Our Genome Editing Tools

Introduction

recent years, novel genome editing techniques have been developed. These techniques use bacterial nucleases which are guided by small RNA molecules to a specific site in the genome to cleave the DNA. This causes a double strand break. This break can be repaired in cells, but the most common system (non homologous end joining (NHEJ) is error prone and results in small insertions or deletions (InDels) of nucleotides at the site of the double strand break. The bacterial nucleases are now used to specifically edit the genome to our liking.

There are multiple methods to perform these genomic edits:


Acronyms

Cas9

Nuclease type II used for genome editing. We work with Cas9 from S. pyogenes and several mutants of this nuclease.

CRISPR

Clustered regularly interspaced short palindromic repeats.

Single guide RNA

Cas9 are guided to a specific site in the genome by a combination of two RNA molecules. One which recognizes the target DNA and a second trans activation cr(CRISPR)RNA (TracrRNA). These have been combined into a single guide (sg) RNA for easy use of the system.

Off target effects

Many reports show that genome editing via Cas9 mediated mutation has off target effects where Cas9 does not only cut the desired sequence. The major off targets are indicated

Short guide RNA

Cas makes use of an RNA which recognizes the target DNA and a trans activation cr(CRISPR)RNA (TracrRNA). These have been combined into a small guide (sg) RNA.

Seed sequence

First 7 nucleotides after PAM sequence, vital for correct binding of Cas9, total guide is required for correct nuclease activity.

PAM

Protospacer adjacent motif, Cas9 recognizes NGG, Cas9-VRQR-HF1 recognizes NGAN, Cpf1 recognizes NTTT.

on the Zhang website when designing the guide. These can be sequenced to test for the likeliest off targets. However, whole genome sequencing is the only way to exclude off target effects in your final cell/organism. Off target effects are reduced when the nickase mutant of Cas9 is used or when the Cas9-HF1 mutant form of Cas9 is used (to be reproduced).


Generation of knockouts/mutants

To acquire knockouts/mutants of specific genes there are two Cas9 mediated mutation strategies:

  1. The first uses the WT Cas9 in combination with one guide RNA. Cas9 then cleaves the DNA blunt ended at the site of the guide RNA. Non-homologous end joining then repairs the DNA. This is an error prone DNA repair mechanism. Also, as long as the PAM sequence is still intact, Cas9 will keep cleaving the DNA, making it likely that an error will occur in the DNA repair. This will result in integrations or deletions of DNA (InDels). The amount of nucleotides added/removed in InDels is random (most likely between 1-6 bp). By chance 66% of InDels will result in out of frame reading frame and likely resulting in a premature STOP codon. This will lead to truncation of the protein or nonsense mediated decay and a (null) mutant. The latter needs to be determined for each founder. Also, the guide may bind other places in the genome. These will also be cleaved and mutated. Therefore, the absence of other mutations in the founder(s) need to (should) be verified by NGS.
    Note: New variants of Cas9 have been and are currently generated which minimize off target effects. Cas9-HF1 and Cas9-HF4 are in house, as is eSpCas9. Also a variant which recognizes a different PAM sequence (NGAN) (Cas9-VRQR-HF1) is currently in house.
  2. The second strategy makes use of a mutant form of Cas9, the nickase mutant (D10A). This mutant does not cleave the DNA blunt ended, but generates a nick in the DNA. By combining two guide RNAs in opposite directions (~50 bp spaced), this region in the genome will be deleted. By using two guides that can only delete when they are closely spaced, the specificity improves and off target effects can be minimized.
  3. The third strategy makes use of a base editor version of Cas9. A kinase dead or nickase version of Cas9 is fused to cytidine deaminase and specifically converts C or G to A or T nucleotides. This requires that the desired mutation is part of the guide sequence. This strategy is not very efficient (improved when using the nickase version of Cas9 (BE3), but no or very little off targets are reported and no scar is left in the DNA.
CAS9 Nickase

Generation of insertional mutants

To generate insertional mutants, cleavage with WT Cas9, and a subsequent homologous driven repair (HDR) mechanism to introduce a specific mutation or gene (or both) is used. Not only a guide RNA and Cas9 are introduced in the cells/embryos, but also a repair DNA molecule which has the specific mutation or insert. We have successfully used several repair constructs.

  1. A small repair construct with 50-100 bp homology arms on each side of the guide RNA cleavage point. This is ordered as an oligo (maximum 200 nt oligos can be ordered). The desired mutation is introduced with one of the homology arms.
  2. A small integration construct with a STOP codon cassette in hard to target sites in the genome. Some loci more often result in InDels which do not alter the reading frame, not resulting in STOP codons. The integration of a stop codon cassette then always results in a STOP codon in every reading frame. A 20 bp overhang (homology arm) on each side of the cleavage point is used in combination with the 35 bp cassette. This is ordered as an oligo.
  3. A large repair construct with 300 bp-1 kb overhang with a specific point mutation and an antibiotics selection cassette framed by LoxP sites. This is subcloned. This form of integration is preferred in difficult to target cells. Do keep in mind that lox sites will leave a scar in the genome, so either a truncation mutation is generated or the selection cassette is placed in an intron. However, always check the transcripts for correct splicing. Novel, scar-less approaches, are currently being developed.

Alternative PAM sequence

CAS9A strong benefit of the mouse model has always been the possibility to generate gene specific, endogenously regulated, reporter models. This is done by introducing a (fluorescent) reporter or Cre protein in the 3’UTR of a gene in combination with an IRES or a V2A sequence. This ensures that the transcript ratio between gene and reporter is equal and to an endogenous level. The 3’UTR of a gene is by definition AT rich. However, Cas9 makes use of a PAM sequence which is CG rich and therefore very unsuitable to target the 3’UTR of most genes. This can be circumvented by using alternative nucleases or Cas9 nuclease variants which recognize different PAM sequences. For instance, Cpf1 recognizes PAMs which are AT rich and makes a staggered cut.

Transcriptional regulation

CRISPR/Cas9 is not only used to make mutations or integrations in the genome, but can also be modified to regulate transcription. A kinase dead form of Cas9 is then used (Cas9d) which is unable to cut the DNA but is only recruited to the site recognized by the sgRNA. When Cas9 is then fused to activator proteins like VP16, transcription blocking proteins, like KREB or even (de)methylation enzymes transcription can be altered.