CRISPR/Cas9 Construct Design Part 2 – Designing the Repair Template

PAM (Protospacer Adjacent Motif)

A short DNA sequence (typically NGG for SpCas9) required for Cas9 binding; Cas9 unwinds DNA upon binding a PAM, then checks crRNA complementarity

Protospacer

The region in the target DNA (approximately 20 nucleotides) that is recognized by the crRNA; located immediately upstream of the PAM

crRNA spacer

The 20 nucleotides at the 5' end of the crRNA that must be complementary to the DNA upstream of the PAM for Cas9 to create a DSB

Non-Homologous End Joining (NHEJ)

A DSB repair pathway that does not require a homologous template; the two broken ends are ligated together, usually resulting in small insertions or deletions (indels) at the repair site

Homology Directed Repair (HDR)

A DSB repair pathway that uses a homologous DNA template (such as a sister chromatid or exogenous donor DNA) for accurate repair; extremely accurate, almost always restoring original sequence

When NHEJ predominates

NHEJ is the fastest DSB repair pathway and can occur in all phases of the cell cycle, but it predominates in G1 phase; it is the most common way DSBs are repaired in most cells, especially mammals

When HDR is effective

HDR repair pathways are effective only during S, G2, and M phases in somatic cells; they are inhibited during G1; during meiosis, HDR is available especially during prophase

NHEJ proteins

Ku proteins (Ku70 and Ku80) which bind to DSBs as a heterodimer and recruit DNA-dependent protein kinases; these kinases then recruit accessory factors including a DNA polymerase that can add nucleotides and a DNA ligase that joins the broken ends

HDR proteins

1. MRE11-Rad50-Nbs1 (MRN complex) which binds to DSBs and promotes resection;

2. CtIP which works with MRN;

3. exonucleases (including MRE11) that resect 5' ends creating 3' overhangs;

4. RPA proteins which accumulate on and protect ssDNA;

5. Rad52 or BRCA2 (depending on species) which displace RPA;

6. and RAD51 (eukaryotic RecA homolog) which forms a nucleofilament that invades duplex DNA to form a D-loop

HDR proteins 1

MRE11-Rad50-Nbs1 (MRN complex) which binds to DSBs and promotes resection;

CtIP which works with MRN;

HDR proteins 2

exonucleases (including MRE11) that resect 5' ends creating 3' overhangs;

RPA proteins which accumulate on and protect ssDNA;

HDR proteins 3

Rad52 or BRCA2 (depending on species) which displace RPA;

and RAD51 (eukaryotic RecA homolog) which forms a nucleofilament that invades duplex DNA to form a D-loop

Ku proteins (Ku70/Ku80)

Proteins that recognize DSBs and promote NHEJ;

they bind to DSBs as a heterodimer and recruit DNA-dependent protein kinases, which recruit accessory factors including a DNA polymerase and ligase

MRN complex (MRE11-Rad50-Nbs1)

A hallmark of DSBs repaired by HDR; MRN recruits CtIP, and MRE11 (an exonuclease) promotes resection of 5' ends, creating single-stranded 3' overhangs that displace Ku and inhibit NHEJ

RPA proteins

Proteins that immediately accumulate on single-stranded DNA created during HDR; RPA-bound ssDNA cannot pair with other ssDNA, protecting it from spurious matches but preventing further repair until RPA is displaced

RAD51

A recombinase (eukaryotic homolog of RecA) that displaces RPA from ssDNA to form a RAD51-ssDNA nucleofilament; mediates homology search by invading duplex DNA and facilitating base-pairing with complementary sequence

D-loop

The three-stranded intermediate formed when RAD51-ssDNA invades duplex DNA and pairs with complementary sequence; the common starting point for all HDR pathways

Repair template (donor DNA)

Exogenous DNA added to cells that contains the desired DNA sequence change or "edit"; includes homology arms matching sequences adjacent to the DSB

Homology arms

DNA sequences in the repair template that match the DNA sequence adjacent to the DSB; used by the cell's HDR machinery to accurately repair the break using the donor DNA as a template

Optimal homology arm length for dsDNA repair template in C. elegans

60 nucleotides on both sides of the insert is sufficient when using double-stranded DNA from a plasmid as the repair template

Optimal homology arm length for ssODN in C. elegans

35 nucleotides on both sides of the insert is optimal when using single-stranded oligodeoxyribonucleotides (ssODNs)

Efficiency of repair vs distance from DSB

The efficiency of repair decreases with increasing distance between the insert site and the DSB; the tag insertion site does NOT have to be exactly at the DSB location

