PITCh KnockIn

An easy and efficient method for gene knock-in

What are the PITCh systems ?

 The PITCh (Precise Integration into Target Chromosome) systems are the alternative methods of CRISPR-Cas9- and TALEN-mediated knock-in 1 .


Advantages

 These systems enable seamless gene knock-in with extremely short homologous sequences (10–40 bp), which can be easily added to the donor vector by PCR and
In-Fusion cloning 1 .

 The PITCh knock-in is based on one of the repair mechanism of DNA double strand break,microhomology-mediated end-joining (MMEJ). Since MMEJ is independent on homologous recombination (HR), which is mainly used for gene knock-in, PITCh system can be utilized in cells and organisms with low HR frequency.

Applications

 The PITCh systems have currently been applied in cultured cells (HEK293T, HeLa, HCT116, CHO), silkworms, frogs, zebrafish, and mice.







Web Application :
"PITCh designer 2.x"

PITCh designer 2.x is a free online tool to design
sgRNA and homology arms for gene knock-in.

 This tool only needs sequence around the target region. After the sequence submission, followed by the selection of the target base, the application automatically designs sgRNA, microhomologies, and primers for constructing a donor vector for PITCh knock-in and for genotyping.

Go to the PITCh designer 2.x >>>


Procedure

1. Paste sequence :


  Paste the FASTA-formatted sequence around the target region, and click submit.

[NOTICE]
  • The acceptable length of the sequence is 100-15,000[bp].
  • The target base should not be located on either end of the sequence.
    The tool requires 48-bp flanking sequences on the both sides of the target base regardless of the lengths of microhomologies.
PITCh designer 2.x :

1. Paste sequence :

Options :

 You can use design options for the PITCh knock-in.



1 : Reading frame

 Three reading frames or 'No frame' can be selected (as shown in the right figure).

Options :
  • Frame 1 (default)
  • Frame 2
  • Frame 3
  • No frame
...

2 : Adjustment of reading frame

 PITCh designer 2.x offers two methods to prevent frameshifting.

Options :
  • 5'C-insertion (default)

    C-insertion mode fills in cytosine base(s) to adjust the reading frame.
    The number of fill-in bases depends on the selected frame and the position of knock-in.
  • Codon deletion

    Codon deletion mode removes extra base(s).
...

3 : Knock-in cassette

 The tool designs the primers for the construction of a targeting vector to knock-in the following two preset cassettes or user-specifyed sequence.

Options :
  • User defined insert
...

The plasmids for EGFP2APuroR knock-in are available from Addgene:
https://www.addgene.org/browse/article/16395/

4-6 : Microhomology lengths and PAM sequence requirement


4,6 : Lengths of left/right microhomologies
 The acceptable length of each microhomology arm is 10-40[bp]
(The default length is 40 bp.)

5 : PAM sequence requirement
 PAM sequence can be defined using IUB codes.
(The default sequence is 5'-NGG-3' ; SpCas9)

*PITCh system using Cpf1 is unconfirmed.
...
IUB codes 2
Code Definition Mnemonic
G G Guanine
A A Adenine
T T Thymine
C C Cytosine
R A, G puRines
Y C, T pYrimidines
M A, C aMino
K G, T Keto
S C, G Strong
W A, T Weak
H A, C, T not G
B C, G, T not A
V A, C, G not T
D A, G, T not C
N A, C, G, T aNy
PAM sequences 3
Species PAM sequence ( 5' -> 3' )
Streptococcus pyogenes (SP) ; SpCas9 NGG
Staphylococcus aureus (SA) ; SaCas9 NNGRRT or NNGRR(N)
Neisseria meningitidis (NM) ; NmCas9 NNNNGATT
Streptococcus thermophilus (ST) ; St1Cas9 NNAGAAW

7 : Genomes of target organism

 The tool can show CRISPRdirect or GGGenome URL link to check sgRNA and primer specificity against genomic DNA, if you choose genome sequence.
 CRISPRdirect and GGGenome perform exhaustive searches against genomic sequence. Approxmately 600 genomes are recorded in these tools.
...

8 : Parameters of genotyping primers

 The tool helps the users to set parameters (Tm or GC%) for the primers and automatically designs primers for genotyping.

 This automated tool designs the primers according to the following procedure.

  1. Perform exhaustive primer design in the distance of 100-200 bp from the corresponding positions.
  2. Select primers according to the user's setting.
  3. The optimized primers are chosen using rankings based on range of parameters and primer length.
    4. *The longer primers have a higher priority in the primers with the same Tm and GC%.
...

2. Select target :

 The application displays nucleotide buttons, which are color-coded according to their targetability and molecular species.

Color Targetability Base
Green Adenine
Red Thymine
Blue Guanine
Yellow Cytocine
Light Blue Any
Gray Out of range Any

 After selecting the desired position, the webtool begins to design the sequences for PITCh knock-in.

2. Select target:

3. Show result :

 The design results consist of sequence information and graphical images of knock-in and donor vector construction ( as shown in the left figure ).

Downloadable items
  • Summary
    • The designed sequences are exported to tab-delimited text or CSV format, which can be viewed in Microsoft Excel.

  • Whole sequence of donor vector
  • Graphical image of knock-in
  • Graphical image of donor vector construction

3. Show result :

Primer viewer

 The PITCh designer 2.x can not only automatically select best primer pair for genotyping but help users to manually choose it on this viewer ( as shown in the right figure ).
 This viewer can show primers for left junction / right junction / out-out PCR. By clicking on the arrow button on the left button form, you can change the primer type.

 After clicking the left button for selecting primer pair, the viewer displays sequence information at the bottom of the graphical map. The sequence information can also be download by clicking the "DOWNLOAD SEQENCE" button.

Primer viewer :

Efficiency annotation

 The PITCh designer 2.1 can analyze the knock-in design and adds "efficiency annotations" to the knock-in design. We showed that the group of annotated knock-in design significantly achieved the high efficiency of precise knock-in compared with the no annotation group in HEK293T cells ( as shown in the right figure ).
 The sequence features, which contribute to the gain of precise knock-in efficiency, will be shown in Desirable labels. On the other hand, Undesirable labels indicate the feature causing less knock-in efficiency.

 The features were determined based on our previous research (Nakamae et al., 2022). The PITCh designer searches sequence context resulting in the mutation patterns affecting the knock-in efficiency.

Efficiency annotation: