOTE: mCherry(-) animals can also be singled from these P₀ plates, but the probability of identifying correctly edited animals is much lower (Figure 2A).
3. Allow individual F₁s to self-fertilize and lay eggs for 1 - 2 days at room temperature.

7. Single Worm PCR and Genotyping

1. After 1 - 2 days of egg-laying, transfer the F₁s into individual PCR strip tube caps containing 7 µL of Worm Lysis Buffer using a worm pick (Table 2, Figure 1D, day 4 - 5).
2. Centrifuge PCR tubes at maximum speed for 1 min at room temperature to bring animals to the tube bottom, and freeze tubes at -80 °C for 1 h.
   NOTE: Worms can be stored at -80 °C indefinitely.
3. Lyse frozen worms in a thermocycler using the following program: 60 °C for 60 min, 95 °C for 15 min, 4 °C hold.
4. Set-up PCR mastermix as shown in Table 3.
5. Add 21 µL of PCR mastermix into clean PCR tubes, and then add 4 µL of worm lysis from step 3. Mix well using a pipette and run PCR program following the manufacturers guidelines (see Materials Table).
6. Purify PCR reactions using a DNA Clean and Concentrate kit, following the manufacturer instructions (see Materials Table). Elute DNA by adding 10 µL water to the spin column.
7. Set-up restriction enzyme mastermix as shown in Table 4.
8. Add 10 µL of the restriction enzyme mastermix to each cleaned PCR reaction and incubate for 1 - 2 h at 37 °C¹³.
9. Separate digested PCR products on a 1.5% agarose gel run at 120 V using 1x Tris-acetate-EDTA (TAE) buffer¹⁴.

8. Identification and Sequence Verification of Edited Animals

1. Examine enzyme digested samples for the presence of bands that indicate potential edited animals¹⁴ (Figure 1D, day 4 - 5).
   NOTE: Load an N2 control to identify potential indel mutations which can be observed as shifts in band size and/or unexpected and complex restriction enzyme digestion banding patterns.
2. After identifying potential edited animals, use the remaining 3 µL of worm lysis and repeat the PCR reaction. Do not clean or restriction enzyme digest the PCR reaction after the program is complete. Load the PCR reaction on a 1% agarose gel, separate and extract the band using a gel extraction kit¹⁵ (see Materials Table).
3. Sanger sequence¹⁶ gel extracted DNA using the F1 primer to identify correctly edited animals (Figure 1A)
   NOTE: Heterozygote reads will contain overlaid peaks due to the presence of the wild type and engineered allele.
4. To obtain homozygous animals from verified heterozygous edited animals, single 8 - 12 F₂ animals and perform steps 7 - 8.

Results

Mutations in human superoxide dismutase 1 (SOD1) account for ~10 - 20% of familial amyotrophic lateral sclerosis, a devastating neurodegenerative disease that invariably leads to paralysis and death¹⁷. Human SOD-1 is an evolutionarily conserved protein sharing 55% identity and 70% similarity with the C. elegans SOD-1 protein (Figure 1B). To demonstrate the simplicity, feasibility, and efficiency of the CRISPR-Cas9 RNP...

Discussion

The CRISPR-Cas9 system is a powerful and effective tool for precisely modifying the genome of model organisms. Here, we demonstrate that chimeric sgRNAs²⁰ coupled with ssODN HDR templates enable the highly efficient generation of genomic point mutations in C. elegans. Importantly, we demonstrate that RNP delivery produces high editing efficiency when fluorescence is used as a co-selection marker, highlighting the ease and reliability of the technique.

The major...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CRISPRevolution sgRNA  EZ Kit	Synthego Inc.		chimeric sgRNA
Nuclease-free TE	Synthego Inc.	provided with the sgRNA kit EZ kit
Nuclease-free water	Synthego Inc.	provided with the sgRNA kit EZ kit
4 nmole Ultramer DNA Oligo	Integrated DNA Technologies		ssODN HDR template
Alt-R S.p. HiFi Cas9 Nuclease 3NLS	Integrated DNA Technologies	1078728	Cas9 protein
pCFJ90	Addgene	19327	Pmyo-2::mCherry Marker Plasmid
KCl	Sigma	P5405
HEPES	Sigma	H4034
DNA Clean & Concentrator	Zymo Research	D4004
Zymoclean Gel DNA Recovery Kit	Zymo Research	D4002
Q5 Hot Start High-Fidelity 2X Master Mix	New England Biolabs	M0494L
Proteinase K	Sigma	P2308
Glass Borosilicate Glass Micropipettes	Sutter Instruments	BF100-78-10	OD: 1.0mm. ID: 0.78mm
Trizma Hydrochloride	Sigma	T5941
MgCl2	Sigma	M2393
NP-40	Sigma	74385
Tween-20	Fisher Scientific	BP337-100
RNaseZap Decontamination Solution	Fisher Scientific	AM9780