Skip to content

Synonymous PE

Introduce Synonymous Mutation in pegRNA

Additioal edit for efficient prime editing

Prime editing efficiency is determined by various factors beyond RT-PBS. One well-known factor is the inhibitory effect by the mismatch repair (MMR) system. To prevent prime editing efficiency inhibition by MMR, a strategy of introducing additional mutations into pegRNA can be used (Chen et al., 2021, Cell).

However, introducing additional mutations may not only reduce the impact of MMR but also decrease the activity of pegRNA. Moreover, if the target site for genome editing lies within the protein coding sequence (CDS) region, it is necessary to introduce synonymous (silent) mutations that do not affect protein function. Considering these factors, designing and selecting which mutations to introduce additionally into pegRNA can be a rather cumbersome task.

SynonymousPE module in GenET

GenET's SynonymousPE provides the functionality to easily design pegRNAs with additional mutations introduced. Specifically, it identifies possible synonymous mutations that align with the CDS, making it useful for biological research applications.

Furthermore, SynonymousPE is directly compatible with DeepPrime, allowing for the creation of pegRNAs with synonymous mutations directly from pegRNAs designed in DeepPrime. 

from genet.predict import DeepPrime
from genet.design import SynonymousPE

# DeepPrime pipeline
seq_wt   = 'CTTGCCTGTCTCTGTGGGCTGAAGGCTGTTCCCTGTTTCCTTCAGCTCTACGTCTCCTCCGAGAGCCGCTTCAACACCCTGGCCGAGTTGGTTCATCATCATTCAACGGTGGCCGACGGGC'
seq_ed   = 'CTTGCCTGTCTCTGTGGGCTGAAGGCTGTTCCCTGTTTCCTTCAGCTCTACGTCTCCTCCAAGAGCCGCTTCAACACCCTGGCCGAGTTGGTTCATCATCATTCAACGGTGGCCGACGGGC'

pegrna = DeepPrime('ABL1_ex4_pos21G_A', seq_wt, seq_ed, edit_type='sub', edit_len=1)

pe2max_output = pegrna.predict(pe_system='PE2max', cell_type='HEK293T')


# Select a pegRNA record that you want to add synonymous mutation 
dp_record = pe2max_output.iloc[9]

# Setup SynonymousPE input parameters 
synony_pegrna = SynonymousPE(dp_record, ref_seq=seq_wt,
                            frame=0, cds_start=45, cds_end=121)

# print selected RTT containing synonymous mutation
print(synony_pegrna.extension)

You can find more detailed information about the usage of DeepPrime in the documentation of the genet.predict module documentation. In the provided example, a synonymous mutation was introduced into one of the many designed pegRNAs. Since each pegRNA may have different regions and lengths for RTT, the types of additional mutations that can be introduced may also vary.

It is recommended to first select appropriate pegRNAs based on DeepPrime scores and then further optimize them using SynonymousPE.

SynonymousPE's input parameters

de_record: pd.Series
An object containing information about the pegRNA. When designed using DeepPrime, if you retrieve a specific index from the resulting DataFrame, you can use it directly.

ref_seq: str
The DNA sequence information that serves as the target for the pegRNA. SynonymousPE assumes the presence of a CDS within this sequence and introduces synonymous mutations according to predefined rules. The genetic sequence used as the reference for frame, cds_start, and cds_end is defined below.

frame: int
This value represents the frame of the reference sequence (ref_seq). It is determined by the codon frame of the CDS and is expressed as 0, 1, or 2. For example, if the CDS sequence starts from the beginning of a codon (3bp), you would input frame as 0. It's crucial to input the correct frame, as an inaccurate frame could lead to synonymous mutations resulting in a completely different amino acid sequence.

codon_frame

cds_start: int
ref_seq Indicates the position where the CDS starts in the ref_seq.

cds_end: int
ref_seq Indicates the position where the CDS ends in the ref_seq.

adj_rha: bool
When a synonymous mutation occurs in the RHA of the RT template, the efficiency of prime editing may significantly decrease if the RHA becomes too short. As an alternative, you can artificially extend the length of the RT template. adj_rha is an option to specify whether to extend the length of the RT template by the same amount as the shortening of the RHA when it becomes too short (default: True). If adj_rha is set to False, regardless of where the synonymous mutation occurs, the length of the RT template remains the same as that of the original pegRNA.

SynonymousPE input examples

The information regarding the frame and CDS positioning in SynonymousPE can be somewhat complex to grasp at first use.