Why repair template must alter PAM or crRNA binding sequence

Unless the tag disrupts the PAM or the crRNA complementary sequence, the repaired DNA will be vulnerable to re-cutting by CRISPR-Cas machinery still present in the cell

Synthesis-Dependent Strand Annealing (SDSA)

An HDR mechanism for gene conversion where resected DSB ends pair with complementary strands in the donor, are extended by DNA synthesis, then withdraw and anneal back at the locus

Linker sequence

A small sequence of 2-3 amino acids (DNA sequence GGATCG) inserted between the protein of interest and the tag to allow both proteins to fold properly; prevents direct abutment that could disrupt tertiary structure

N-terminal tag linker placement

If tagging the N-terminus, the linker sequence should go between the tag and the start of the protein (tag-linker-protein)

C-terminal tag linker placement

If tagging the C-terminus, the linker sequence should go between the protein and the tag (protein-linker-tag)

Why tag at the protein ends rather than middle

Adding a tag to the N- or C-terminus is less likely to disrupt protein structure, function, and expression pattern compared to adding it in the middle of a protein

GFP tag disadvantage

GFP is a relatively large protein, making it more likely to disrupt protein function and expression compared to small peptide tags

GFP tag advantage

GFP is auto-fluorescent and can be viewed in vivo without fixing worms, allowing observation of dynamic protein localization in living, developing C. elegans (which are transparent)

Silent mutation

A change in DNA sequence that does not change the amino acid sequence of the protein; can be used to alter the PAM or protospacer while preserving protein function

Codon usage frequency table

A table indicating which codons encode which amino acids and their tRNA frequency in C. elegans; when changing codons, change to a codon with similar or higher usage frequency to maintain expression levels

Prioritizing nucleotide changes to disrupt Cas9 recognition

Mismatches proximal to the PAM are more likely to disrupt Cas9 function; prioritize altering nucleotides closest to the PAM. Some scientists alter every possible nucleotide in the crRNA-binding region

Repair template final components

Tag sequence (the change being made), linker sequence, possibly changes to protospacer or PAM, and 60 nucleotide homology arms upstream and downstream of all modifications

How to determine if a PAM contains a wobble nucleotide

Determine the open reading frame by counting forwards from START or backwards from STOP (depending on N- or C-terminal tagging); check if the PAM's GG nucleotides are in the third (wobble) position of a codon

UTR changes affecting gene function

Changes in UTRs can affect mRNA stability, localization, translation efficiency, and binding of regulatory proteins; 3'UTRs are particularly important for post-transcriptional regulation

How to predict if a UTR change is neutral

Compare DNA sequences of homologous genes across species; regions that are evolutionarily conserved are more likely to have functional significance; non-conserved regions may tolerate changes better

NHEJ vs HDR for precise genome edits

Homology Directed Repair (HDR) can be exploited to make precise edits; NHEJ typically causes random indels. Both pathways could potentially be used, but HDR is used for precise insertions

Required components of a repair template for making a missense mutation

Regions of homology to the target at both ends of the repair template, one nucleotide that differs from target DNA at the desired mutation site, and perhaps changes to ensure Cas9 does not recut the DNA

Why make a silent mutation in the PAM site of repair template

To prevent Cas9 from recutting the repaired DNA while preserving the amino acid sequence of the protein

Alternative if unable to make silent mutation in PAM

Alter the nucleotide sequence of the protospacer region (the crRNA binding site); make at least 4 silent changes, prioritizing nucleotides proximal to the PAM

GFP sequence optimization

The egfp (enhanced GFP) sequence used in this experiment is optimized for C. elegans codon usage to ensure proper expression

ApE software

A plasmid Editor software used to annotate DNA sequences; can highlight features like start codons (green), stop codons (red), PAM sites, and crRNA binding sites

Where to insert C-terminal tag in unc-32

Insert the tag sequence immediately before the stop codon, with the linker sequence between the unc-32 coding sequence and the GFP tag

Repair template homology arm definition

The terminal 60 nucleotides on both the 5' and 3' ends of the repair template must be identical to the target DNA sequence; these flank any changes made (tag, linker, PAM alterations)

SDSA from Paix et al. 2017

Showed that as long as the insert is within 10-15 nucleotides of the DSB, the polarity of the repair template did not affect success rate; resection creates 3' overhangs that pair with donor DNA

dsDNA vs ssODN for large insertions

For larger edits (>3000 nucleotides like GFP), double-stranded donor DNA works with higher efficiency; for shorter edits (<50 bases), single-stranded oligodeoxyribonucleotides (ssODNs) are more efficient