Example 1:

Example_1

from genet.design import SynonymousPE

# pegRNA record & ref_seq
dp_record = dp_record
ref_seq   = 'CTCTACGTCTCCTCCGAGAGCCGCTTCAACACCCTGGCCGAGTTGGTTCATCATCATTCAACGGTGGCCGACGGGCTCATCACCACGCTCCATTATCCAGCCCCAAAGCGCAACAAGCCCA'

# Setup SynonymousPE 
synony_pegrna = SynonymousPE(dp_record, ref_seq=ref_seq, frame=0, cds_start=0, cds_end=121)

Example 2:

Example_2

from genet.design import SynonymousPE

# pegRNA record & ref_seq
dp_record = dp_record
ref_seq   = 'CTACGTCTCCTCCGAGAGCCGCTTCAACACCCTGGCCGAGTTGGTTCATCATCATTCAACGGTGGCCGACGGGCTCATCACCACGCTCCATTATCCAGCCCCAAAGCGCAACAAGCCCACT'

# Setup SynonymousPE 
synony_pegrna = SynonymousPE(dp_record, ref_seq=ref_seq, frame=2, cds_start=0, cds_end=121)

Example 3:

Example_3

from genet.design import SynonymousPE

# pegRNA record & ref_seq
dp_record = dp_record
ref_seq   = 'TTGCCTGTCTCTGTGGGCTGAAGGCTGTTCCCTGTTTCCTTCAGCTCTACGTCTCCTCCGAGAGCCGCTTCAACACCCTGGCCGAGTTGGTTCATCATCATTCAACGGTGGCCGACGGGCT'

# Setup SynonymousPE 
synony_pegrna = SynonymousPE(dp_record, ref_seq=ref_seq, frame=1, cds_start=44, cds_end=121)

SynonymousPEs mutation Priority

When introducing a 1bp point mutation into the pegRNA RT template, there may be multiple synonymous mutations. SynonymousPE assigns priority scores to each possible mutation and selects the RT template with the highest priority mutation for additional incorporation, providing it as .extension. The criteria for determining the priority of SynonymousPE are as follows.

  • Priority 1: If there is a synonymous mutation that eliminates the PAM (GG), it is assigned the highest priority.
  • Priority 2: When introduced into the Left Homology Arm (LHA), mutations closer to the intended prime-edit position are assigned higher priority.
  • Priority 3: When introduced into the Right Homology Arm (RHA), mutations closer to the intended prime-edit position are assigned higher priority.

stack: Introduction of synonymous mutations

This functionality allows for the insertion of multiple synonymous mutations into a single RT template. By default, SynonymousPE selects the highest priority mutation among those introducing only one synonymous mutation into the RT template and provides it as .extension. However, if you wish to introduce two or more synonymous mutations simultaneously, you can use the .stack method.

from genet.design import SynonymousPE

# Create a SynonymousPE object
synony_pegrna = design.SynonymousPE(dp_record, ref_seq=seq_wt, frame=0)

# Generate an RT template with up to three synonymous mutations introduced
three_syn = synony_pegrna.stack(num=3)

.stack It incorporates possible synonymous mutations in order of assigned priority. When each synonymous mutation is introduced individually, there is no change in the amino acid sequence. However, when two mutations are introduced simultaneously, there may be a change in the amino acid sequence. In such cases, the process moves on to the next priority mutation.

Objects Available for Retrieval in SynonymousPE

Below are the objects available for accessing key results.

Name Type Description
.extension str Selected RT-PBS sequence containing recommanded synonymous mutation.
.synonymous pd.DataFrame Table of available synonymous mutations and corresponding informations for given pegRNA.
.mutations pd.DataFrame Table of all available additional mutations and corresponding informations for given pegRNA.
.rec pd.Series Record of pegRNA from input.

다음과 같이 값을 확인할 수 있다. 

from genet.design import SynonymousPE

# Create a SynonymousPE object
synony_pegrna = design.SynonymousPE(dp_record, ref_seq=seq_wt, frame=0)

# Check for available synonymous mutations for introduction
synony_pegrna.synonymous

Results:

Codon_WT Codon_Mut Codon_MutPos Codon_RefStart Codon_RefEnd RTT_DNA_frame RTT_DNA_Strand AminoAcid_WT AminoAcid_Mut Silent_check Mut_pos Mut_refpos PAM_Mut Priority Edit_class RTT_DNA_Mut
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTCGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC 5 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 5 62 CG 1 PAM_edit CCGTCGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTAGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC 5 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 5 62 AG 2 PAM_edit CCGTAGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTTGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC 5 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 5 62 TG 2 PAM_edit CCGTTGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAAGAGCCGCAGTCAGATCCTAGCGTCGAGCCC 8 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 8 65 GG 15 RHA_edit CCGTGGAAGAGCCGCAGTCAGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAACCGCAGTCAGATCCTAGCGTCGAGCCC 11 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 11 68 GG 18 RHA_edit CCGTGGAGGAACCGCAGTCAGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCCCAGTCAGATCCTAGCGTCGAGCCC 14 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 14 71 GG 20 RHA_edit CCGTGGAGGAGCCCCAGTCAGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCACAGTCAGATCCTAGCGTCGAGCCC 14 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 14 71 GG 21 RHA_edit CCGTGGAGGAGCCACAGTCAGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCTCAGTCAGATCCTAGCGTCGAGCCC 14 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 14 71 GG 21 RHA_edit CCGTGGAGGAGCCTCAGTCAGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAATCAGATCCTAGCGTCGAGCCC 17 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 17 74 GG 24 RHA_edit CCGTGGAGGAGCCGCAATCAGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCTGATCCTAGCGTCGAGCCC 20 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 20 77 GG 26 RHA_edit CCGTGGAGGAGCCGCAGTCTGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCCGATCCTAGCGTCGAGCCC 20 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 20 77 GG 27 RHA_edit CCGTGGAGGAGCCGCAGTCCGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCGGATCCTAGCGTCGAGCCC 20 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 20 77 GG 27 RHA_edit CCGTGGAGGAGCCGCAGTCGGATCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGACCCTAGCGTCGAGCCC 23 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 23 80 GG 30 RHA_edit CCGTGGAGGAGCCGCAGTCAGACCCTAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGATCCAAGCGTCGAGCCC 26 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 26 83 GG 32 RHA_edit CCGTGGAGGAGCCGCAGTCAGATCCAAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGATCCGAGCGTCGAGCCC 26 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 26 83 GG 33 RHA_edit CCGTGGAGGAGCCGCAGTCAGATCCGAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGATCCCAGCGTCGAGCCC 26 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 26 83 GG 33 RHA_edit CCGTGGAGGAGCCGCAGTCAGATCCCAGCGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGATCCTAGTGTCGAGCCC 29 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 29 86 GG 36 RHA_edit CCGTGGAGGAGCCGCAGTCAGATCCTAGTGTCGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTGGAGCCC 32 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 32 89 GG 38 RHA_edit CCGTGGAGGAGCCGCAGTCAGATCCTAGCGTGGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTAGAGCCC 32 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 32 89 GG 39 RHA_edit CCGTGGAGGAGCCGCAGTCAGATCCTAGCGTAGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTTGAGCCC 32 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 32 89 GG 39 RHA_edit CCGTGGAGGAGCCGCAGTCAGATCCTAGCGTTGAGCCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAACCC 35 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 35 92 GG 42 RHA_edit CCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAACCC
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCG 38 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 38 95 GG 44 RHA_edit CCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCG
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCA 38 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 38 95 GG 45 RHA_edit CCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCA
GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCC GCCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCT 38 58 97 1 + VEEPQSDPSVEP VEEPQSDPSVEP TRUE 38 95 GG 45 RHA_edit CCGTGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCT

Below are the objects containing parameters and other detailed information used for design within the code.

Name Type Description
.sID str Sample ID from pegRNA record.
.rtpbs_dna str Reverse complementary DNA sequence of RT-PBS.
.pbs_dna str Reverse complementary DNA sequence of PBS.
.rtt_dna str Reverse complementary DNA sequence of RT template
.rtt_len int Length of RT template
.wt_seq str WT target sequence (74nt)
.ed_seq str Prime-edited target sequence (74nt)
.edit_pos int Position of intended prime editing. Start from nick site.
.ref_seq str Reference target sequence from input.
.frame int Frame of coding sequence (CDS) from input.
.cds_start int Start position of CDS
.cds_end int End position of CDS
.strand str Strand of pegRNA. It should '+' or '-'.
.rtt_frame int Frame of RT template.
.codon_le int Length of left end (LE) of codon sequence.
.codon_re int Length of right end (RE) of codon sequence.
.codon_RTT str Codon sequence with RT template.

You can check the values as follows. 

from genet.design import SynonymousPE

synony_pegrna = design.SynonymousPE(dp_record, ref_seq=seq_wt, frame=0)
synony_pegrna.ref_seq